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ENVIRONMENTAL RISK MANAGEMENT AUTHORITY NGÄ KAIWHAKATÜPATO WHAKARARU TAIAO

Use of Antibiotic Resistance Marker Genes in Genetically Modified Organisms

Deborah Read ERMA New Zealand

December 2000 IBSN 0-478-21515-0 $9.95 excl GST

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Foreword This is the first in a series of reference reports on key HSNO issues, which ERMA New Zealand wishes to produce. The idea behind this so called ‘generic issues programme’ is that we need to take time to properly research and consider matters which affect many HSNO applications. There either isn’t the time or resourcing to do so in the context of individual applications, or the relevance is very restricted for individual applications. The current report provides a good example. Antibiotic resistance marker genes have been commonly used in the development of GMOs, and the issue of what concerns this might raise comes up repeatedly. The thorough analysis in this report should enable such concerns in the future to be considered more quickly, more thoroughly but at lower cost than in the past. This makes sense for everyone. The pattern followed in the development of these reports will differ from case to case. However, there is a commitment to making the process open, to enable a wide range of people to contribute or comment if they have views or information to offer. A second project in this series is underway, and it is to develop a better approach to dealing with Māori cultural concerns over genetic modification. The subject matter is quite different and the project will thus develop in its own way. Regretfully no more projects in this series will be undertaken for the time being because we do not have sufficient funding for this. However I am personally very keen to see this programme restarted if funding issues can be restored. Bas Walker Chief Executive

ER-GI-01-1 12/00

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Acknowledgements This report was prepared by Dr Deborah Read, Senior Public Health Advisor, ERMA New Zealand. The report was externally peer reviewed by: Dr Marion Healy,

Australia New Zealand Food Authority

Associate Professor Brian Jordan,

Massey University Dr Maggie Brett,

Limited, and

Institute of Food, Nutrition and Human Health,

Kenepuru Science Centre, Institute of Environmental Science and Research

Professor Barry Scott,

Institute of Molecular BioSciences, Massey University.

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Table of Contents Foreword ........................................................................................................................ 2 Acknowledgements ....................................................................................................... 3 Table of Contents........................................................................................................... 4 Terms of Reference ....................................................................................................... 9 Preface.......................................................................................................................... 10 Method .......................................................................................................................... 10 Summary ...................................................................................................................... 11 Evaluation of the implications of the use of antibiotic resistance marker genes in genetically modified organisms ........................................................................................................................ 11 Antibiotics in New Zealand........................................................................................................ 11 Horizontal gene transfer ............................................................................................................ 12 Antibiotic resistance genes and human health....................................................................... 12 Antibiotic resistance marker genes and the environment ..................................................... 14 Comparative analysis of the risks of antibiotic resistance occurring by other means .......... 14 Pros and cons of alternative selectable markers, including removal of antibiotic resistance marker genes from GMOs for release ........................................................................................... 16 Recommendations of agencies and their basis about the use of antibiotic resistance marker genes ................................................................................................................................................ 16 Conclusions about the use of antibiotic resistance marker genes in GMOs, in the context of applications under Part V of the Hazardous Substances and New Organisms (HSNO) Act 1996 ................................................................................................................................................... 17

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Introduction ......................................................................................................... 18 What is gene technology? .............................................................................................................. 18 What is a selectable marker? ......................................................................................................... 19 Antibiotic resistance marker genes............................................................................................... 19 What antibiotic resistance genes are used as markers? ............................................................ 20 Kanamycin resistance ............................................................................................................... 20 Hygromycin resistance .............................................................................................................. 22

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Streptomycin resistance............................................................................................................ 22 Ampicillin resistance ................................................................................................................. 23 Others .......................................................................................................................................... 23

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Antibiotics and antibiotic resistance ................................................................. 25 What are antibiotics used for? ....................................................................................................... 25 Mechanisms of antibiotic resistance............................................................................................. 25 Development of antibiotic resistance............................................................................................ 25 The public health impact of antibiotic resistance ........................................................................ 27 Antibiotics in New Zealand............................................................................................................. 27 Aminoglycosides........................................................................................................................ 27 Other antibiotics ......................................................................................................................... 29 Antibiotic resistance in New Zealand ............................................................................................ 30 Is antibiotic resistance lost? .......................................................................................................... 31

3.

Antibiotic resistance marker genes in food ...................................................... 32 Introduction...................................................................................................................................... 32 The concept of substantial equivalence ....................................................................................... 32 The general basis for assessment of the potential health impact of marker genes and their gene products .................................................................................................................................. 33 Antibiotic resistance genes and food safety ................................................................................ 33 Direct consequences of the antibiotic resistance gene ......................................................... 33 Direct consequences of the gene product encoded by the antibiotic resistance gene ..... 34 Indirect consequences of the effects of the antibiotic resistance gene or its gene product ...................................................................................................................................................... 36 Horizontal gene transfer in humans .............................................................................................. 37 Potential gene transfer to oral micro-organisms .................................................................... 37 Potential gene transfer to gut epithelial cells or micro-organisms....................................... 37 Inactivation of antibiotic by the gene product ........................................................................ 42

4.

Horizontal gene transfer mechanisms in bacteria............................................ 44

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Transduction .................................................................................................................................... 44 Conjugation...................................................................................................................................... 45 Transformation ................................................................................................................................ 45 Evidence for gene transfer between bacteria............................................................................... 46 Prevalence of antibiotic resistance genes.................................................................................... 47

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Ecological issues ................................................................................................ 49 Introduction...................................................................................................................................... 49 Antibiotic resistance and weediness ............................................................................................ 49 Gene transfer .............................................................................................................................. 49 Antibiotics in soil........................................................................................................................ 50 Pleiotrophic effects ......................................................................................................................... 51 Use of antibiotic as a herbicide...................................................................................................... 51 Horizontal gene transfer in the environment................................................................................ 52 Prerequisites for transformation .............................................................................................. 53 Persistence of DNA .................................................................................................................... 53 Transformation of soil micro-organisms ................................................................................. 54 Approaches to evaluate possible horizontal gene transfer of plant DNA to soil microorganisms ................................................................................................................................... 58

6. The New Zealand and international approach to antibiotic resistance marker genes ............................................................................................................................ 62 The New Zealand approach to antibiotic resistance marker genes ........................................... 62 Environmental Risk Management Authority (ERMA).............................................................. 62 Antibiotic Resistance Expert Panel .......................................................................................... 62 Australia New Zealand Food Authority (ANZFA) .................................................................... 62 The approach of other countries and international organisations to antibiotic resistance marker genes ................................................................................................................................... 63 Australia ...................................................................................................................................... 63 Norway......................................................................................................................................... 64 United Kingdom.......................................................................................................................... 64

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Codex Alimentarius Commission ............................................................................................. 66 European Commission .............................................................................................................. 66 Nordic Working Group on Food Toxicology and Risk Assessment ..................................... 66 United States............................................................................................................................... 67 World Health Organisation ........................................................................................................ 68

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Alternative strategies to antibiotic resistance marker genes.......................... 70 Characteristics of a selectable marker.......................................................................................... 70 Choice of a selectable marker........................................................................................................ 70 Disadvantages of antibiotic resistance marker genes ................................................................ 70 Herbicide resistance ....................................................................................................................... 71 Metabolic markers ........................................................................................................................... 72 Other selectable markers in plants................................................................................................ 74 Other selectable markers in mammalian cells ............................................................................. 74 Other markers in yeast and micro-organisms.............................................................................. 75 Alternatives to the selection of antibiotic resistance genes in bacteria.................................... 76 Elimination of the selectable marker ............................................................................................. 76 No selectable marker or reporter gene .................................................................................... 76 Reporter gene only..................................................................................................................... 77 Inactivation of the selectable marker gene.............................................................................. 77 Removal of the selectable marker gene................................................................................... 78 Modulation of gene expression ................................................................................................ 81 Intron-containing antibiotic resistance genes ........................................................................ 81

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Conclusion........................................................................................................... 83

9.

References ........................................................................................................... 86

10. Glossary ............................................................................................................... 96 11. Appendices .......................................................................................................... 97 Appendix I: Evaluation of the kanamycin resistance gene ......................................................... 97

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Impact of the gene product NPTII ............................................................................................. 97 Impact of the DNA in food ......................................................................................................... 98 Impact on the efficacy of antibiotics ........................................................................................ 99 Impact on the environment ..................................................................................................... 100 Appendix II: Genetically modified insect resistant maize ......................................................... 101 Appendix III: Summary of submissions ...................................................................................... 104

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Terms of Reference

Terms of Reference 1. The objective is to produce a reference report for the Environmental Risk Management Authority that: • Evaluates the implications of the use of antibiotic resistance marker genes in genetically modified organisms (GMOs). This will include the technical mechanisms available for the transfer of antibiotic resistance to other organisms, the likelihood of occurrence and under what circumstances, and the practical effects of such transfers, ie the level and nature of risk presented. The influence of the antibiotics involved will be considered. • Provides some degree of comparative analysis of the risks of antibiotic resistance occurring by other means, whether that represented by GMOs is additive/cumulative and is significant in that context. • Considers the pros and cons of alternative selectable markers, including removal of antibiotic resistance marker genes from GMOs for release. This will include other risks that may be increased by the alternatives. 2. The work will include a review of the relevant scientific literature, will include informed inputs from sources within New Zealand and will consider the recommendations of other agencies, and their basis, about the use of antibiotic resistance marker genes. 3. The final report will draw conclusions about the use of antibiotic resistance marker genes in GMOs, in the context of applications under Part V of the Hazardous Substances and New Organisms (HSNO) Act 1996. It will also include identification of the unresolved issues and the level of certainty/uncertainty that can be attributed to the current state of knowledge.

Use of Antibiotic Resistance Marker Genes in GMOs

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Preface and Method

Preface As most of the scientific literature focuses on antibiotic resistance marker genes in relation to genetically modified (GM) plants the emphasis in this report is on plants, in particular food crops, rather than other GMOs. The focus of the report is also on the implications of the use of antibiotic resistance genes in GMOs for human health rather than the environment. An overview of horizontal gene transfer in the environment is however included since gene transfer events in the environment indirectly effect humans as a result of resistant micro-organisms contaminating food or water.

Method A literature search of on-line bibliographic databases was undertaken using DIALOG for publications in English concerning antibiotic resistance marker genes and alternative marker strategies. The search period covered 10 years up until October 1999. Bibliographies of identified papers were also examined. Some references published after the search period that have come to the author’s attention have also been reviewed. The final peer reviewed draft was available for public comment. Submissions are summarised in Appendix III.


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Summary

Summary Risk assessment methods address the known and foreseeable risks derived from experience and from extrapolation from experience. Risk assessment cannot offer an absolute guarantee of safety because it is always carried out under some uncertainty and is limited by the state of available knowledge. Antibiotic resistance marker genes are used to select transformed bacteria during the initial cloning and manipulation of genes in the bacterium Escherichia coli (E. coli) and to select the few transformed cells following introduction of the gene construct into the recipient host organism. Most antibiotic resistance marker genes confer resistance by inactivating the antibiotic through modifying enzymes. The most frequently used antibiotic resistance genes are the ampicillin resistance gene (bla) for bacterial transformation and the kanamycin resistance gene (nptII) for plant transformation. Antibiotic resistance marker genes serve no useful purpose in the GMO. Key points from the report that address each of the Terms of Reference are summarised below. Evaluation of the implications of the use of antibiotic resistance marker genes in genetically modified organisms Antibiotics in New Zealand Annual antibiotic use in New Zealand is estimated to be 74.9 tonnes. Human use accounts for about 53 percent of this amount. In addition to antibiotic use in humans and animals about 1.2 tonnes of streptomycin is used annually in horticulture (Antibiotic Resistance Expert Panel, 1999). The main antibiotics currently used in New Zealand that are potentially affected by the use of antibiotic resistance marker genes in GMOs are the aminoglycoside (eg kanamycin, neomycin) and beta-lactam (eg ampicillin) antibiotics. The estimated annual use of aminoglycosides in New Zealand is 2,242 kg (35 kg humans; 2,207 kg animals) (Antibiotic Resistance Expert Panel, 1999). The amount used in molecular genetics research is estimated as only hundreds of grams (B Scott, personal communication, May 2000). Kanamycin is used only in serious systemic infections and when the infecting micro-organism is resistant to other antibiotics. It is a reserve agent for tuberculosis. It is not absorbed by mouth and has been given orally to reduce gut micro-organisms (eg preoperative bowel preparation). Neomycin is used topically in the management of skin, eye and ear infections. It is poorly absorbed by mouth and has been given orally to reduce gut micro-organisms (eg preoperative bowel preparation). Although neomycin has limited clinical use it is an important antibiotic in veterinary medicine. Resistance mediated by the nptII gene is also demonstrable for some other aminoglycosides and although these are not in widespread clinical or veterinary use they are still used for infections resistant to other antibiotics.


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Summary

Ampicillin belongs to the clinically important group of antibiotics called the beta-lactams that include the penicillins and the cephalosporins. The beta-lactams, in particular the penicillins, account for about 67 percent of the estimated annual human antibiotic use (Antibiotic Resistance Expert Panel, 1999). It is given orally as well as intravenously or intramuscularly and is used in the treatment of a variety of infections. Ampicillin is also used in veterinary medicine. Beta-lactam antibiotics account for 26 percent of the animal antibiotic use in New Zealand (Antibiotic Resistance Expert Panel, 1999). Horizontal gene transfer Horizontal gene transfer has been reported between distantly related bacteria, and from bacteria to yeast, mammalian cells and plant cells. The few examples of transfer from plants to bacteria indicated by DNA sequence comparisons and the lack of experimental confirmation suggest that the frequency of evolutionary successful gene transfer from plants to bacteria is extremely low. However this inference is based on a small number of experimental studies and indications in the scientific literature. Detection of horizontal gene transfer events is difficult due to the limitations of the techniques available. Unequivocal proof requires isolation of the putative transformed bacteria for thorough genetic characterisation. The rate of gene transfer from plants to bacteria is insignificant compared to gene transfer between micro-organisms. Almost any type of bacterium has the potential to transfer DNA to any other type of bacterium if it contains a broad host range gene transfer element. Antibiotic resistance genes and human health The presence of the antibiotic resistance gene by itself is not associated with any adverse health effects. There is in vitro evidence that free DNA in human saliva is capable of transforming a naturally competent human oral bacterium (Mercer et al, 1999). Since the regions preceding the stomach are likely to have the highest concentrations of intact DNA entering with the diet further research is needed to establish whether transformation of oral bacteria occurs at significant frequencies in vivo. Although most ingested DNA is likely to be degraded and diluted in the human gastro-intestinal tract, natural transformation of gut epithelial cells or micro-organisms cannot be completely ruled out. Research in mice indicates that DNA can survive digestion and uptake by gut epithelial cells occurs, however at levels of DNA intake unlikely to be encountered in a normal diet (Schubbert et al, 1997). The mechanism of DNA uptake by gut epithelial cells is unknown and its significance is unclear. If DNA uptake does occur in humans critical factors are the presence of regulatory sequences that allow gene expression and the presence of selective pressure. Without selective pressure it is highly Use of Antibiotic Resistance Marker Genes in GMOs

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Summary

unlikely that genes taken up by gut epithelial cells would be expressed even if they were integrated into the genome. Homology between the prokaryotic DNA sequences in an antibiotic resistance marker gene and the recipient host’s DNA is more likely to be found in gut micro-organisms which are prokaryotic than in gut epithelial cells which are eukaryotic. The probability of integration and expression of a marker gene is therefore greater in gut micro-organisms than in gut epithelial cells. Transformation is considered the only natural mechanism that can be involved in gene transfer from plants to gut micro-organisms. Transformation requires access to free DNA that is present at the time and place in which competent bacteria develop or reside. Conditions in the mammalian intestine are probably more conducive to gene transfer than conditions found elsewhere in nature. High concentrations of bacteria mean encounters between different types of bacteria occur readily and residence time in the intestine is long enough to provide opportunities for gene transfer. However there has been little direct evidence to support this hypothesis. The introduction of bacterial genes, bacterial regulatory sequences and bacterial origins of replication into GM plants increases the degree of sequence homology between GM plant DNA and the genomes of competent bacteria. It has been hypothesised that this could increase the probability of DNA transfer from plants to bacteria by favouring homologous recombination in the recipient host (Paget et al, 1998; Gebhard and Smalla, 1998). If gene transfer and expression were to occur it is most likely from viable GM food micro-organisms (or released GM micro-organisms that can be unintentionally ingested) followed by raw GM plant material or the uncooked seed of GM plants. It is least likely from highly processed GM food microorganisms, plant or animal material. Viable GM micro-organisms may remain intact through the gut and gene transfer could be achieved through conjugation with gut micro-organisms. It is generally considered that the probability of antibiotic resistance genes being transferred from GM plant material or other eukaryotes to either gut epithelial cells or micro-organisms is extremely low given the complexity of sequential steps required for gene transfer and gene expression. However few experimental data are available to support this theoretical assessment. Rare transfer events can be amplified very rapidly under selective pressure. The health impact would be significant if a gene conferring resistance to a clinically important antibiotic was transferred and expressed in a pathogenic micro-organism normally treated with that antibiotic. Assuming that antibiotic resistant bacteria have not previously colonised the gastro-intestinal tract there is no risk of compromised therapeutic efficacy of the antibiotic unless GM food containing the particular antibiotic resistance gene is consumed at the time the antibiotic is administered orally. From what is known about mechanisms of horizontal gene transfer between organisms and the survival of intact DNA following processing and digestion it is concluded that the risk is highest for


13

Summary

ingested viable GM micro-organisms, in particular if the antibiotic resistance gene confers resistance to a clinically important antibiotic, and lowest for highly processed GM food. Antibiotic resistance marker genes and the environment Laboratory and field studies have been unable to confirm gene transfer occurs from plants or other eukaryotes to naturally occurring soil or plant-associated bacteria. Two studies using artificially introduced homology between plant and bacterium DNA have shown transfer of antibiotic resistance marker genes from plants to bacteria under optimised laboratory conditions (Gebhard and Smalla, 1998; de Vries and Wackernagel, 1998). In the event that transfer from the plant genome to soil micro-organisms did occur in most cases there would be no selective pressure. Exceptions are streptomycin use in horticulture or when manure is used as fertiliser following in-feed antibiotic use for animal growth promotion and prophylaxis. Soil is a reservoir of micro-organisms with the capacity to produce antibiotics. There is a lack of information about the extent of selective pressure of different antibiotics in soil and the soil conditions that promote such selective pressure. Extrapolation of transformation frequencies from microcosm studies to the environment could be misleading because the concentrations of transforming DNA in situ are not known. Lack of information on the prevalence of naturally competent bacteria in the environment, the frequency of transformation and environmental factors triggering transformation impairs predictions of the extent of horizontal gene transfer from plants to micro-organisms. Comparative analysis of the risks of antibiotic resistance occurring by other means Antibiotics are used to eliminate bacteria and maintain the health of humans; animals, including fish; and some plants. Global selective pressure has occurred as the same antibiotics are used in all three areas. Antibiotics have the undesirable side effect of selecting for the growth and spread of otherwise rare resistant micro-organisms. The extent of antibiotic resistance depends on the presence of the antibiotic and a resistance gene, the transfer and expression of the resistance gene, and spread of resistant bacteria. Given the presence of both the antibiotic and a resistance gene, antibiotic resistant bacteria will be selected and propagated. Resistance genes can be transferred between bacteria of the same or unrelated species. Bacteria that develop resistance to one agent in a class typically show cross-resistance to other antibiotics in the same class.


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Summary

The major source of the development and spread of antibiotic resistant micro-organisms in humans is the human use (and often overuse) of antibiotics in both the community and hospitals. Within hospitals person-to-person transmission is aided if infection control practices are less than ideal. Some antibiotic resistant bacteria occur naturally in the environment but many are a result of contamination with human and animal excreta in sewage, slurry and manure. Antibiotic resistance is therefore also acquired through ingestion of resistant micro-organisms from animals or soil contaminating food or water. Enteric human pathogens (eg Salmonella) are commonly acquired from animals and in some instances these micro-organisms acquire resistance to antibiotics used for growth promotion, prophylactic or therapeutic use in animals. Gaps in information about patterns of antibiotic resistance in New Zealand prevent the conclusion that antibiotic use in animals is exacerbating antibiotic resistance in human pathogens. Most overseas data support the concept that antibiotic resistance appeared only after the emergence of strong selective pressure resulting from the massive use of industrially made antibiotics in human and veterinary medicine and as food supplements for farm animals. Antibiotic resistance is common in human commensal gut micro-organisms even in the absence of concurrent or recent antibiotic consumption (Levy et al, 1988). Such resistant bacteria are a reservoir of resistance genes that are potentially transferable, directly or indirectly, to human pathogens. There are geographical variations within and between countries in the incidence of specific antibiotic resistance. Global movement of people, breeding stock and food means resistance in one area can spread to another. Limited monitoring of some zoonotic micro-organisms from human, animal and food sources and data from some hospitals indicate that there is a low level of acquired antibiotic resistance in New Zealand compared to the United States, the United Kingdom and the European Union (Antibiotic Resistance Expert Panel, 1999). Untreatable infections due to bacteria that are resistant to all available antibiotics are still exceptional but do occur. In New Zealand hospitals it is not uncommon to encounter bacterial infections that are resistant to all but one or two antibiotics (Lang and Blackmore, 1999). It is more likely that antibiotic resistance genes would be introduced into gut micro-organisms through transfer between naturally occurring ingested contaminating micro-organisms and gut micro-organisms than through transfer from DNA released during digestion of GM plant or animal material. The probability that transfer occurs between ingested GM micro-organisms and gut microorganisms is likely to be the same as between non-GM micro-organisms and gut micro-organisms. The potential impact of the use of antibiotic resistance genes in GMOs on the prevalence of antibiotic resistance, though additive, is far less significant than the impact of the current use of antibiotics in humans and animals in New Zealand. However antibiotic resistance is receiving increasing scrutiny nationally and internationally, and there are an increasing number of strategies being implemented with the aim of curbing all antibiotic use. Use of Antibiotic Resistance Marker Genes in GMOs

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Summary

Pros and cons of alternative selectable markers, including removal of antibiotic resistance marker genes from GMOs for release Alternative strategies to the use of antibiotic resistance genes in GMOs include no selectable marker or reporter gene, a reporter gene only, inactivation of the selectable marker gene and removal of the selectable marker gene. As yet these approaches are far from routine or from being generally applicable. Use of alternative markers, in particular genes concerned with various metabolic pathways, or subsequent removal or inactivation of the antibiotic resistance gene is becoming more common. Recently developed new marker strategies are based on the use of selectable genes that give the transformed cells a metabolic advantage compared to the non-transformed cells which are starved with a concomitant slow reduction in viability (eg the GUS gene and the manA gene with cytokinin glucuronides and mannose respectively as selective agents). Removal of selectable marker genes allows the same markers to be used repeatedly in subsequent transformations into the same host and minimises the amount of foreign DNA to that involved in conferring the desired traits. This will be important as successive rounds of genetic modification become more prevalent. Recommendations of agencies and their basis about the use of antibiotic resistance marker genes There appears to be an emerging international consensus to evaluate each GMO containing antibiotic resistance marker genes on a case-by-case basis. Marker genes that encode resistance to clinically important antibiotics should not be present in food or feed in view of the potential for gene transfer and expression, albeit rare, to occur. Viable GM food micro-organisms should not contain antibiotic resistance marker genes since gene transfer to gut micro-organisms could occur as a result of conjugation. The difference of opinion between countries about the presence of antibiotic resistance marker genes in GMOs arises mainly over the question of whether extremely low but non-zero risks of increased antibiotic resistance are acceptable. Irrespective of the scientific conclusions removal of the antibiotic resistance gene from the final GM plant or use of alternative strategies is now being recommended whenever feasible (Donaldson and May, 1999; European Commission, 1999; WHO, 2000). Elimination of antibiotic resistance marker genes could have positive effects on consumer acceptance by alleviating perceived risks.


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Summary

Conclusions about the use of antibiotic resistance marker genes in GMOs, in the context of applications under Part V of the Hazardous Substances and New Organisms (HSNO) Act 1996 The scientific literature on the use of antibiotic resistance marker genes in GMOs is characterised by many opinions and relatively few data. Each GMO containing antibiotic resistance marker genes should be evaluated on a case-by-case basis. Of particular relevance is the clinical importance of the antibiotic and the probability that the antibiotic would be encountered. The potential contribution to antibiotic resistance from antibiotic resistance marker genes is likely to be very small in comparison to that arising from antibiotic use in medicine and agriculture, and insignificant in terms of health impact unless the antibiotic is clinically important in New Zealand. Limited data exist on the patterns of antibiotic resistance in New Zealand. Studies that include selective pressure during exposure of competent bacteria with DNA are needed. Although there is information about phenotypic resistance few studies have identified the gene responsible. Monitoring should be at the genotypic rather than phenotypic level. Since all resistance genes originate from micro-organisms monitoring should distinguish a gene that has been transferred from a GM plant. Relatively little consideration has been given to the potential cumulative consequences of a rare event in a scenario of widespread cultivation of GM plants and ingestion of raw and unprocessed GM plant material by millions of people and animals. The overall importance of a value to be protected, such as human health, for the overall risk assessment and the resulting regulatory constraints is frequently a matter of science policy rather than a scientific issue (Doblhoff-Dier et al, 1999). Although there is no scientific evidence that antibiotic resistance genes have transferred from GM plants to pathogenic micro-organisms, and the probability of such an event is considered to be extremely low, a precautionary approach could be adopted that recommends use of an alternative marker such as the manA gene or removal of the antibiotic resistance gene from the final GMO. Such an approach would take into account public perception of the role of antibiotic resistance marker genes in antibiotic resistance.


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Introduction

1.

Introduction

What is gene technology? 1

Gene technology involves the isolation and subsequent introduction of discrete DNA segments containing the gene(s) of interest into a recipient organism. It is also used to control, increase or turn off specific functions within an organism.

2

Gene technology is characterised by the capacity to transfer genes between unrelated species and to specify the genes that will be transferred. Its application to animals is still in its infancy compared with its use in plants and micro-organisms.

3

Using special enzymes DNA from the donor organism that contains the desired gene is cut into shorter fragments that are then separated and purified. The gene is then removed using enzymes that cut the DNA in defined places.

4

In order to ensure that the desired gene is incorporated into the host cell nucleus as efficiently as possible a carrier system or vector is normally used. A typical vector is made up of a circular piece of DNA from a bacterium or virus that is cut using enzymes so that the gene can be inserted. The vector carrying the gene is multiplied, usually in bacterial cells, to produce a sufficient quantity of the gene construct. Cells are transformed by insertion of the gene construct.

5

The vector must contain a promoter that enables the gene to function in the host. More recently careful selection of the promoter is starting to allow gene expression to be targeted (eg to the leaves and roots of plants).

6

Bacteria are transformed by direct DNA uptake. Plant cells are transformed by insertion of the gene construct into the cell nucleus by one of several methods including: • direct DNA uptake mediated by polyethylene glycol treatment or electroporation; • micro-injection of DNA; • particle bombardment (firing tiny particles coated with the DNA); and • use of the soil bacterium Agrobacterium tumefaciens as a vehicle to carry the DNA.

7

Micro-injection into embryos is the standard method of transformation for mammals. The embryo is transferred to a recipient mother and in a small proportion of injected embryos the introduced DNA is integrated into the animal’s genome. Particle bombardment is also used to transform animal cells.

8

Transformation is an inefficient process. The frequency with which transformed cells are obtained varies from species to species and with various transformation methods but in plants is often as low as 1 in 10,000 or 1 in 100,000 of the cells treated (WHO, 1993).

9

Recovery of the transformed cells depends on selection for the rare transformation event and against the non-transformed cells. This is achieved by using a selectable marker.


18

Introduction

What is a selectable marker? 10

A marker is used to indicate a successful genetic alteration in a cell in the early stages of research. Markers are vital to the transformation process but usually secondary to the objective of developing a GMO. The exception is herbicide resistance genes where the gene is the marker and the herbicide resistant plant also has agronomic value. Expression of selectable marker genes gives an almost absolute advantage to GMOs in the laboratory. Generally no selective conditions are expected outside the laboratory with the exception of herbicide resistance genes.

11

The selectable marker allows the researcher to readily identify and select the few transformed cells and saves having to assay for incorporation of the desired gene by more complex and expensive methods.

12

There are three classes of selectable marker genes. They are: • genes that confer resistance to antibiotics • genes that confer resistance to herbicides, and • genes concerned with various metabolic pathways.

Antibiotic resistance marker genes 13

During the 1980s antibiotic resistance genes were introduced as markers for selection. The ampicillin resistance gene is the most common antibiotic resistance marker for bacterial (E. coli) transformation whereas for plant transformation markers largely comprise genes resistant to the aminoglycoside antibiotics.

14

As a result of the presence of the antibiotic resistance gene the few cells that contain the introduced DNA are able to grow and multiply in the presence of the antibiotic unlike the surrounding non-transformed cells that either die or have severely inhibited growth. If plants, the transformed cells are subsequently regenerated into GMOs.

15

Antibiotic resistance genes are used at two stages during the genetic modification of eukaryotes. The first stage is in bacteria during the development of the gene construct containing the genes to be introduced into the eukaryote and the second stage is in the selection of transformed eukaryotic cells following introduction of the gene construct.

16

The bacterium E. coli is commonly used as the host organism during the development of the gene construct. To allow identification of E. coli cells containing the construct it is necessary to use a marker gene that is functional in the bacterium. It carries bacterial promoters that will not be expressed or needed in an eukaryotic cell. The marker gene does not end up in the GM plant or animal genome unless transformation methods using the entire construct are used.

17

An antibiotic resistance marker gene may be included with the desired gene in the construct that is introduced into an eukaryotic cell. Both genes will have eukaryotic promoters to allow expression in eukaryotic cells. Although the marker gene is required only immediately after


19

Introduction

the genetic modification procedure to facilitate identification of transformed cells it may remain in most, if not all, cells of the resulting GM plant or animal, and be expressed. 18

Not all of the antibiotic resistance genes are equally effective for selecting transformed cells due to the level of sensitivity of the plant species or variety, or the ability of the gene to protect the transformed cells from the effects of the selective agent (WHO, 1993).

19

As a component of the genetic modification the antibiotic resistance gene(s) must be taken into account when considering the release of the GMO and/or its product. Depending on the nature of the organism, release may cause the gene and its product to be present in the environment on a large scale. This has to be considered in the context of whether, and to what extent, the gene is already present in the environment from other sources.

20

Antibiotic resistance genes are the most prevalent selectable marker genes. They were used in 31 of the 52 US Food and Drug Administration (FDA) consultations regarding GM crops to the end of 1997. The kanamycin resistance gene (nptII) accounted for 27 of these and the hygromycin resistance gene (hpt) the rest. Of the 30 consultations that were completed by the end of 1997, in two cases nptII had been segregated out and the final GM variety did not contain the resistance gene.

21

In 19 of the 52 FDA consultations the GM crop had one or more antibiotic resistance genes under a bacterial promoter incorporated into the plant genome. In most cases the ampicillin resistance gene (bla) was involved. In some cases only partial fragments of the genes were incorporated and therefore the genes were not functional (US FDA, 1998).

22

The presence of antibiotic resistance genes in GMOs in New Zealand is currently confined to GMOs developed in or imported into containment, and some imported processed GM food.

What antibiotic resistance genes are used as markers? 23

Choice of antibiotic resistance marker genes that have been used in genetic modification has been influenced by availability and familiarity.

Kanamycin resistance 24

The most commonly used selectable marker is the kanamycin resistance gene from a transposon (Tn5) from E. coli K12.

25

The kanamycin resistance gene was one of the first markers to be developed and was available from many laboratories. It is widely used as a selectable marker in the transformation of organisms as diverse as bacteria, yeast, plants and animals. It was the first marker used in plant genetic modification and the first GM food plant, the delayed softening tomato Flavr Savr, commercialised in 1994, contains this gene. Many hectares of GM crops containing this gene are being grown commercially in North America, Europe, Asia and Australia (cotton only) (Conner, 1997).


20

Introduction

26

At least 18 genes have been cloned which encode aminoglycoside modifying enzymes conferring resistance to kanamycin (Nap et al, 1992). The enzymes catalyse three types of reactions – acetylation, adenylation and phosphorylation. Four are acetyltransferases, three are adenyltransferases and nine phosphotransferases. Of these the most commonly used is aph(3')-IIa (or aphA-2, also called nptII) which encodes aminoglycoside 3'-phosphotransferase II (APH(3')II, also called neomycin phosphotransferase II (NPTII)). Detailed biosafety evaluations have been published on the nptII gene and its product (see Appendix I).

27

There is no structural similarity among the different classes of genes and enzymes. There are even differences among the members of each type of enzymes (Karenlampi, 1996). The relative occurrence of aminoglycoside modifying enzymes shows local and temporal variations in populations. They are found in a variety of bacterial species and also in the antibiotic-producing actinomycetes (Nap et al, 1992).

28

NPTII is not novel to humans because NPTII producing kanamycin resistant bacteria are often present in the normal gut microflora. Cell death and lysis of these bacteria would result in exposure of the immunocompetent cells of the gastro-intestinal tract to the same protein as is present in the GMO (Karenlampi, 1996).

29

Many aminoglycosides are phosphorylated by NPTII but it does not confer resistance to all aminoglycosides because of widely different phosphorylation rates for the different substrates (Redenbaugh et al, 1993).

30

NPTII confers resistance to kanamycin, neomycin, paromomycin, ribostamycin, butirosin, gentamicin A and B and geneticin (G418). Of these, kanamycin, neomycin and paromomycin are used in human medicine. It does not confer resistance to gentamicin that is used therapeutically, as gentamicin A and B are minor components of the commercial drug (US FDA, 1994). Geneticin is used only for in vitro experimentation. Resistance is not conferred to amikacin although enzymatic activity for this substrate is detectable in vitro. The resistance profile of another enzyme (APH(3')-VI) includes amikacin (Karenlampi, 1996). The gene used in the Flavr Savr tomato encodes an enzyme that confers resistance only to neomycin, kanamycin and geneticin (Redenbaugh et al, 1994).

31

Kanamycin is normally used as the selective agent.

32

It has been suggested that neomycin phosphotransferase might modify intracellular phosphorylation when introduced into mammalian cells. However several researchers have expressed the kanamycin resistance gene in human cells and have introduced them into people via gene therapy without reported adverse effects (Karenlampi, 1996).

33

There are no safety data on the other identified kanamycin resistance genes. It is possible that several might be useful as selectable markers because of their ability to detoxify kanamycin in the plant. The effects of these genes in a food plant can be expected to be similar to nptII unless there are different substrates for the various enzymes (Karenlampi, 1996).

34

Practically the kanamycin resistance gene provides excellent selection because of the properties of the antibiotic kanamycin and the resistance gene. With kanamycin, cell death is slow enough to permit expression of the added marker gene and inactivation of the antibiotic


21

Introduction

to occur before cell death. With some selective agents, exposure to the selective treatment must be delayed to ensure that cells with the gene will survive. As a result cells are left longer in tissue culture which is less desirable as somaclonal variation may occur. 35

Hygromycin and some of the herbicide resistance markers have similar advantages (WHO, 1993).

Hygromycin resistance 36

Hygromycin resistance genes aph(4)-Ia (or hph) and aph(4)-Ib (or hyg), also referred to as hpt encode an aminoglycoside 4-phosphotransferase APH(4)-I, also called hygromycin phosphotransferase. The aph(4)-Ia gene has been isolated from a strain of E. coli and aph(4)-Ib has been isolated from a hygromycin B producing strain of Streptomyces.

37

The enzyme confers resistance to the aminoglycoside hygromycin B, an inhibitor of protein synthesis in prokaryotic and eukaryotic cells.

38

There is no detailed safety evaluation available for these genes. Hygromycin is not in clinical use therefore the potential for inactivation of an oral dose of antibiotic by consuming plant material containing the marker gene simultaneously with the antibiotic is not an issue. It is used in veterinary medicine (Karenlampi, 1996).

Streptomycin resistance 39

The streptomycin resistance gene provides a phenotypic marker facilitating non-lethal screening of transformed cells based on differential colour. Sensitive plants are pale (but not killed) as streptomycin inhibits protein synthesis and growth of the non-transformed plant cells. The marker is used where preservation of both transformed and non-transformed cells is an advantage. A problem in using this marker is that plants may also acquire streptomycin resistance by mutation.

40

Several aminoglycoside modifying enzymes are known to confer resistance to streptomycin. Of these two are adenyltransferases (eg ANT(3")-I (gene ant(3")-Ia or aadA )) and two are phosphotransferases(eg APH(6)-I (genes aph(6)-Ia, aph(6)-Ib, aph(6)-Ic, aph(6)-Id)). The source of the genes is the micro-organism that produces the antibiotic and resistance factors present in bacteria.

41

ANT(3")-I confers resistance to streptomycin and spectinomycin.

42

There is no safety evaluation available for the streptomycin resistance genes. Although the genes are not novel for humans as resistance factors carrying them are common and found at high frequency in bacteria from clinical isolates, evaluation of allergenicity in humans and pleiotrophic effects from the enzyme in different plants is required (Karenlampi, 1996).


22

Introduction

Ampicillin resistance 43 The ampicillin resistance gene (bla, also referred to as amp) encodes a beta-lactamase enzyme. Resistance to the clinically important group of antibiotics known as the beta-lactams, which includes the penicillins and cephalosporins, is most frequently due to beta-lactamases. 44

The bla gene is present in many plasmids derived from the E. coli plasmid vector pBR322 including those of the pUC family which were used in the development of GM insect resistant maize (Malik and Saroha, 1999). Appendix II discusses the controversy around the use of this gene in GM insect resistant maize.

45

The bla gene on the pUC18 vector encodes an early form of beta-lactamase that has subsequently undergone extensive evolution to form novel beta-lactamases with potent activity against newer broad-spectrum beta-lactams. If such mutations occurred in the resistance gene used as a selectable marker, and in the rare event that this gene was then transferred and expressed in micro-organisms (eg Neisseria meningitidis carried in the human nasopharynx), this could have implications for the treatment of serious bacterial infections (eg meningococcal meningitis) (OECD, 2000a).

Others 46

Other antibiotic resistances that have been used include tetracycline, rifampicin, amikacin, chloramphenicol, bleomycin and puromycin.

Tetracycline resistance 47 The tetracycline resistance gene (tet) is more abundant and widespread than the kanamycin resistance gene. This is partly attributed to many years of use of tetracycline as a growth promotant in animals and extensive use in human and veterinary medicine. The gene encodes a protein that causes the active efflux of tetracycline from a bacterial cell (Pittard, 1997). Chloramphenicol resistance 48 The chloramphenicol resistance gene (cat) encodes chloramphenicol acetyltransferase. Resistance has been widely reported although its prevalence has tended to decline where use of the antibiotic has become less frequent (Reynolds, 1996). Amikacin resistance 49 The amikacin resistance gene confers resistance to a clinically important aminoglycoside antibiotic. It has been used in a potato developed by Avebe to generate only the starch mylopectin.

50

The European Commission Scientific Committee on Plants did not recommend the GM potato for Commission approval for sale and cultivation throughout the European Union on the grounds that the resistance gene could pose a risk to human and animal health (Scott, 1998).


23

Introduction

Rifampicin resistance 51 The rifampicin resistance gene encodes an altered sub-unit of RNA polymerase and transcription is no longer inhibited by rifampicin. Resistance occurs as a result of random mutations at frequencies of about 10-9 in normal bacterial populations (Pittard, 1997).

52

Resistance to this antibiotic is not widespread but easily selected by the use of rifampicin. It is used in the treatment of tuberculosis and leprosy in combination with other drugs to delay or prevent the development of resistance.

Bleomycin (phleomycin) resistance 53 Sources of the bleomycin resistance gene are Streptoalloteichus hindustanus and E. coli. It is rarely used in plant transformation. Bleomycin causes DNA breakdown resulting in the death of rapidly growing cells and hence has a limited use in cancer treatment. It is not used as an antibiotic (WHO, 1993). Puromycin resistance 54 The puromycin resistance gene (pac) from Streptomyces alboniger encodes puromycin acetyltransferase. This enzyme inactivates puromycin produced by Streptomyces alboniger. The gene has been used as a marker for the selection of transformed mammalian cells (Vara et al, 1986).

55

Puromycin is a synonym for a tetracycline, achromycin, used topically in human medicine (US Registry of Toxic Effects of Chemical Substances, 2000).

56

Streptomycin and chloramphenicol resistance genes are used as scoreable markers (or reporter genes). These marker genes are normally used to detect the presence or assay the level of expression of a transferred gene in a modified organism.

57

The application of reporter genes is generally confined to the laboratory or small-scale field trials and is not primarily directed towards full-scale commercial use. However it has been predicted that in the long term crops will be considered for commercial release while still carrying reporter genes (Metz and Nap, 1997). The most frequently used reporter gene, the beta-glucuronidase gene (GUS), is not an antibiotic resistance gene.


24

Antibiotics and antibiotic resistance

2.


What are antibiotics used for? 58

Antibiotics are used both internally and externally in humans and animals to control, prevent and treat bacterial infections, and in animals to enhance growth and feed efficiency in situations where animals are intensively reared (eg chickens, pigs, some fish). They are also used to maintain the health of some plants. Many of the same antibiotics are used in all three areas.

59

Antibiotics have the undesirable side effect of selecting for the growth and spread of otherwise rare resistant micro-organisms. Laboratory studies have demonstrated that antibiotics increase the frequency of gene transfer between micro-organisms. Antibiotics can also increase the mutation rate of micro-organisms thereby increasing the probability that a new resistance determinant will arise (Heinemann, 1999).

Mechanisms of antibiotic resistance 60

Bacteria become resistant by one of the following mechanisms: • production of enzymes that inactivate the antibiotic • alteration of the cellular target, or • active removal of the antibiotic from the bacterial cell (Antibiotic Resistance Expert Panel, 1999).

Development of antibiotic resistance 61

Resistance to antibiotics resulting in therapeutic failure for bacterial pathogens was first encountered soon after the introduction of antibiotics in the 1940s. Since then resistance has emerged in response to the introduction of new or modified antibiotics although the time taken has varied. Resistance has emerged for all known antibiotics in use (Levy, 1998). For most antibiotics and classes of antibiotics antibiotic resistance genes have also entered the bacterial population in the settings where antibiotics are used (eg hospitals, farms).

62

The major factors that contribute to resistance are the antibiotic itself and the resistance genes being selected for. When an antibiotic is used to treat an infection the bacteria most sensitive to the drug die or their growth is inhibited. Bacteria that have or acquire the ability to resist the antibiotic persist and replace the sensitive bacteria. These bacteria may directly cause human infections resistant to treatment.

63

Bacteria can also become resistant indirectly when resistance genes are passed on from other bacteria by mechanisms that allow the transfer of genetic material. Antibiotic resistance genes on plasmids and transposons flow to and from Gram-positive and Gram-negative bacteria, and among bacteria that inhabit very different ecological niches. As a result resistance can be


25


transferred between non-pathogenic and pathogenic bacteria, and from bacteria that usually inhabit the gastro-intestinal tract of animals to those that infect humans. 64

Whenever an antibiotic is used bacteria will develop resistance, either by mutation, gene transfer, or a combination of the two. Mutation occurs at a frequency of between 1 in 106 and 1 in 1012 bacteria (JETACAR, 1999). Gene transfer is more common than mutation. Some resistance genes are located on plasmids and are transferable whereas others (eg streptomycin, rifampicin, fluoroquinolones) are located on the chromosome and are not transferable.

65

There is conflicting evidence with respect to the presence of antibiotic resistance in bacterial pathogens before the advent of antibiotics. However it is likely that most of the antibiotic resistance genes were already present. The biochemical diversity found among the types of resistance mechanisms implies a diversity of origins. The most likely source of the resistance genes is antibiotic-producing micro-organisms (eg Streptomyces) (Benveniste and Davies, 1973; Trieu-Cuot et al, 1987). This has yet to be established conclusively (Davies, 1997).

66

The evolution and spread of antibiotic resistance depends on the selective pressure exerted in the bacterial environment. Selective pressure is a general concept that refers to many factors that create an environment and allow micro-organisms with novel mutations or newly acquired characteristics to survive and proliferate. Micro-organisms resistant to antibiotics were resistant before antibiotics were used but were not able to differentially proliferate; thus both survival and proliferation are essential. Antibiotic exposure selectively amplifies resistant bacteria and the resistance genes they carry. This increases the prevalence of resistant bacteria in the total bacterial population and results in large reservoirs of resistant bacteria and resistance genes where formerly they were rare. Resistance to specific antibiotics varies geographically with considerable variation both between and within countries.

67

In small spatial compartments low level resistant bacteria may be selected by very small quantities of antibiotics such as those created by antibiotic-producing micro-organisms, those present in food as contaminants, or those present in the human body. Antibiotic concentrations are often low in some compartments where the density of micro-organisms is high (eg colon, oropharynx). Selection of low level resistant variants may increase the possibility of further evolution towards higher resistance levels. Since antibiotic selection is only part of the environment that results in the selection of antibiotic resistant bacteria it is not always possible to correlate the use of antibiotics and resistance to antibiotics (Baquero et al, 1998).

68

Most data support the concept that antibiotic resistance appeared only after the emergence of strong selective pressure resulting from the massive use of industrially made antibiotics in human and veterinary medicine and as food supplements for farm animals.


26


The public health impact of antibiotic resistance 69

The extent of the clinical antibiotic resistance problem depends on the presence of the antibiotic and a resistance gene, the spread of resistant bacteria and the cell-to-cell spread of the resistance gene. Given the presence of both the antibiotic and a resistance gene, drug resistant bacteria will be selected and propagated (Levy, 1997).

70

The prevalence of antibiotic resistant bacteria and the number of antibiotics to which they are resistant are increasing because of the use of antibiotics. As a consequence the morbidity and mortality of previously treatable bacterial diseases is increasing. Essential lifesaving antibiotics are becoming less effective and there are fewer alternatives available for treatment (JETACAR, 1999).

71

During the last decade the development of new antibiotics has become more difficult, expensive and uncommon. This contrasts with previous decades following the discovery of penicillin in 1940.

72

Emergence of resistance and control of antibiotic resistant pathogens are now major challenges in both hospitals and the community. Multi-drug resistant pathogens such as penicillin-resistant pneumococci, vancomycin-resistant enterococci, and methicillin-resistant staphylococci have emerged as well as a variety of multi-resistant Gram-negative organisms (eg Pseudomonas aeruginosa, Acinetobacter baumanii) (Levy, 1998).

73

Evaluation of the human health impact of antibiotic resistance depends on the importance of the antibiotic or antibiotic class in medicine and potential human exposure (direct and indirect) to resistant bacteria, in particular those that are human pathogens. However bacteria that are not usually pathogenic may cause infections in some people (eg those who are hospitalised or immunocompromised).

Antibiotics in New Zealand 74

Annual antibiotic use, excluding ionophores,1 in New Zealand is estimated to be 74.9 tonnes. Human use accounts for about 53 percent of this amount. In addition to antibiotic use in humans and animals about 1.2 tonnes of streptomycin is used annually in horticulture in New Zealand (Antibiotic Resistance Expert Panel, 1999).

75

The main antibiotics currently used in New Zealand that are potentially affected by the use of antibiotic resistance marker genes in GMOs are discussed below.

Aminoglycosides 76

Aminoglycoside antibiotics exert their effect on bacteria by binding to bacterial ribosomes and inhibiting protein synthesis. Phosphorylation of the antibiotics by the aminoglycoside modifying enzyme neomycin phosphotransferase interferes with binding and thus prevents

1

Ionophores have a different mode of action to other groups of antibiotics and are not used in human or veterinary medicine. They are not known to select cross-resistance to antibiotics used in human or veterinary medicine.


27


the antibiotic from inhibiting protein synthesis. In this way cells that contain the kanamycin resistance gene and express neomycin phosphotransferase are resistant to the action of some aminoglycoside antibiotics. 77

The estimated annual use of aminoglycosides in New Zealand is 2,242 kg (35 kg humans; 2,207 kg animals) (Antibiotic Resistance Expert Panel, 1999). The amount used in molecular genetics research is estimated as only hundreds of grams (B Scott, personal communication, May 2000).

78

In contrast in the Netherlands approximately one kilogram of kanamycin is used annually in molecular genetics, 100 kg in humans and an estimated 20,000 kg of neomycin and 10,000 kg of kanamycin in animals (Nap et al, 1992).

Kanamycin 79 Kanamycin is produced by the actinomycete Streptomyces kanamyceticus. It is active against strains of Gram-negative bacteria, excluding Pseudomonas species, as well as some strains of Staphylococcus and Mycobacterium, although resistant strains are widely distributed. A decline in use has meant that resistance has become less prevalent.

80

It is used only in serious systemic infections and when the infecting micro-organism is resistant to other antibiotics. It is a reserve agent for tuberculosis, in particular multi-drug resistant tuberculosis the prevalence of which has increased dramatically in the last decade.

81

Kanamycin is given intramuscularly or intravenously in the treatment of severe infections often in combination with another agent. It is not absorbed by mouth and has been given orally to reduce gut micro-organisms (eg preoperative bowel preparation, hepatic encephalopathy). The main side effects are ototoxicity and nephrotoxicity.

Neomycin 82 Neomycin is produced by the actinomycete Streptomyces fradiae. It is active against many strains of Gram-negative bacteria, excluding Pseudomonas species, and against many strains of Staphylococcus aureus. Its chemical properties are similar to kanamycin.

83

It is used topically in the management of skin, eye and ear infections. It is poorly absorbed by mouth and has been given orally to reduce gut micro-organisms (eg preoperative bowel preparation, hepatic encephalopathy, selective decontamination of the gastro-intestinal tract). It is not used parenterally or systemically because of its ototoxicity and nephrotoxicity.

84

Although neomycin has limited clinical use it is an important antibiotic in veterinary medicine.

Streptomycin 85 Streptomycin is produced by Streptomyces griseus. It is active against Mycobacterium tuberculosis as well as against many Gram-negative bacteria, excluding Pseudomonas aeruginosa. It is used in the treatment of tuberculosis in combination with other agents, and for other severe infections. It is not absorbed by mouth. The main side effect is ototoxicity.

86

Streptomycin is also used in horticulture and veterinary medicine.


28


87

The streptomycin resistance gene also confers resistance to spectinomycin. Spectinomycin is used as an alternative agent in the treatment of uncomplicated gonorrhoea but is not available in New Zealand.

Other aminoglycosides 88 The nptII gene also confers resistance to paromomycin. Paromomycin has a similar antibacterial spectrum to neomycin and is given orally in the treatment of protozoal infections such as amoebiasis. It is not available in New Zealand.

89

Gentamicin or tobramycin are the antibiotics of choice in the treatment of life-threatening Gram-negative infections and are often used in association with other antibiotics eg betalactams. With the continuing emergence of aminoglycoside resistance amikacin and netilmicin should be reserved for severe infections resistant to other aminoglycosides. Amikacin, and to a lesser extent netilmicin, are not affected by most of the aminoglycoside modifying enzymes eg NPTII that inactivate the aminoglycosides.

Other antibiotics Ampicillin 90 Ampicillin is effective against Gram-positive and some Gram-negative bacteria. It has a broader spectrum of activity than benzylpenicillin and is used in the treatment of a variety of infections.

91

Ampicillin is a beta-lactam antibiotic. Beta-lactam antibiotics are the largest group of antibiotics and include the penicillins and the cephalosporins. All have related structures and the same mechanism of action. The beta-lactams, in particular the penicillins, account for about 67 percent (26,289 kg) of the estimated annual human antibiotic use (Antibiotic Resistance Expert Panel, 1999).

92

It is given orally as well as intravenously or intramuscularly and is often given with an aminoglycoside for broad spectrum empirical therapy (Reynolds, 1996).

93

Ampicillin is also used in veterinary medicine. Beta-lactam antibiotics account for 26 percent of the animal antibiotic use in New Zealand (Antibiotic Resistance Expert Panel, 1999).

Tetracyclines 94 The group of tetracycline antibiotics has a broad spectrum of activity against bacteria and some protozoa. Tetracyclines have been used in the treatment of a large number of infections. Although use has become more restricted with the emergence of resistance a tetracycline is the usual antibiotic of choice in some infections (eg chlamydial and mycoplasmal infections).

95

They are usually given orally.

96

Tetracyclines are also used in veterinary medicine.


29


Rifampicin 97 Rifampicin is bactericidal against a wide range of micro-organisms and interferes with nucleic acid synthesis by inhibiting RNA polymerase (Reynolds, 1996).

98

It is important in the treatment of tuberculosis and contacts of Haemophilus influenzae meningitis and meningococcal disease to eradicate nasopharyneal carriage of H. influenzae and Neisseria meningitidis respectively. This latter use is particularly important in New Zealand which has been experiencing an epidemic of meningococcal disease for the last decade.

99

It is given orally as well as intravenously.

Chloramphenicol 100 Chloramphenicol is a broad-spectrum antibiotic effective against both Gram-negative and Gram-positive bacteria as well as some other organisms (eg mycoplasmas). Use is limited by its toxicity but it is used particularly in typhoid and other salmonellal infections and in the treatment of bacterial meningitis. It is used orally or intravenously and widely used topically in eye infections (Reynolds, 1996).

Antibiotic resistance in New Zealand 101

The potential contribution of antibiotic resistance acquired from consumption of antibiotic resistance marker genes in GMOs needs to be considered in the wider context of antibiotic resistance in New Zealand.

102

Untreatable infections due to bacteria that are resistant to all available antibiotics are still exceptional but do occur. In New Zealand hospitals it is not uncommon to encounter bacterial infections that are resistant to all but one or two antibiotics (Lang and Blackmore, 1999).

103

The major source of the development and spread of antibiotic resistant micro-organisms in humans is the human use (and often overuse) of antibiotics in both the community and hospitals. Within hospitals person-to-person transmission is aided if infection control practices are less than ideal.

104

Antibiotic resistance is also acquired through ingestion of resistant micro-organisms from animals or soil contaminating food or water. Enteric human pathogens (eg Salmonella) are commonly acquired from animals and in some instances these micro-organisms acquire resistance to antibiotics used for growth promotion, prophylactic or therapeutic use in animals. Global movement of people, breeding stock and food also means resistance in one area can spread to another.

105

Gaps in information about patterns of antibiotic resistance (eg in enteric micro-organisms isolated from food animals) in New Zealand prevent the conclusion that antibiotic use in animals is exacerbating antibiotic resistance in human pathogens. Limited monitoring of some zoonotic micro-organisms from human, animal and food sources by the Institute of Environmental Science and Research and data from some hospitals indicate that there is a


30


low level of acquired antibiotic resistance in New Zealand compared to the United States, the United Kingdom and the European Union (Antibiotic Resistance Expert Panel, 1999). The Antibiotic Resistance Expert Panel considered that vigilance is required to ensure that in the event that resistant strains of enteric zoonotic micro-organisms such as Campylobacter, Enterococcus and pathogenic E. coli are introduced or emerge, action can be taken to limit their spread. 106

There are no data on the antibiotic resistance patterns of bacteria found on fruit that may be a result of streptomycin use in horticulture.

107

Whilst the potential contribution to antibiotic resistance from antibiotic resistance marker genes is likely to be very small in comparison to that arising from antibiotic use in medicine and agriculture, and insignificant in terms of health impact unless the antibiotic is clinically important in New Zealand, it adds to the wider public health problem of increasing antibiotic resistance. A number of strategies proposed by recent advisory bodies (eg European Commission, 1998, 1999; JETACAR, 1999; Antibiotic Resistance Expert Panel, 1999) have been or are being implemented to curb antibiotic use and halt further escalation of antibiotic resistance.

Is antibiotic resistance lost? 108

Once resistance appears it is likely to decline slowly, if at all, once no antibiotic is present. There are no counter-selective measures against resistant bacteria. The slow loss of resistance is linked to poorly reversible genetic and environmental factors (Levy, 1998). Resistance genes may acquire new functions and also become maintained by factors other than the antibiotic (Heinemann et al, 2000).

109

Expression of antibiotic resistance in bacteria may involve a fitness cost that is disadvantageous compared with susceptible bacteria when no antibiotic is present and resistance would therefore be gradually lost. This idea is supported by some laboratory experiments and by a low prevalence of resistance in human and animal populations not exposed to antibiotics. If this is the case then improving the management of antibiotics should reduce the prevalence of resistance. However Schrag and Perrot (1996) demonstrated that although resistance may initially impose a fitness cost, natural selection can result in compensatory mutations that markedly reduce this cost by restoring physiological functions impaired by resistance without altering the level of bacterial resistance. This suggests that reduced antibiotic use may not lead to a decrease in the current prevalence of resistant bacteria. Resistance can be maintained in bacterial populations in the apparent absence of specific antibiotic selection for several years.

110

To restore efficacy to earlier antibiotics and maintain the success of new agents that are introduced use needs to assure an ecological balance that favours the predominance of susceptible bacteria (Levy, 1997).


31

Antibiotic resistance marker genes in food

3.


Introduction 111

If food from GMOs becomes widely available there is the potential for antibiotic resistance genes to be present in many everyday items in the diet. As a small number of different marker genes are currently used on a regular basis it is feasible that the same marker proteins may be present in several different GM components of the human diet thereby increasing the total consumption of the marker gene protein (Kok et al, 1994). It is also foreseeable that future GM food crops may contain several marker genes accumulated in consecutive transformation events (Karenlampi, 1996).

112

However some antibiotic resistance marker genes, in particular the kanamycin resistance gene, are not novel to the food supply. Such antibiotic resistance genes may be present in contaminating bacteria on or in food.

113

A misconception occasionally associated with the introduction of GM food is that the presence of antibiotic resistance marker genes means the GMO produces antibiotic. This is incorrect. Consumers do not receive a dose of antibiotic when they eat such GM food.

114

Food safety regulation of GM food has been discussed by international expert committees resulting in a number of advisory reports proposing different strategies for risk assessment. The food safety of marker genes is discussed in most of these reports.

The concept of substantial equivalence 115

Assessment of the safety of food and food components derived by gene technology is based on the concept of substantial equivalence that was elaborated by the Organisation for Economic Cooperation and Development (OECD) in 1993 and subsequently endorsed by the Food and Agriculture Organisation (FAO) and the World Health Organisation (WHO) in 1996. The concept of substantial equivalence compares the GMO to its traditional counterpart as a means of identifying novel aspects that may affect its food safety. If a GM food product is considered to be substantially equivalent to an analogous conventional food product no additional safety concerns are expected.

116

Further evaluation is based on identified differences or, where no counterpart has been previously consumed as food, on its composition and properties.

117

The safety assessment of GM food is by definition within the limits of current knowledge (Jones, 1999). Marker genes are dealt with in the same way as other inserted genes.

118

Factors that influence whether the presence of a marker gene affects its substantial equivalence to a conventional counterpart include: • whether the marker encodes a protein product and, if so at what levels it would be expected in the food, what its function is, and whether there are concerns about its safety at the predicted levels;


32


• • 119

whether the marker encodes resistance to a clinically useful antibiotic and, if so does ingestion at the same time as use of the antibiotic interfere with its clinical efficacy; and the probability of horizontal transfer of resistance genes to pathogens on or in the food or in the consumer’s gastro-intestinal tract (OECD, 1993).

International consensus has not yet been obtained as regards interpretation and use of substantial equivalence (Pascal, 1999).

The general basis for assessment of the potential health impact of marker genes and their gene products 120

The structure and function of the gene and its expressed product are first compared to other genes and proteins in order to evaluate the novelty of the marker.

121

If the gene is novel to humans the assessment also needs to consider the: • structure of the gene and its product • function of the gene product, and • quantity of the gene and its product.

122

This includes the level of the gene and its product in food and the estimated daily intake, and stability of the gene and its product in the gastro-intestinal tract (Karenlampi, 1996).

123

Other factors to consider are the availability of any required cofactor in the gastro-intestinal tract and use of the antibiotic among those populations that eat the food.

Antibiotic resistance genes and food safety 124

Possible food safety problems from the use of antibiotic resistance genes include: • direct consequences of the gene, • direct consequences of the product encoded by the gene, • indirect consequences of the effects of the gene or its product, • possibility of horizontal gene transfer from ingested GMOs (and/or derived foods or food components) into gut epithelial cells and/or gut micro-organisms, and • inactivation of antibiotic by the gene product.

Direct consequences of the antibiotic resistance gene 125

DNA is present in the cells of all living organisms, including every plant and animal used for food by humans or animals. The genes of incidental food contaminants are also consumed. The large amount of DNA that passes the gastro-intestinal tract daily indicates that DNA itself is not intrinsically toxic to humans.


33


126

The DNA that makes up an antibiotic resistance gene has no unusual composition compared to other genes (composed of four nucleotides common to all genes in all organisms in varying amounts) and its presence poses no more health risk than the other DNA that is ingested.

Direct consequences of the gene product encoded by the antibiotic resistance gene 127

The extent of normal exposure to protein variants is not well known but is easily in the tens of thousands, and probably in the order of 100,000. An eukaryotic cell contains 5,000 to 10,000 different polypeptides that must be degraded to produce the amino acids required for growth. When this number is multiplied by a factor to account for tissue-specific differences and by the number of different species that are eaten the number of proteins in the diet becomes very large. Genetic polymorphism also contributes to the total dietary protein array (Kessler et al, 1992). These variants do not arise as totally new proteins but as incremental changes of what was present previously.

128

Proteins derived from marker genes differ from proteins derived from other introduced genes in a GM plant in two aspects. The same protein may be present in a number of GM foods in the diet resulting in increased total marker gene protein consumption because of the small number of marker genes currently used regularly. In addition as few tissue-specific or developmental stage-specific promoters are yet available the gene will often be expressed in more tissues and for a longer time period than necessary (Kok et al, 1994). It is however likely that tissue-specific or developmental stage-specific promoters will become the norm.

129

Most proteins rapidly degrade upon consumption and exposure to the mammalian digestive tract. The gastro-intestinal tract is specifically designed to digest ingested dietary proteins by conversion to amino acids and small peptides that are absorbed by the intestinal tract.

130

If there is any risk from ingestion it should in most cases correlate with the potency of functional protein available in the gastro-intestinal tract. This depends on the estimated daily intake (and therefore the level of the protein in food) and stability of the protein in the gastro-intestinal tract (Karenlampi, 1996). Food that potentially carries the greatest risk is food consumed fresh (ie uncooked or unprocessed).

131

A protein is likely to be safe for consumption if based on experience proteins of the same function have been safely consumed at similar levels. Proteins that are not functionally similar to proteins known to be safely consumed need to be assessed relative to their potential toxicity and allergenicity. The source, amino acid sequence and function of the gene product can be used to identify proteins that would raise a safety concern.

132

Gene sequence information and evolutionary studies of proteins give useful guidance for safety evaluations. Knowledge of the DNA sequence allows the use of computer algorithms to identify other proteins with related sequences. An immunologically significant sequence identity with known allergens requires a match of at least eight contiguous amino acids. Criteria for comparing amino acid sequences may change or evolve over time with additional research and insight into the molecular structure of allergens. Public domain sequence databases include GenBank, EMBL, PIR and SwissProt.


34


133

Physico-chemical and biological properties of the gene product can be compared to properties of known allergenic proteins as a means of predicting allergenic potential. With the exception of identifying known allergens transferred from allergenic sources there is currently no single predictive property that can conclusively determine allergenic potential. The key prerequisite for food protein allergenicity is resistance to heat denaturation and proteolytic degradation. Typically allergens are 10 to 70 kDa in molecular weight and are often glycosylated but there are exceptions to this generalisation (Metcalfe et al, 1996). Relative abundance may also suggest a protein is allergenic. In general the proteins are not likely to exceed 0.1 percent of the total soluble protein content of the GM plant material (Metz and Nap, 1997).

134

Demonstration of the lack of amino acid sequence homology to known protein toxins or allergens and their rapid proteolytic degradation under simulated mammalian digestive conditions is considered appropriate to confirm safety. Demonstration of proteolytic digestion under both gastric and intestinal conditions supports the expectation that the protein is likely to be degraded during food consumption and digestion (FAO and WHO, 1996). To elicit an allergenic response the protein must survive the acid and proteolytic environment of the gastro-intestinal tract to reach and be absorbed through the intestinal mucosa and trigger a series of IgE-mediated responses.

135

In vitro testing is however not identical to the physiological conditions in the gastro-intestinal tract. The conclusions drawn from such testing may not always give clear evidence on the possible toxic or allergenic potential of peptides formed as breakdown products in the test system. The absence of homology of a protein’s primary structure to a known allergen also does not exclude the presence of allergenic epitopes formed by its secondary or tertiary structure (OECD, 2000a).

136

Validated animal models are not yet available for evaluation or prediction of a novel protein’s allergenicity or possible unintended effects. Traditional animal feeding studies are designed to assess safety of substances that are an insignificant component of the diet such as food additives. Such studies are inadequate for testing whole foods that are a substantial dietary component due to the difficulty of feeding animals adequate doses of the test food. In many cases meaningful information is unlikely to be produced (OECD, 2000a). Experiments on the protein alone can provide more meaningful information than experiments on the whole food.

137

New and better methods to evaluate GM foods are needed (Metz and Nap, 1997). Although not considered necessary for the kanamycin resistance gene because of its evaluation to date (see Appendix I) and history of human exposure, further evaluation is required for some other resistance marker genes eg hygromycin, streptomycin (Karenlampi, 1996). Consideration needs to be given to whether long term feeding studies are necessary to provide greater information on potential allergenicity and toxicity (The Royal Society, 1998). Re-examination of the methods for testing allergenicity and toxicity was also a recommendation from the recent OECD conference on the scientific and health aspects of GM foods (Krebs, 2000).

138

Many antibiotic resistance genes used in genetic modification are present on commercially available plasmids whose characteristics are known and can therefore be taken into account


35


in the assessment. For example, a gene with bacterial regulatory sequences that remains in a construct with a high copy number bacterial replicon may dictate higher expression levels if it is expressed in the GMO than those found from replicons currently present in nature (ACNFP, 1996). Indirect consequences of the effects of the antibiotic resistance gene or its gene product 139

There are no characteristics of marker genes or their products that suggest that their site of insertion into the plant genome will result in specific secondary and/or pleiotrophic effects that may in some way alter any of the organism’s toxicological or ecological characteristics. Possible secondary effects in the plant due to insertion need to be determined on a case-bycase basis as they are expected to be highly dependent on the host plant and the site of insertion of the marker gene. Secondary effects can be assessed by comparing key substances to establish substantial equivalence (WHO, 1993).

140

Few data are available with respect to GM plants containing a selectable marker gene and no methods yet exist to approach these issues in an undisputed way. Prediction can be attempted to a limited extent. Pleiotrophic effects are therefore very difficult issues for safety assessments. At present, for both the toxicology and ecology of GM plants it is unclear whether unpredictable pleiotrophic effects, such as changes in a metabolite(s) or plant growth and development, do occur to the extent that any effects can be measured in a meaningful way. With respect to safety and biosafety regulations, general considerations seem to allow the conclusion that putatively pleiotrophic effects will be of no or only minor importance. However this view is controversial (Metz and Nap, 1997).

141

The food safety risks associated with development of GM plants need to be considered in the context of the risks associated with plants developed using conventional breeding methods. It has been argued that as gene technology is more precise and allows better characterisation of the changes occurring developers are better placed to assess safety than when using conventional methods (Smith, 2000).


36


Horizontal gene transfer in humans Potential gene transfer to oral micro-organisms 142

The regions preceding the stomach (ie mouth and oesophagus) are likely to have the highest concentrations of intact DNA entering with the diet. Free DNA has been shown in vitro to survive for 10 minutes (between 35 and 61 percent had been degraded) in human saliva and to be capable of transforming a naturally competent human oral bacterium (Streptococcus gordonii) to erythromycin resistance (Mercer et al, 1999). Further research is needed to establish whether transformation of oral bacteria can occur at significant frequencies in vivo.

143

Pollen and other airborne sources such as dust from dry milling are also a potential source of exposure to DNA from GM plants. The United Kingdom’s Advisory Committee on Novel Foods and Processes (ACNFP) considers the likely levels of exposure and potential gene transfer to oral and respiratory tract bacteria from GM plants in its assessment (OECD, 2000a). No references were found in the peer reviewed scientific literature that discussed this risk.

Potential gene transfer to gut epithelial cells or micro-organisms 144

Although most of the ingested DNA will be degraded and diluted, natural transformation of gut epithelial cells or micro-organisms cannot be excluded. It is conceivable that microenvironments exist where DNA is not degraded or that certain dietary components protect against degradation (Flint and Chesson, 1999).

145

It is generally considered that the probability of antibiotic resistance genes being transferred from GMO material to either gut epithelial cells or micro-organisms is extremely low. However few experimental data are available to support this theoretical assessment (Kok et al, 1994).

Degradation of DNA 146 Any gene is unlikely to be intact or functional after processing and/or cooking. In unprocessed food the gene is likely to be intact when consumed but DNA is rapidly broken down under normal gastro-intestinal conditions into fragments usually too small to be functional.

147

Research in mice has however indicated that DNA can survive digestion. About two to four percent of orally ingested foreign DNA was detected in the gastro-intestinal tract of mice and fragments were detected in the faeces between one and seven hours after feeding. For humans of average weights between 50 and 80 kg, an intake of about 50 to 80 mg of DNA in the daily food would be needed to parallel the situation simulated in the mouse experiments (Schubbert et al, 1994). This is unlikely to be encountered in a normal diet. For example, Calgene Inc estimated the daily amount of ingested kanamycin resistance gene from fresh Flavr Savr tomatoes was 0.33-1 pg (Karenlampi, 1996).

148

Although proteins are broken down to smaller peptides and amino acids by digestive enzymes the possibility that the protein encoded by the gene remains intact needs to be considered. In vitro models (eg Minekus et al, 1995) have been developed to simulate the


37


human gastro-intestinal tract to study the fate of ingested compounds. They can be modified to simulate atypical conditions (eg high gastric pH) that may be encountered in some people that might affect protein degradation. 149

Seeds from some GM plants and some viable GM micro-organisms are notable exceptions to DNA degradation. In seeds protected by a resilient seed coat (eg tomato) the DNA remains intact through the gut but is not available for gene transfer. Even if transfer were to occur expression is unlikely unless the regulatory sequences on the transferred sequence are functional in the gut epithelial cell or gut micro-organism.

Potential DNA uptake 150 One of the public concerns associated with the introduction of GM food is the possibility that genes from GMOs may be taken up by those eating such food and become part of their genetic makeup. A cell would have to take up the DNA in question and integrate it into its own genetic material. The view that DNA is unlikely to enter human cells is supported by the limited success of gene therapy, even when conditions for transfer are optimised, carried out during the last decade (Donaldson and May, 1999). The acquired gene must also have the ability to be expressed in the cell (eg genes from bacteria are normally not expressed if transferred to an eukaryotic cell).

151

Cells that may come into direct contact with the gene are the epithelial cells of the mucous membrane that covers the surfaces of the gastro-intestinal tract. These cells are terminally differentiated (ie do not divide) and have a relatively short life span of seven days.

152

There is some recent evidence in mice to show possible DNA uptake. Although after a single feeding episode of phage M13 DNA in mice more than 95 percent of the foreign DNA was lost after passing through the stomach, the findings suggest transport of very small quantities of foreign DNA through the intestinal wall and Peyer’s patches to peripheral blood leucocytes (white blood cells) and into several organs (spleen, liver). Fragments were found in about 0.1 percent of the peripheral blood leucocytes up to eight hours after feeding and in the spleen and liver up to 18 hours after feeding. The mechanism of foreign DNA uptake by the gut epithelial cells is unknown. Foreign DNA was also detected up to 18 hours after feeding in the caecum. However no foreign DNA was found in intestinal bacteria (Schubbert et al, 1997).

153

Similar results to the study above were obtained by Schubbert et al using a plasmidcontaining gene for the green fluorescent protein (GFP) that was orally administered to mice. It was detected in the intestinal contents, liver, spleen and kidney up to eight hours after feeding and in the intestinal wall three to eight hours after feeding.

154

When foreign DNA was fed to pregnant mice DNA fragments were detected in various organs of fetuses and newborn mice. Foreign DNA was not found in all the cells of the fetus suggesting that the DNA entered the fetus transplacentally rather than by germ line transmission (Schubbert et al, 1998).

155

There is some in vitro evidence that DNA can be transferred from invasive strains of bacteria (Shigella flexneri and E. coli) to mammalian cells provided that they are able to penetrate the host cell (Courvalin et al, 1995; Grillot-Courvalin et al, 1998). This direct gene transfer


38


showed a broad host cell range and the vectors were stably inherited and expressed by the cell progeny. 156

To date DNA uptake has not been reported for other DNA sources such as food and the significance of the findings of Schubbert et al is unclear. DNA is consumed daily by most people in most of the food (eg fruit, vegetables, cereals, meat) that they eat.

157

There are no published reports on the transfer, integration or expression of genes in human gut epithelial cells (US FDA, 1998). Even if transformed, gut epithelial cells have a rapid turnover and since the germ line is unaffected transformed cells could not be maintained or spread in the human population. Many gut micro-organisms carry antibiotic resistance genes but no problem associated with transfer to gut epithelial cells has ever been identified (ACNFP, 1994).

158

Some experts participating in consultation carried out by the FDA cautioned that it should be assumed that DNA can get into the gut epithelial cells but the critical factor is the lack of selective pressure. Without selective pressure it is highly unlikely that genes taken up by these cells would be expressed even if they were integrated into the genome (US FDA, 1998).

Potential transfer to gut micro-organisms 159 Homology between sequences in an antibiotic resistance marker gene that is prokaryotic in origin and the recipient’s DNA is more likely to be found in gut micro-organisms which are prokaryotic than in gut epithelial cells which are eukaryotic. The probability of integration and expression of a marker gene is therefore greater in gut micro-organisms than in gut epithelial cells.

160

Potential gene transfer scenarios to gut micro-organisms include from: • micro-organisms in or on food • GM food micro-organisms • unintentional ingestion of GM micro-organisms released into the environment, and • GM food (plant or animal).

161

For gene transfer to occur the following events are required: • release of DNA from cells or tissue • survival of DNA in the gastro-intestinal tract, including exposure to gastric acid and nucleases, • recipient micro-organisms are competent for transformation • recipient micro-organisms bind the DNA to be transferred • DNA penetrates the cell wall and translocates across the cell membrane • DNA survives the restriction or modification system developed by the microorganism to degrade foreign DNA, and • DNA is integrated into the host genome or plasmid (ACNFP, 1996).

162

If transfer is successful the gene must be expressed for it to have any impact.


39


163

Viable GM micro-organisms may remain intact through the gut and gene transfer could be achieved through conjugation with gut micro-organisms. Conjugation between lactic acid bacteria and related species present in the gut has been recorded in vitro (ACNFP, 1994).

164

No data are available concerning the natural transformation of bacteria in the human gastrointestinal tract. The transit time of food in the mouth, pharynx, oesophagus, stomach and duodenum is rapid and conditions in the stomach will prevent any natural transformation. The most relevant location for possible gene transfer therefore is the lower part of the small intestine (ileum) and the colon in which consumed food stays a relatively long time (Nap et al, 1992).

165

There is concern about the potential consequence of transfer of an introduced gene from GM food to gut micro-organisms in such a way that the gene can be successfully integrated and expressed and impact on human health. The probability of transfer in the gastrointestinal tract has to be assessed in the light of the nature of the GMO and the characteristics of the gene construct.

166

Conditions in the mammalian intestine are probably more conducive to gene transfer than conditions found elsewhere in nature as the environment is warm, wet and contains abundant nutrients. Concentrations of bacteria are so high that encounters between different types of bacteria occur readily and residence time in the intestine is long enough to provide many opportunities for gene transfer. However there has been little direct evidence to support this hypothesis (Salyers, 1993).

167

When an antibiotic resistance gene is integrated into the plant genome the codon usage may have been altered for more efficient expression in the plant and the gene may have picked up the plant’s methylation patterns. If this DNA is now taken up by a bacterium it would be recognised as foreign and degraded by the micro-organism’s restriction enzymes thus making integration and expression even more unlikely (US FDA, 1998).

168

Since uptake is usually not sequence-specific an antibiotic resistance gene would also be competing for transfer into a bacterium with the rest of the DNA in the plant genome and DNA from other sources in the diet (US FDA, 1998).

169

The probability of integration into the genome of a gut micro-organism depends on the degree of homology of the foreign DNA with that of the genomic DNA. At least 20 base pairs in a complete homologous DNA sequence are required for significant recombination at both ends of the foreign DNA. Antibiotic resistance genes are of bacterial origin and there may be sufficient homology to allow integration of such genes into gut (or rumen) bacteria under favourable circumstances. They are also propagated in E. coli and as a result there is likely to be sufficient homology for plasmid or chromosomal integration into E. coli, Enterobacter, Salmonella, Shigella or Pseudomonas to be feasible. It is possible that other less well understood processes such as illegitimate recombination could also lead to plasmid or chromosomal integration into gut (or rumen) bacteria (ACNFP, 1996).

170

The probability that foreign DNA would persist in a micro-organism is significantly enhanced under conditions that exert selective pressure viz oral therapeutic use of the relevant antibiotic. The antibiotic would not provide selective pressure if the gene is under


40


the control of an eukaryotic promoter and therefore unable to be expressed in microorganisms (FAO and WHO, 1996). Rearrangements could however bring a prokaryotic promoter in front of the gene leading to its expression (US FDA, 1998). 171

In January 1999 the New Scientist reported research in the Netherlands indicating that some DNA can reach the large intestine intact and survive long enough for transfer to occur. Using a computer-controlled artificial gut Havenaar and coworkers found that DNA from GM bacteria containing an antibiotic resistance gene remains intact in the simulated large intestine for a half-life of six minutes. If the modified bacteria were normally in the gut (eg Enterococcus) each bacterium had a one in 10 million chance of transferring DNA containing an antibiotic resistance gene to an indigenous gut bacterium. This suggests many bacteria would be transformed given there are approximately 1014 bacteria present in the gastrointestinal tract. Transfer of antibiotic resistance was not detected from micro-organisms not normally present in the gut (eg Lactobacillus) or the Flavr Savr tomato. The transfer rate increased ten-fold when some normal gut micro-organisms were killed such as would occur during antibiotic therapy (MacKenzie, 1999). To date this research has not been published in the peer reviewed scientific literature.

172

Much of the literature relating to gene transfer relates to transfer in environments other than the human gut. There are differences between the gastro-intestinal tract and other environments such as soil that need to be taken into account in assessing possible food safety concerns. These include: • free DNA for uptake is continuously degraded in the gastro-intestinal tract; • there are no authenticated reports of bacterial transformation in the human gastrointestinal tract; • DNA degradation begins well before the arrival of material at the critical sites for transformation (ie lower small intestine, caecum and colon); • DNA degradation rapidly fractionates the DNA to sequences smaller than needed for proper expression; • for effective uptake of DNA by gut bacteria the plant or animal DNA should undergo recombination and be expressed in the recipient bacteria.

173

In the case of bacteria a gene is most likely to be transferred if it is on a broad host range gene transfer element such as a plasmid or transposon. Resistance to chloramphenicol, streptomycin, ampicillin and neomycin is located on plasmids and transposons. The least transmissible genes are those that are integrated into the chromosome and not linked with gene transfer elements. For such genes to be transferred they must be acquired by a transmissible element which requires strong selective pressure (Salyers, 1999).

174

A GM micro-organism intended for environmental release, where antibiotics are unlikely to be encountered in concentrations sufficient to exert selective pressure, would be unlikely to make a non-transmissible antibiotic resistance gene capable of transfer to other microorganisms. In the unlikely event that transfer did occur selection would be needed for it to be incorporated and expressed in the new host. In contrast a GM micro-organism administered to animals that were eating antibiotic-supplemented feed or to humans exposed to antibiotics might experience the selective pressure needed to foster gene transfer. It is of note that naturally occurring resistant strains (eg probiotic strains of Lactobacillus) could present as great


41


or greater hazard as GM micro-organisms because their resistance genes are more likely to be on transmissible elements (Salyers, 1999). 175

There is a relatively greater probability of transfer to gut micro-organisms from ingested GM micro-organisms. If transfer of the intact gene occurred it is possible that the DNA could become functional as the same or similar promoters used for expression in the GM microorganism may also be present in the gut micro-organism. It is also possible that intact plasmid vectors from GM micro-organisms could be transferred to and maintained in gut micro-organisms, and although less likely transposition could be achieved by systems used in the recipient species (ACNFP, 1994).

176

Functional antibiotic resistance genes in GM probiotic bacteria would be expected to persist in the gut since probiotic bacteria are intended to survive in and colonise the gut. The extent to which they would compromise the use of the corresponding antibiotic would depend inter alia on the level of gene expression and the extent of any cross-resistance to other antibiotics (ACNFP, 1994).

177

In nature antibiotic resistance is widespread among contaminating micro-organisms found in or on food. It is more likely that antibiotic resistance genes would be introduced into gut micro-organisms through transfer between naturally occurring ingested contaminating microorganisms and gut micro-organisms than through transfer from DNA released during digestion of GM plant or animal material. The probability that transfer occurs between ingested GM micro-organisms and gut micro-organisms is likely to be the same as between non-GM micro-organisms and gut micro-organisms (ACNFP, 1994).

Inactivation of antibiotic by the gene product 178

Under certain circumstances such as ingestion of viable GM micro-organisms there is a low but finite probability that antibiotic resistance genes could be transferred. If transfer occurs and the transformed micro-organisms survive, colonise the gut and express the protein (whether or not it is expressed in the plant) an increase in gut micro-organisms resistant to the specific antibiotic could result. Given sufficient selective pressure and because of the short generation times of bacteria clonal expansion of the transformed bacteria could occur.

179

The gene involved may possibly interfere with antibiotic therapy either by the coincident consumption of food containing the gene product with oral doses of the antibiotic or by the transfer of the resistance gene to pathogenic gut micro-organisms treated with the particular antibiotic.

180

The probability that all of the prerequisite events will occur resulting in a public health problem is very unlikely but cannot be excluded. The clinical use and importance of the antibiotic to which the gene confers resistance is therefore a central part of the safety assessment.

181

Clinically important antibiotics include vancomycin and other glycopeptides, macrolides, fluoroquinolones, gentamicin, amikacin, and amoxycillin and later derivatives of beta-lactam antibiotics.


42


182

A WHO workshop (1993) concluded that it is necessary to identify all oral uses of the antibiotic and possible gastro-intestinal conditions (eg high gastric pH) that may interfere with the normal degradation of DNA, and to determine the necessary enzyme cofactors and their presence in the gut. Calculation of the extent of potential inactivation of an oral dose of the antibiotic should be based on the maximum intake of the GM food that could be consumed during oral antibiotic therapy.

183

Estimation of the extent of the antibiotic’s use indicates the therapeutic consequences should the resistance become prevalent in bacterial pathogens. The potential for cross-resistance also has to be taken into account as many genes are effective against antibiotics with similar structures and mechanisms. Although the antibiotic used for selection may be rarely used it is possible others in the group may be of therapeutic importance (eg amikacin). The prevalence of the resistance gene in natural populations is also important (ACNFP, 1996).

184

Theoretical calculations of the probability of a transfer event based on a number of assumptions have shown that the chances of this occurring are extremely small and probably insignificant when compared with naturally occurring resistance. Experiments to test whether these assumptions and calculations are correct although difficult to perform would be extremely valuable. Studies with human volunteers could be considered (Kok et al, 1994). Preliminary results from a study by Heritage and coworkers of gene transfer from GM maize to chicken gut bacteria have found no evidence of transfer of the ampicillin resistance gene to normal flora (Coghlan, 2000). This study will also investigate whether gene transfer occurs from GM maize to sheep gut bacteria.

185

The types of experiments carried out should be commensurate with the importance of the antibiotic that may be compromised. While an in vitro model is sufficient for an antibiotic that is relatively unimportant clinically, studies in animals may be warranted for important antibiotics. Some scientists have suggested that it could be concluded that transfer does not occur if a large number of animals were fed GM plants containing an antibiotic resistance gene under strong selective pressure and new resistant micro-organisms with this genotype were not detected (US FDA, 1998).


43

Horizontal gene transfer mechanisms in bacteria

4.


186

The significance of horizontal gene transfer for bacterial evolution was not recognised until the 1950s when multi-drug resistance emerged on a global scale (Ochman et al, 2000).

187

Bacteria transfer resistance genes through three main mechanisms. DNA can enter the cell on plasmids through cell-to-cell contact between two cells (conjugation), by bacteriophage introduction (transduction), or by cellular uptake of free (extracellular) DNA (transformation). Gene transfer crosses species and genus barriers and groups of resistance genes tend to travel together. This has contributed largely to the efficiency by which antibiotic resistance has spread.

188

The relative importance of the gene transfer mechanisms in nature is not known. The specific requirements of each mechanism suggest different probabilities for their occurrence in different natural habitats. Conjugation has the greatest requirements. Each transfer mechanism is also characterised by its specific host range.

189

Barriers to gene transfer between bacterial species include different microhabitats, host ranges of genetic exchange vectors and barriers that block the establishment of the acquired genetic information.

190

To become replicable and stably inherited, transferred DNA must become integrated into the recipient chromosome. Efficiency of integration by homologous recombination depends on the genomic sequence divergence between species. Once recombination is initiated genetic integrity is controlled by the mismatch-repair system that inhibits recombination.

191

The frequency of recombination in natural populations depends upon the recombination rate and natural selection. If the transformants are selectively disadvantageous they will be lost from the population and never observed. If they are selectively neutral most may be lost but some may be retained though at a lower frequency than those that are selectively advantageous. The rate of interspecies recombination is very low but the rare transformants can survive in situations of strong selective pressure (Matic et al, 1996).

192

Genes that do not provide a meaningful function also tend to be lost from bacterial genomes (Ochman et al, 2000).

Transduction 193

Transduction can occur in a wide range of bacteria. In transduction bacteriophages pick up genetic material from one bacterial cell and place it in another. As part of their life cycle bacteriophages attach to bacteria and inject their DNA. This DNA then serves as the blueprint for making more copies of the bacteriophage which are released from the infected bacterium and go on to infect other cells. Sometimes some of the new particles carry bacterial instead of viral DNA and deliver it to a second bacterium that incorporates it into its chromosome. Bacteriophages are capable of transferring whole plasmids and pieces of chromosomes between hosts. Transduction depends on the survival of the transducing


44


particles but not of the donor bacterium. Most bacteriophages infect only one species of bacteria and most bacteriophages in the wild infect only bacteria that are native to the bacteriophage’s habitat (Miller, 1998). Conjugation 194

Conjugation requires cell-to-cell contact and is a process by which plasmids or transposons transfer DNA from donor to recipient cells. It occurs in both Gram-positive and Gramnegative bacteria and can involve distantly related species. Some plasmids can transfer DNA between very unrelated species: between Gram-negative and Gram-positive bacteria, from bacteria (E. coli) to yeast, and from bacteria (A. tumefaciens) to plants (Droge et al, 1998). Conjugation requires live metabolically active donor and recipient cells.

195

The main limitation is the host range in which the element can express its genes and replicate. Since many of the conjugative elements exhibit a broad host range of transfer and autonomous replication, conjugation is regarded as an important factor for gene transfer among bacteria.

196

Conjugal transfer of resistance genes between the normal microflora of animals and humans can be demonstrated under laboratory conditions (Shoemaker et al, 1992). Bacterial gene transfer studies in model ecosystems or in the environment have identified environmental hot-spots, particularly the phytosphere, where conditions increased the probability of conjugative transfer among bacteria (Pukall et al, 1996; Droge et al, 1998).

Transformation 197

Transformation is the process by which a competent bacterial cell takes up, integrates and expresses foreign DNA. Transformation in both Gram-negative and Gram-positive bacteria requires that the free DNA remains stable and that potential recipient cells become competent to take it up (ie the recipients must display specialised surface proteins that bind to the DNA and internalise it).

198

Transformation does not require a living donor cell because release of DNA during death and cell lysis suffices to provide free DNA. Therefore it can potentially occur with DNA from any source. Transformation in bacteria is assumed to be limited to those situations where the incoming DNA can reconstitute to form a self-replicating entity (eg plasmid), or where there is sufficient sequence homology with the recipient chromosome to allow insertion by recombination.

199

Currently only about 40 species are known to be capable of developing the competence for transformation (Gebhard and Smalla, 1998). The natural competence to act as recipients has been identified in several genera including Acinetobacter, Haemophilus, Pneumococcus, Streptococcus, Bacillus, Pseudomonas and Neisseria. Since some of these bacterial species are promiscuous in their uptake of free DNA, gene transfer by transformation between even distantly related bacteria is possible (Droge et al, 1998).


45


200

Experiments under controlled conditions suggest there may be species-specific and even strain-specific responses to environmental factors with regard to competence development and transformation efficiency. How long the competent state of cells can be maintained once it is achieved is important for the overall probability that a cell will become transformed.

201

There is no specific model micro-organism that is representative of other naturally transformable bacteria in a given habitat.

Evidence for gene transfer between bacteria 202

Little is known about natural transformation among gut bacteria for most gut species. An in vivo experiment by Griffith in 1928 in mice in which the non-pathogenic form of S. pneumoniae was transformed by dead cells from the pathogenic form was the first demonstration of gene transfer by transformation. There is no other direct experimental evidence for transformation in vivo. However results of co-cultivation experiments suggest that substantial intra- and interspecies transfer of virulence determinants via transformation occurs in Neisseria species. An increasing body of nucleotide sequence data of chromosomal genes in transformable species further suggests the occurrence of frequent genetic exchange within and between species (Lorenz and Wackernagel, 1994).

203

There is also evidence in several naturally transformable bacteria (eg Neisseria meningitidis) of antibiotic resistance arising from transfer of DNA, including from different species, and formation of mosaic genes from the original susceptible alleles and introduced resistant alleles. These mosaic genes express enzymes with decreased affinity for the antibiotic (Maiden, 1998).

204

Transfer of a plasmid containing an antibiotic resistance gene between Enterococcus faecalis and Lactobacillus species has been demonstrated in the murine intestinal tract (McConnell et al, 1991). Although this gene transfer event involved members of minor populations of intestinal bacteria it shows that such transfers do occur in the intestine.

205

Discovering that antibiotic resistance genes in two different genera of bacteria are 99 percent identical at the DNA sequence level suggests that a horizontal transfer event occurred sometime during the past million years but not that the transfer event was recent. In contrast the finding of 100 percent sequence identical tetQ genes in two different human colonic Bacteroides species suggests transfer has occurred recently. However more systematic studies are needed to establish that such transfers occur frequently and to determine whether their frequency is related to the widespread use of antibiotics (Salyers, 1993).

206

Failure to demonstrate transfer of a particular marker gene from a bacterial donor to a bacterial recipient under laboratory conditions does not mean that it is non-transmissible. Transfer could be regulated and if so the proper inducer may not have been used. Alternatively the marker gene could be on an element that is mobilised by some other gene transfer element that is not present in the particular donor (Salyers, 1993).

207

From laboratory studies transformation and conjugation are recognised as the gene transfer mechanisms that have the broadest host range. It has been argued that in the environment


46


transformation may be a rare event except under defined conditions whereas conjugation may be favoured because the genetic material to be transferred is protected from attack by free nucleases. In addition conjugal DNA transfer has been shown to partially overcome host restriction systems due to the transfer of single stranded DNA that may be refractory to nuclease activity in the recipient. 208

Understanding of the mechanisms and factors involved in gene transfer in natural bacterial populations is very limited (Davies, 1997). Probably not only the physical characteristics of the population but also other factors play important roles in natural gene transfers. For example, some antibiotics have been shown to promote conjugation of resistance genes.

209

Low doses of tetracycline increased conjugative transfer of a transposon conferring resistance to kanamycin, erythromycin and tetracycline from E. faecalis to Listeria monocytogenes in the gastro-intestinal tract of mice (Doucet-Populaire et al, 1991).

Prevalence of antibiotic resistance genes 210

Resistance genes reside in commensal (normally harmless) bacteria as well as pathogens. Some antibiotic resistant bacteria occur naturally in the environment but many arise by contamination with human and animal excreta in sewage, slurry and manure. This in turn contaminates water and agricultural land to become a source of resistant bacteria for animals and humans. They may be ingested as contaminants of water and food or in the case of animals by licking their environment. Even people not receiving antibiotics or on a vegetarian diet may be colonised by large numbers of resistant bacteria (Linton, 1986).

211

Enterococci and streptococci that contain resistance plasmids or transposons are common in the gastro-intestinal tract of humans and animals. These bacteria might serve as a reservoir of resistance genes for other bacteria (Doucet-Populaire et al, 1991).

212

In addition a number of antibiotics are contaminated with DNA encoding antibiotic resistance from the producing organism (Webb and Davies, 1993).

213

A US study found high level aminoglycoside resistance in aquatic environmental isolates of enterococci (Rice et al, 1995). The occurrence of high level resistance is an example of acquired rather than intrinsic resistance.

214

A study by Smalla et al (1993) showed that resistance to kanamycin is widely distributed among culturable bacteria isolated from different habitats. Since culturable bacteria represent only a minor proportion of the bacterial populations of the tested habitats the total DNA extracted directly from environmental samples was used to get information about the natural occurrence of the nptII gene and the transposon Tn52 in non-culturable bacteria. Bacteria may be naturally non-culturable or become so as a result of environmental stress. This approach gives information on the presence of a certain sequence in a sample but not on the localisation of the detected sequence (ie in culturable, non-culturable or dead bacteria, or in free DNA).

2

The nptII gene is commonly associated with transposon Tn5.


47


215

Bacteria containing Tn5 or nptII were not found in soil and were primarily obtained from sewage samples. Evidence for the occurrence of nptII was obtained for sewage, pig manure slurry, river water and some soil samples via polymerase chain reaction (PCR) analysis of environmental DNA extracts. Tn5 was not detectable via PCR in any of these extracts but it was found in soil samples taken from a field two and four weeks after release of a Tn5containing GMO. Although the kanamycin resistance phenotype is widespread among environmental bacteria Smalla et al (1993) concluded that Tn5 or nptII are scarce.

216

Using a marker-rescue system de Vries and Wackernagel (1998) found nptII without the need for amplification by PCR in all four soil samples tested.

217

Henschke and Schmidt (1990) found a high proportion of antibiotic resistant strains among soil bacteria.

218

A US study of the natural frequency of antibiotic resistance genes (to seven different antibiotics) in the commensal gut microflora of people in the community and in hospital found resistance was common in the absence of concurrent or recent antibiotic use. In more than a third of the faecal samples resistant strains constituted a major proportion of the total microflora (Levy et al, 1988).

219

In people with no recent history of antibiotic use at least 10 percent of the total microflora was resistant to at least one antibiotic in 63 percent of samples. Thirty-eight percent of the samples showed resistance(s) in half or more of the microflora. This level of resistance may be the result of slow loss of resistance genes acquired from previous antibiotic use, ingestion of resistant bacteria in food, or other unknown factors that favour persistence of resistance genes.

220

Individuals taking antibiotics produced more samples with a higher proportion (≥ 50 percent) of resistant bacteria and these samples also had a significantly greater number of different resistance genes.

221

Data from a multiple sample group revealed changes in resistance patterns were very common and occurred frequently within a two week period. Ninety percent of individuals showed a gain and/or loss of one or more detectable resistances (Levy et al, 1988).

222

About 106 to 108 E. coli are generally found per gram of human faeces. Given about 100g of faeces is excreted per day, an individual with a 10 percent frequency of a resistance gene produces 107 to 109 resistant bacteria per day. Even at a one percent resistance level relatively large quantities of resistance genes are being excreted. This represents a large reservoir of resistant bacteria and resistance genes potentially transferable, directly or indirectly to human pathogens (Levy et al, 1988). Notably this study preceded the release of GMOs containing antibiotic resistance genes into the US food chain. This refutes the claims of Ho who links the commercialisation of gene technology to the spread of antibiotic resistance. No direct evidence is provided for the hypothesis but the claim is deduced from the evidence that horizontal gene transfer spreads antibiotic resistance (Ho, 1998).


48

Ecological issues

5.

Ecological issues

Introduction 223

Specific ecological concerns with respect to antibiotic resistance marker genes relate to the possibility that if the gene is expressed in the plant antibiotic resistance might result in a plant or one of its wild relatives becoming a weed, or might disturb the ecological relationships of the plant in another unknown way. The antibiotic resistance gene could also potentially be transferred from the GMO to soil micro-organisms. Any increase in antibiotic resistant soil micro-organisms could lead to a potential increase in human exposure to antibiotic resistant micro-organisms from ingesting them as contaminants of food and water.

Antibiotic resistance and weediness Gene transfer 224

The antibiotic resistance gene might spread from the GM plant to sexually compatible species or to other organisms that as a result become a weed. Gene transfer by sexual hybridisation to plants of the same or related species depends on the degree of sexual compatibility between the donor and recipient species as well as the physical distance between the two, the duration of pollen viability, and factors such as temperature. The donor and recipient plants should produce receptive flowers at the same time in the presence of any necessary pollinating agent. If fertilisation successfully occurs it may not always result in a plant that is able to grow successfully, or if a plant is produced then it may not compete well with other species in the environment. If the resulting plant produces pollen that goes on to fertilise other plants then the inserted gene will reach an equilibrium in the overall population if there is no selective advantage for the plants that contain it.

225

The spread of an antibiotic resistance gene to relatives via pollen depends on a variety of ecological and genetic factors and stochastic events. Factors concerning the plant itself and the ecological relationships with other species of that plant at the particular location need to be taken into account to be able to estimate the probability of cross-pollination. Interspecies gene transfer is much less frequent than intervarietal exchanges (Thuriaux, 1999). It is prudent to assume that an antibiotic resistance gene may spread by cross-pollination in some conditions and at some locations with a certain probability that can be estimated (Nap et al, 1992).

226

There is little information relating to the transfer distances of pollen from GM plants containing marker genes and the dynamics of its spread in a natural population (Harding, 1999).

227

Antibiotic resistance would only be able to contribute to the weed characteristics of a plant if the environment exerts selective pressure. Even then it may have no effect on weediness. Similarly, relatives that receive the gene would only become less controllable than the parent plant in the context of selective antibiotic conditions.


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Ecological issues

228

Monocotyledonous plants are already highly resistant to kanamycin probably because it cannot enter the cell in sufficient amounts. Kanamycin resistance may therefore develop as a result of spontaneous mutations and does not seem to require the transgene. As a result phenotypic kanamycin resistance should not be considered a novel characteristic for an ecosystem and will not contribute to enhanced weediness of a plant or its relative (Nap et al, 1992).

229

The only situation in which antibiotic resistance feasibly contributes to a selective advantage of the GM plant is when selective antibiotic concentrations are found in the environment as a result of natural production by soil micro-organisms or addition of antibiotic to soil.

Antibiotics in soil 230

Soil is a reservoir of micro-organisms with the capacity to produce antibiotics. Kanamycin and neomycin are produced by soil micro-organisms but it is unclear whether they are only produced under laboratory and industrial conditions or also in the natural environment. Actual concentrations of antibiotics in natural soils are also unclear and difficult to estimate, since with few exceptions antibiotics are rarely isolated from soil. This is probably a methodological problem since, depending on the specific antibiotic, antibiotics are presumed to bind to varying degrees to soil and hence become inactivated (Nielsen et al, 1998).

231

There is a lack of information about the extent of selective pressure of different antibiotics in soil and the soil conditions that promote such selective pressure. The selective advantage of expressing antibiotic resistance genes in soil is unclear and estimation of the selection of putative transformants receiving antibiotic resistance genes from GM plants is currently not possible (Nielsen et al, 1998).

232

A study by de Oliveira et al (1995) showed evidence for selective pressure exerted by streptomycin in soil influencing the survival of an introduced Pseudomonas fluorescens strain carrying the streptomycin resistance encoding transposon Tn5 and for lack of selection by kanamycin. The effect of streptomycin in soil on the Tn5 carrying bacteria depended on conditions such as soil type.

233

Recorbet et al (1992) assessed the occurrence of a selective advantage associated with antibiotic resistance encoded by nptII by adding kanamycin and neomycin. No selection was found suggesting kanamycin is inactivated via adsorption to soil.

234

Veterinary use is a potential source of antibiotic addition to soil through the application of manure. Kanamycin and neomycin are poorly absorbed in the gastro-intestinal tract and an estimated 97 percent leaves the body unchanged in faeces.

235

Most substances (eg nucleic acids, proteins, clay) can form complexes with aminoglycoside antibiotics and therefore reduce their biological activity. Most, if not all, soils are therefore able to inactivate substantial amounts by adsorption or desorption. Although kanamycin and neomycin are relatively stable compounds it is also likely that the concentration is limited by biodegradation. Consequently it is unlikely that soil will be able to accumulate kanamycin or neomycin in concentrations that are selective for antibiotic resistant plants (Nap et al, 1992).


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Ecological issues

236

It has been argued that particularly in the absence of selection, the burden of an extra but useless gene would reduce the fitness of the plant suggesting that the gene may decrease weediness. Data from laboratory studies and field trials do not support this view (Nap et al, 1992).

Pleiotrophic effects 237

As well as inactivating the antibiotic, the antibiotic resistance gene product could interfere with existing metabolic pathways. Substrate specificity of neomycin phosphotransferase is high and available data show that the enzyme does not interfere with the basic functions of growth and development of a plant. The probability of the kanamycin resistance gene undergoing mutation resulting in a novel and functional enzyme with other substrate specificity in such a way that plant metabolism is disturbed and the plant has a selective advantage has been judged as negligible (Nap et al, 1992).

238

However the effect of the enzymatic activity (eg phosphorylation) of the marker gene in the plant’s biochemical environment is unknown. Plant cells produce a diverse range of metabolites. Many are chemically ill-defined with no known function. Some may have a chemically similar structure to aminoglycoside antibiotics making them possible targets for phosphorylation by neomycin phosphotransferase. No activity of phosphorylation products has yet been identified but the potential of this non-specific phosphorylation to chemically modify plant metabolites not normally phosphorylated and their metabolite breakdown products is likely to vary among plants. As the range of GM plant species increases the potential for non-specific phosphorylation of metabolites is also likely to increase. This may have consequences for insects that come in contact with plant exudates such as sap or pollen (Harding and Harris, 1997).

239

The presence of an antibiotic resistance gene or its product may have pleiotrophic effects that in some way alter any of the ecological relationships of the plant itself, any wild relative derived from outcrossing, or any micro-organism derived from horizontal gene transfer (Metz and Nap, 1997).

Use of antibiotic as a herbicide 240

The presence of kanamycin resistance in crops means it is possible to use kanamycin as a herbicide. Apart from the relatively high costs of production, kanamycin and neomycin are not likely to be used as a herbicide because of their limited efficacy, the intrinsic resistance of various plants, and the likely public unacceptability of such a practice. Even if large amounts were used it is considered that their physico-chemical characteristics would prevent the establishment of selective conditions (Nap et al, 1992).


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Ecological issues

Horizontal gene transfer in the environment 241

Horizontal gene transfer in the environment includes from plant to plant via pollen, from micro-organism to micro-organism, from micro-organism to plant, and from plant (or animal) to micro-organism.

242

Horizontal gene transfer into plants from the soil bacterium Agrobacterium tumefaciens that is a plant pathogen is well documented and occurs in nature. This is the best studied example of gene transfer from a micro-organism to a plant. In contrast, research has not detected transfer of plant DNA to Agrobacterium (OECD, 2000b).

243

As plants do not have any identified mechanism to facilitate broad host range gene transfer (except for pollen hybridisation with related species) the possibilities and barriers of horizontal gene transfer from plants to micro-organisms have been approached within the framework of known mechanisms of horizontal gene transfer within bacteria.

244

Horizontal transfer of genetic information between bacteria has been extensively demonstrated both in vitro and in natural systems (Droge et al, 1999). It is unclear to what extent all possible mechanisms of gene transfer in bacteria have been identified since less than one percent of the bacteria present in the natural environment have been described at species level (Nielsen et al, 1998).

245

Transduction is unlikely as viruses that function in both plants and bacteria, and thereby possibly facilitate gene transfer from plants to bacteria have not yet been identified.

246

Mechanisms that support conjugative gene transfer from higher plants to bacteria are unknown, and transposons that function in both plants and prokaryotes have not been identified (Nielsen et al, 1998).

247

Transformation is a mechanism by which plant DNA could be transferred to microorganisms. Since some competent bacterial species take up free DNA independently of its sequence, transformation theoretically facilitates gene transfer from plants to bacteria. It is considered the only natural mechanism that can be involved in promoting the uptake of plant DNA by bacteria (Bertolla and Simonet, 1999).

248

Several barriers that restrict gene transfer between distantly related organisms in the environment have been proposed. The main ones are probably transfer and establishment barriers (Nielsen et al, 1998).

249

During plant growth or decay or animal feeding on plants the antibiotic resistance gene could become available and transfer to micro-organisms. Plant cells contain DNases that are released when cells are damaged resulting in the breakdown of the plant’s DNA. Soil microorganisms would increase the degradation process and further reduce the probability of transfer from decaying material. If DNA was available then the level of transformation of soil micro-organisms would depend upon the proportion of micro-organisms that were naturally transformable. DNA transfer between micro-organisms is supposed to occur frequently in soil, but the available data are related to conjugative transfer of plasmids. Plant


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Ecological issues

DNA will not be available as plasmids so conjugative DNA transfer can be excluded (Nap et al, 1992). 250

Assessment of potential impact must also consider bacterial ecology. Bacterial populations found in different sites differ markedly in composition, metabolic activities and their environmental conditions (Salyers, 1999). Studies have shown that natural transformation can occur in soils but the major limiting factor is development of competence by bacteria. However there are niches in which bacteria may find more favourable conditions for developing the specific metabolism required for transformation (eg the gut of warm-blooded animals) (Bertolla and Simonet, 1999).

Prerequisites for transformation 251

There are a number of steps for transformation to occur under natural conditions. These are: • release of DNA into the environment; • adsorption onto soil for protection against enzymatic activity; • presence of genetically adapted bacterial genotypes for natural transformation; • appropriate conditions for the development of competence; • efficient adsorption of the DNA to the bacterial cell surface; • efficient DNA uptake; • chromosomal integration via recombination or autonomous replication of the transforming DNA; and • expression of the gene by the recipient bacterium (Bertolla and Simonet, 1999).

252

Transformation requires access to free DNA which must be present at the time and place in which competent bacteria develop or reside.

253

The probability that bacterial transformation will occur is greater the slower the rate of turnover of the DNA in soil. Free DNA in soil is derived from lysis of cells either following the death of plants, animals and micro-organisms or as a result of excretion of plasmid or chromosomal DNA by some micro-organisms. Most free DNA is rapidly degraded but a proportion escapes as a result of adsorption onto soil particles. DNA degradation rates differ significantly among various habitats.

254

Both plant and bacterial DNA have been shown to persist in soil for weeks or months. Persistence of DNA in soil over time does not necessarily imply that the DNA is in a physical or chemical condition that makes it available for transformation. However bacterial DNA adsorbed to soil particles has been shown in microcosm experiments to be able to transform competent bacteria (Sikorski et al, 1998).

Persistence of DNA 255

Different abiotic and biotic factors seem to affect the persistence of free DNA in soil making general predictions on the persistence of plant DNA in soils difficult. Plant DNA can persist for months in soil in particular those rich in organic matter and clay on which nucleic acids can adsorb without inhibiting their availability to competent bacteria. Adsorption provides partial protection from degradation by nucleases. This is considered the most important


53

Ecological issues

factor determining DNA persistence in the environment. In addition, high bacterial activity accompanied by an enhanced presence of bacterial DNase affects the persistence of free DNA in soil (Gebhard and Smalla, 1999). 256

Analysis of DNA extracted directly from soil samples by PCR showed long term persistence of GM plant DNA in soil samples collected at the release site of GM sugar beet. The soil samples were taken about six months after GM sugar beet was incorporated into the soil after shredding. None of the more than 2,000 kanamycin resistant bacteria screened for the presence of nptII by dot blot hybridisations contained the GM plant DNA (Smalla et al, 1994).

257

Paget et al (1998) carried out a field experiment with GM tobacco containing a bacterial gentamicin resistance marker gene (aacC1). The fate of plant DNA in soil was monitored for up to three years. Most previous studies have relied on laboratory systems. Results indicated that plant DNA is released into the soil and can persist there for months but no longer than three years under natural conditions. (Results were negative three years after harvesting the GM tobacco). The gene was detected by PCR in all soil samples suggesting that soil contains non-culturable bacteria containing sequences very similar to the aacC1 gene.

258

Under most experimental conditions DNA is initially degraded at a high rate. In controlled laboratory systems using mixtures of ground plant tissue and soil a small proportion of the added DNA resisted degradation and nptII was detectable for several months. This DNA may have been adsorbed to soil particles and therefore protected from complete degradation. Low temperature and low moisture were found to have stabilising effects on DNA in nonsterile soil (Widmer et al, 1996).

259

An investigation of the persistence of nptII in the field showed differences in DNA stability that may be related to the different plant types and different experimental conditions (GM tobacco leaves were buried and residual GM potato plant litter was obtained from the soil surface). Results indicated that residual plant tissue might be a location where relatively large amounts of detectable DNA persist for several months at a field site (Widmer et al, 1997).

260

Gebhard and Smalla (1999) have demonstrated long term persistence of GM sugar beet DNA under field conditions (up to two years) and in soil microcosms with introduced free GM sugar beet DNA (up to six months).

261

Research findings of rapid initial degradation of plant DNA in different soil and field systems indicate that a possible transfer frequency of the kanamycin resistance gene and most likely other genes from plants to micro-organisms may be very low and restricted to microhabitats that contain residual plant tissues and DNA adsorbed on soil particles.

Transformation of soil micro-organisms 262

Predictions of the extent of horizontal gene transfer from plants to bacteria are currently hindered by lack of information on the abundance of naturally competent bacteria in the environment, frequency of transformation and environmental factors triggering competence and transformation (Gebhard and Smalla, 1999).


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Ecological issues

263

Induction of competence is usually related to the physiological stage of the cells and/or to accumulation of an environmental factor. Knowledge of environmental factors that affect the regulation of bacterial competence is scarce (Lorenz and Wackernagel, 1994).

264

Adsorption of DNA to the bacterial cell surface is considered the first step of the transformation process.

265

Some soil micro-organisms may be naturally transformable and may take up and incorporate DNA causing genomic rearrangements that might help them occupy particular ecological niches.

266

Recombination events in bacteria with environmentally regulated transient deficiencies in their DNA repair and recombination system or mutations, illegitimate recombination events, homologous recombination, and origin of replication based plasmid rescue are potential means to stabilise DNA from GMOs if transferred to micro-organisms (Nielsen et al, 1998).

267

Transformation rates can be markedly affected by widespread bacterial restriction and modification systems protecting the host DNA against contamination by foreign sequences. The fact that DNA uptake involves a single strand step in most naturally competent bacteria indicates that transforming DNA will escape the restriction systems, allowing successful transformation of the bacterium. Saturating amounts of DNA or inefficient restriction systems could also lead to successful transformation.

268

Stable maintenance of GM plant DNA in bacteria requires linkage to an origin of replication such as by integration of this DNA into the bacterial chromosome, or its autonomous replication based on the presence of replication functions and an origin of replication in the DNA.

269

Homologous recombination can only occur when the donor DNA and the host genome share DNA sequence similarities. The effectiveness of homologous recombination may also vary as a function of the insertion site of the transforming DNA.

270

Recombination is also under the control of the mismatch repair system involved in correction of replication errors and base modifications. This mechanism prevents heterologous recombination and allows the cell to maintain some level of genetic stability (Matic et al, 1996). However one percent of natural isolates of pathogenic and commensal enterobacteria (E. coli and Salmonella species) display mutations that could favour recombination with heterologous transforming DNA (Bertolla and Simonet, 1999). Subpopulations of bacterial communities might show an enhanced frequency for such recombination.

271

Illegitimate recombination events occur in different organisms. Construction of all GMOs is currently based on illegitimate recombination events with random sites of insertion of the genes in the host genome (Nielsen et al, 1998).

272

With the introduction of bacterial genes, bacterial promoter and terminator sequences, and bacterial origins of replication into GM plants, the degree of sequence homology between the genomes of competent bacteria and GM plant DNA increases. The probability of gene


55

Ecological issues

transfer is therefore increased (Gebhard and Smalla, 1998). Homologous recombination between a recombinant sequence in the plant chromosome and the natural sequence in competent bacteria can result in the stable insertion of the DNA. 273

Short regions of homology can mediate recombination that includes incorporation of adjacent non-homologous sequences. Short repetitive sequences are commonly found dispersed in bacterial genomes and although speculative, these may, if integrated into the genomes of GMOs also mediate the transfer of adjacent non-homologous gene sequences to bacteria. Vector development that facilitates the transfer of large DNA fragments may introduce longer bacterial sequences in GMOs (Nielsen et al, 1998).

274

Recently a new class of mobile genetic elements in bacteria, the gene cassettes, have been described. Gene cassettes are usually found integrated adjacent to integrons which can mediate the expression of the cassettes and their movement. Many antibiotic resistance genes have been identified as functional gene cassettes including some selectable markers in GMOs (Hall, 1997). Although not generally used as cassettes in GM work use of a gene cassette in a GMO may circumvent the requirement for homologous recombination based stabilisation of DNA in bacteria since integration of the cassette can be encoded by the integron.

275

Stabilisation of GM DNA in bacteria is also feasible if the plant DNA contains replication functions and a bacterial origin of replication facilitating its autonomous replication. Construction of GM monocotyledonous plants such as cereals, rice and maize is usually facilitated by electroporation or the use of particle guns that result in the integration of whole plasmids with intact replication functions. If fragments of such DNA become recircularised following their uptake in bacterial recipients they might become stabilised by a plasmid rescue-like mechanism.

276

Due to DNA cloning methods, eukaryotic genes inserted into GMOs do not normally have introns which probably enhances their expression if transferred downstream of promoters in bacteria. Although the promoters inserted into GMOs usually display low activity in prokaryotic hosts some are also active in bacteria (eg the frequently used cauliflower mosaic virus 35S promoter expresses in E. coli). Insertion of whole plasmids into the GMO may lead to the presence of bacterially expressed vector sequences like the ampicillin resistance gene located on the vector pUC18.

277

Random insertion of protein encoding sequences from GM DNA into existing regulatory sequences in the genome of the bacterium may also mediate gene expression after gene transfer from plants.

278

Uptake and recombination with GM DNA fragments rather than whole genes might also influence gene expression and variability in bacteria. For example, if a deletion is restored in an antibiotic resistance gene, or its expression is upregulated after recombination, this would lead to a stronger antibiotic resistance in the bacterium. Similarly recombination may also alter the specificity of the enzyme conferring the antibiotic resistance (Nielsen et al, 1998).

279

A concentration of DNA per hectare from GM tomatoes was calculated assuming inter alia that 10 percent of the DNA was released and 10 percent of the released DNA was intact. With this concentration and assuming 10 percent of soil micro-organisms are transformable,


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Ecological issues

five percent of which are competent and a natural transformation frequency of 0.01 percent, a transformation frequency of 8.7 x 10-12 was estimated. If gene transfer results in 8.7 x 10-5 transformants per gram of soil containing 107 viable micro-organisms of which 1600 were already kanamycin resistant, the contribution to natural kanamycin resistance would be 5.4 x 10-6 percent (Nap et al, 1992). 280

Transfer of the kanamycin resistance gene will not immediately result in kanamycin resistance because the gene carries regulatory sequences that will generally not work in microorganisms. Upon transfer, recombination therefore needs to occur to make the gene functional, the micro-organism should be present in a natural environment in which a selective advantage for kanamycin resistance occurs, and should be able to outcompete the kanamycin resistant organisms already present. The probability for the subsequent occurrence of all these events is negligible and as a result it was concluded that horizontal transfer of the kanamycin resistance gene will not alter or disturb a soil ecosystem (Nap et al, 1992).

281

Calgene Inc calculated the probability of transfer of the kanamycin resistance marker gene from the Flavr Savr tomato to indigenous soil micro-organisms. The theoretical gene transfer model was developed based on the putative transfer from the plant to B. subtilis which is naturally transformable but shows no homology to the GM DNA. Alternatively transfer of DNA from the plant to A. tumefaciens was considered. It is not naturally transformable but contains the plasmid carrying the same T-DNA border regions inserted into the plant genome.

282

The models estimated a worst case scenario (eg 100 percent of soil bacteria are transformable, every fragment of the plant DNA in soil contains nptII, the transformed gene is expressed after integration into the recipient genome and the gene product is active and stable in the bacterial cells) or the more likely scenario (eg only 10 percent of soil bacteria are transformable, the nptII gene constitutes 10-5 of the tomato genome, the gene is not always integrated and expressed). The estimates for the Bacillus type transformation system were 9 x 105 transformants per acre in the worst case scenario and two transformants per acre in the more likely case. Estimates were three orders of magnitude less for the Agrobacterium type transformation system. From these calculations it was concluded that in the worst case kanamycin resistant Bacillus transformants will constitute about 10-7 of the kanamycin resistant soil bacteria and Agrobacterium transformants will constitute 10-10 of the kanamycin resistant soil bacteria (Droge et al, 1998).

283

Transformation frequency of competent E. coli from GM insect resistant maize containing the ampicillin resistance gene was estimated to be 1 in 6.8 x 1019. Some experts consulted by the FDA said if transformation were to occur it would be more likely in experiments using competent bacteria in the laboratory than in nature because competent bacteria have the highest transformation frequency. If transformation was not observed in the laboratory in either Gram-negative or Gram-positive bacteria the results suggest that gene transfer may not occur in the natural environment to the extent that health or safety concerns would arise. Other experts stated that they did not have much confidence in an in vitro experiment because it does not reflect the complex natural ecosystem. In addition a monoculture of E. coli is an artificial system that is not a strong basis on which to assess risk (US FDA, 1998).


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Ecological issues

Approaches to evaluate possible horizontal gene transfer of plant DNA to soil micro-organisms 284

Although experimental approaches in both field and laboratory studies have not been able to confirm the occurrence of gene transfer to naturally occurring bacteria a few studies have shown transfer of marker genes from plants to bacteria based on homologous recombination. These results and the few examples of gene transfer indicated by DNA sequence comparisons suggest that the frequency of stable gene transfer from plants to bacteria is extremely low. However this inference is based on a small number of experimental studies and indications found in the literature (Nielsen et al, 1998).

Comparison of DNA sequences 285 A few cases suggestive of horizontal gene transfer from plants to bacteria have been identified after comparison of DNA sequences between plants and bacteria. The low number of examples suggests that the frequency of stable gene transfer from plants to bacteria is extremely low (Droge et al, 1998). Screening of bacteria from environmental samples (fields or microcosms with introduced GM plants) 286 Only about 10 percent of soil bacteria are assessable via cultivation techniques. The expected low frequency of transfer under natural conditions impedes screening bacteria from environmental samples as the number of putative transformants has been suggested to be below the limit of detection. Some of the biases involved in the isolation of bacteria from soil can be circumvented by analysing total DNA extracted from soil samples (eg Smalla et al, 1993).

287

The few studies of gene transfer from GM plants to bacteria in soil in natural (eg Paget et al, 1998) or soil microcosm conditions have not been able to show such transfer, indicating that transfer did not occur, or transfer frequencies and expression were too low to be detected, or the techniques used were not appropriate for its detection.

Experimental studies under optimised laboratory conditions 288 Gene transfer from GM plants to bacteria has only been investigated experimentally with the hypothesis that such gene transfer takes place by transformation. Reported studies have all been done in the laboratory with readily culturable, Gram-negative, soil or plant-associated bacteria.

• Studies that have been conducted to assess the potential for gene transfer from GM plants to soil or plant-associated micro-organisms: (1)

Horizontal gene transfer from GM potato to the plant pathogen Erwinia chrysanthemi was not detected under conditions mimicking a natural infection. Gradual stepwise alteration of laboratory conditions to natural conditions revealed a gradual decrease of the potential transformation frequency from 6.3 x 10-2 (one transformant per 630 bacteria) under optimal laboratory conditions to a calculated 2.0 x 10-17 under natural conditions. The latter estimate is far below the detection limit. However the natural competence of Erwinia was low and the presence and stability of released plant DNA with transforming activity from the lysed potato was not demonstrated (Schluter et al, 1995).


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Ecological issues

(2)

Broer et al (1996) used the plant pathogen A. tumefaciens as a recipient for GM tobacco. Gene transfer was not detected. Transformation frequency was found to be below the detection limit (6 x 10-12). Development of competence for natural transformation has not been shown for A. tumefaciens and its ability to take up linear DNA was not shown in this study.

(3)

Acinetobacter are naturally competent bacteria and characterised in vitro by high transformation rates. Nielsen et al (1997) did not detect any transformant using Acinetobacter and GM potato and GM sugar beet. Frequencies decreased to 10-11 in vitro and less than 10-16 in soil far below the detection limit. There was no sequence similarity between the two genomes which could prevent homologous recombination occurring. The donor DNA also did not possess any functional replication origin to permit the potentially circularised DNA to replicate autonomously.

(4)

Gebhard and Smalla (1998) and de Vries and Wackernagel (1998) developed a model based on the construction of recipient strains with a deleted sequence of the marker gene. Under optimised in vitro conditions they demonstrated that plant DNA could successfully transform Acinetobacter and restore an intact gene in the recipient strain that could express kanamycin. Frequencies were low - 5.4 x 10-9 (Gebhard and Smalla, 1998) and 3 x 10-8 (de Vries and Wackernagel, 1998) and decreased to 1.5 x 10-10 when the pure plant DNA solution was replaced by a crushed leave suspension (Gebhard and Smalla, 1998). Transformation might occur in soil if homologous sequences are present in competent bacteria though the frequency is likely to be lower than under laboratory conditions. Transformation might not have been previously demonstrated experimentally because of an absence of homologous sequences in the bacteria or use of less efficiently transformable bacteria (Gebhard and Smalla, 1998).

(5)

The plant pathogen Ralstonia solanacearum is naturally transformable. To overcome natural genetic barriers (only DNA of R. solanacearum transforms this bacterium) GM donor plants and recipient R. solanacearum were constructed to deal with homologous recombination requirements. Transformation did not occur (Bertolla and Simonet, 1999).

289

The only published experimental evidence that demonstrates that gene transfer of heterologous genes occurs from GM plants to naturally occurring soil or plant-associated bacteria are the two studies mentioned above that used artificially introduced homology between the DNA of the plant donor and recipient bacterium.

290

Horizontal gene transfer has been reported from plants to plant-associated fungi. In a study using the pathogenic fungus Aspergillus niger and GM Brassica and GM Datura innoxia plants some fungi acquired antibiotic resistance during co-cultivation but lost this resistance during further cultivation even under selective pressure. One isolate exhibited stable resistance suggesting gene transfer but the mechanism of such a transfer in fungi is unknown (Hoffman et al, 1994).


59

Ecological issues

291

A review by Bertolla and Simonet (1999) concluded that research is a long way from demonstrating that plant-bacterium transfer does occur under natural conditions. Even under optimised conditions such an event only occurs at very low frequencies. Dilution of the marker gene in the genome of GM plants makes the transformation rate lower than that found under optimised laboratory conditions. In addition the GM DNA is potentially transferable only to a very limited number of bacteria (those that develop competence and show tolerance to foreign DNA). When a transfer event does occur it is unlikely to confer any selective advantage to the recipient micro-organism and could even be considered a genetic burden.

292

Studies of gene transfer have mainly been done without selective pressure during the exposure time with DNA. There is a need for studies that incorporate selective pressure during the exposure of competent bacteria with selectable DNA to be designed (Nielsen et al, 1998).

293

Extrapolation of transformation frequencies from microcosms to the environment could be misleading because concentrations of transforming DNA in situ are not known. Transformation frequencies differ considerably among species (Lorenz and Wackernagel, 1994). Conditions influencing occurrence of gene transfer in the natural environment also might remain unidentified in laboratory studies thus generating under- or over-estimates of transformation frequencies.

294

Some experts consulted by the FDA felt it was imprudent to increase the availability of resistance genes in the environment because this may reduce the typical four to five year time lag between first use of a new antibiotic and emergence of resistance in hospitals. Others felt the risk of transfer from plant genome to soil micro-organisms is not significant as there is no selective pressure in most cases. Exceptions include the use of streptomycin as a pesticide in horticulture or use of manure as fertiliser following use of antibiotics as growth promotants in animals (US FDA, 1998).

295

Detection of gene transfer events is difficult due to the limitations of the techniques available. Transfer of antibiotic resistance genes is also difficult to document due to high levels of resistance that already exist. Unequivocal proof of gene transfer requires isolation of the putative transformants for thorough genetic characterisation. However the strategy to monitor the transfer of complete genes of larger DNA fragments might fail because transformation often involves the stable integration of short DNA fragments resulting in gene mosaics.

296

The high prevalence of naturally occurring antibiotic resistant bacteria in the environment has often been used as an argument for the low impact of potential transfer of antibiotic resistance genes, particularly nptII, from GMOs. However the description of a phenotypically observed resistance pattern does not address the natural presence of the antibiotic resistance genes in these bacteria. Many mechanisms can be involved in bacterial antibiotic resistance. A clearer distinction needs to be made between observed phenotype and the corresponding genotype. Although there is information about phenotypic resistance few studies have identified the gene responsible (US FDA, 1998; Salyers, 1999).


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Ecological issues

297

Monitoring should be at the genotypic rather than the phenotypic level and given that all resistance genes originate from micro-organisms it should distinguish a gene that has been transferred from a GM plant (Salyers, 1999).

298

Examining areas with a high concentration of GM plants would increase the chance of finding the rare transfer event. Monitoring markers where the antibiotic is used in animal feed as a growth promotant would also increase the chance of finding a transformant because there would be selective pressure (US FDA, 1998).

299

The present knowledge of bacterial ecology in soil environments is unable to predict and quantify factors in soil that affect the selection of bacterial transformants receiving novel genes. Widespread introduction of GM plants into the environment will generate a continuous exposure of bacteria to high numbers of transgenes and may as a result enhance the probability of the amplification of these genes after integration in bacterial hosts.


61

The New Zealand and international approach to antibiotic resistance marker genes

6.


300

The emphasis in this section is on the approach of national and international regulatory agencies, advisory bodies and other organisations to the potential health rather than environmental impact of the use of antibiotic resistance genes in GMOs.

The New Zealand approach to antibiotic resistance marker genes Environmental Risk Management Authority (ERMA) 301

To date the Environmental Risk Management Authority has not considered any applications for release of a GMO in New Zealand. Field trials of GM sugar beet, potato, sheep, and cattle containing an antibiotic resistance gene have been approved. The gene was either the kanamycin (neomycin) or puromycin resistance gene.

Antibiotic Resistance Expert Panel 302

The Antibiotic Resistance Expert Panel that reported to the Ministry of Agriculture and Forestry Antibiotic Resistance Steering Group on antibiotic resistance and in-feed use of antibiotics in New Zealand agreed with the conclusion of the Australian Joint Expert Technical Advisory Committee on Antibiotic Resistance (JETACAR) report that the probability of antibiotic resistance marker gene transfer to gut micro-organisms is extremely remote. If transfer did occur there would then have to be concurrent or subsequent selection pressure by the presence of the antibiotic in the gut before it could become a health issue. However as a precautionary approach the Panel recommended avoidance of antibiotic resistance marker genes in the development of GMOs intended for wide release (Antibiotic Resistance Expert Panel, 1999).

Australia New Zealand Food Authority (ANZFA) 303

The Australia New Zealand Food Authority considers the possibility of gene transfer and its consequence for human health in its safety assessments of GM foods.

304

It recommends that vectors should be modified to minimise the probability of gene transfer and marker genes that confer resistance to clinically useful antibiotics (eg vancomycin) should not be used (ANZFA, 1998).

305

ANZFA considers the overall risk of gene transfer affecting the clinical use of antibiotics in humans to be effectively zero. However the issue is considered on a case-by-case basis (OECD, 2000a).

306

To date 20 applications seeking approval for GM foods have been received by ANZFA. One application has been withdrawn and a complete safety assessment has been completed for two – insect resistant cotton and glyphosate tolerant soybean. The cotton contains the kanamycin resistance gene (nptII) and streptomycin resistance gene (aad). Only cottonseed oil


62


and cellulose from processed linters, both highly refined products, are used as food. The final modification of soybeans does not include an antibiotic resistance marker gene. 307

In 2000 ANZFA released draft safety assessments for public comment on a further eight applications. Three applications (glyphosate tolerant maize, insect resistant maize and glyphosate tolerant canola) have no antibiotic resistance genes in the final GM plant. Of the other five applications, glyphosate tolerant cotton contains the kanamycin and streptomycin resistance genes; insect resistant potato (with the exception of one line), insect and potato leafroll virus resistant potato and insect and potato virus-Y resistant potato contain the kanamycin and/or streptomycin resistance genes; and high oleic acid soybean contains the ampicillin resistance gene (bla).

308

The aad and bla genes are under the control of bacterial promoters and therefore are not expressed in GM plant cells.

The approach of other countries and international organisations to antibiotic resistance marker genes 309

The stance adopted internationally toward the use of antibiotic resistance marker genes varies according to the level of risk that is regarded as acceptable. Public consultation has not been a feature of these determinations.

Australia Genetic Manipulation Advisory Committee (GMAC) 310 The Genetic Manipulation Advisory Committee is a non-statutory body that oversees development and use of novel genetic manipulation techniques in Australia.3 It provides expert technical advice on specific biosafety matters to organisations using these techniques and to regulatory agencies.

311

Decisions are made on a case-by-case basis.

312

GMAC has concluded that there is no significant biosafety risk associated with the use of the kanamycin (neomycin) resistance gene. The probability that the antibiotic resistance gene could be transferred intact from a GM plant to a pathogenic micro-organism, and expressed in that micro-organism is extremely remote. In addition resistance to the antibiotics is already widespread and the contribution that GM plants would make to existing levels of resistance would be negligible.

Joint Expert Technical Advisory Committee on Antibiotic Resistance (JETACAR) 313 The Joint Expert Technical Advisory Committee on Antibiotic Resistance predominantly examined the relationship between the use of antibiotics in food-producing animals and antibiotic resistant bacteria in animals and humans. However it also considered the use of antibiotic resistance genes as markers in genetic modification. It acknowledged that most GMOs to be released into the environment in the next 10 years will be plants and many of 3

GMAC will be replaced by a statutory authority, the Office of the Gene Technology Regulator in 2001.


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these will contain the kanamycin resistance gene. The Committee concluded that even if improbably kanamycin resistance was transferred to gut micro-organisms in humans or animals it would be of minimal consequence to human health due to the existence already of intestinal kanamycin resistant bacteria (JETACAR, 1999). Norway 314

Legislation on GM foods is divided in Norway between the Ministry of Environment (Gene Technology Act 1993) and the Ministry of Health and Social Affairs (Food Control Act).

315

In Norway since 1997 three GM food plants have been banned from being marketed because of the presence of antibiotic resistance marker genes.

316

Regulations are being developed that will ban the production, import and sale of GM foods and feed that contain antibiotic resistance genes introduced during genetic modification (OECD, 2000a).

United Kingdom Advisory Committee on Novel Foods and Processes (ACNFP) 317 The Advisory Committee on Novel Foods and Processes is an independent expert committee that advises British Health and Agriculture Ministers on the safety of novel foods and processes, including GM food organisms and derived products.

318

In 1991 the ACNFP advised that the developer would need to submit detailed information on the method of selection, including details of any antibiotic resistance marker genes used, and if the genes encoded for resistance to clinically useful antibiotics evidence that they have been removed or inactivated would normally be necessary.

319

The ACNFP concluded that of the potential food safety problems that could arise from the use of antibiotic resistance markers only the possibility of transfer and subsequent expression of the marker genes in gut micro-organisms is of significance. The possibility of such transfer and expression occurring following ingestion is extremely low and if it were to occur is most likely from live GM bacteria used as starter cultures or probiotics. It is less likely to occur from ingested raw GM plant material or from the uncooked seed of GM plants, and least likely from highly processed GM food micro-organisms and plant material.

320

The Committee recommended that GM food micro-organisms intended to be ingested live (eg lactic acid bacteria) should not contain antibiotic resistance marker genes and food or feed from GM plants and non-viable GM micro-organisms should be evaluated on a case-bycase basis (ACNFP, 1994). These recommendations were reaffirmed in a subsequent report (ACNFP, 1996).

321

As decisions are made on a case-by-case basis ACNFP does not publish prescriptive lists of acceptable and unacceptable antibiotic resistance marker genes.

322

Any further increase, however small, in resistant micro-organisms through transfer of antibiotic resistance genes from GM food would be undesirable. Researchers developing


64


GMOs for food should be encouraged to develop and use alternative markers and/or methods to excise the antibiotic resistance genes used (ACNFP, 1994). 323

However the Committee recognises that many GM crops that are currently being developed as a food source were developed before alternative marker genes were available and that commercial considerations have required the continued use of proven selection techniques including the use of antibiotic resistance marker genes.

324

The ACNFP considers that the very low probability of transfer and expression of an antibiotic resistance gene in gut or rumen micro-organisms is of little concern for markers with plant promoters but not those with bacterial promoters. Where transfer is considered to be possible and subsequent expression likely and the potentially affected antibiotic is clinically important (eg ampicillin) use of the marker would be unlikely to be approved.

325

The ACNFP has recommended rejection of three applications submitted to it on the grounds that there was a very small, though finite, risk of transfer of the resistance gene to intestinal micro-organisms of animals fed unprocessed plant material which could compromise human use of the antibiotic. These GM plants were maize containing the ampicillin resistance marker gene and two cottons containing a gene resistant to streptomycin and spectinomycin (OECD, 2000a).

326

The Committee is unlikely to have concerns about markers with bacterial regulatory sequences for which it can be proved that the gene has been disrupted or truncated (ACNFP, 1996).

The Royal Society 327 The Royal Society has also expressed concern about the use of antibiotic resistance marker genes in food and endorsed the ACNFP’s conclusions.

328

Any further increase in the use of such markers in the human or animal food chain would be undesirable and any further increase, however small, in antibiotic resistant micro-organisms through transfer of markers from GM food should be avoided.

329

It is no longer acceptable to have antibiotic resistance genes present in a new GM crop under development for potential food use and researchers should not produce GM plants containing genes that confer resistance to antibiotics that are used to treat infections in animals or humans. Such genes if used in future should be removed at an early stage in development of the GM plant and where possible alternative marker systems should be used (The Royal Society, 1998).

British Medical Association (BMA) 330 A report from the BMA concluded that the use of antibiotic resistance marker genes in GM food is an unacceptable risk, however small, to human health. Since the risk to human health from antibiotic resistance developing in micro-organisms is one of the major public health threats that will be faced in the 21st century, and the risk of antibiotic resistance being passed on to bacteria affecting humans through marker genes in the food chain cannot at present be ruled out, the BMA recommended a ban on their use in GM food. It also recommended


65


further research on the health risks arising from antibiotic resistance (British Medical Association, 1999). Other 331 A report to the UK Ministerial Group on Biotechnology from the British Government’s Chief Medical Adviser and Chief Scientific Adviser recommended that developers of GM food should be encouraged to phase out the use of antibiotic resistance marker genes as soon as is feasible. The report also stated that it is unacceptable for a viable GM micro-organism containing an antibiotic resistance gene to be eaten (Donaldson and May, 1999).

Codex Alimentarius Commission 332

The Codex Alimentarius, an instrument of the FAO and the WHO, develops international food safety standards. Its primary objective is to protect the health of consumers and to ensure fair practices in international food trade. An ad hoc Intergovernmental Task Force on Foods derived from biotechnology has been set up by Codex and met for the first time in March 2000. It has agreed to develop specific guidance on risk assessment of GM foods.

European Commission Scientific Steering Committee on Antimicrobial Resistance 333 The European Commission’s Scientific Steering Committee on Antimicrobial Resistance stated that although the risk of gene transfer is extremely small each plant containing antibiotic resistance marker genes should be evaluated on a case-by-case basis. Emphasis should mainly be on evaluation of the selection pressure acting on the bacterial recipients after possible gene transfer.

334

Although there is no evidence that antibiotic resistance marker genes have transferred from GM plants to pathogenic bacteria and the possibility of such an event has been argued to be remote, the Committee considered it appropriate to recommend that markers should be removed from plant cells before commercialisation whenever feasible. Failure to remove markers should be justified by the developer and use of genes that might have the capacity to express and confer resistance against clinically important antibiotics should be avoided.

335

It also recommended that assessment of the potential for transfer of marker genes from a plant into micro-organisms should be examined more closely (European Commission, 1999).

Nordic Working Group on Food Toxicology and Risk Assessment 336

The concept of a positive list of selectable marker genes refers to a list of acceptable marker genes with respect to biosafety. Development of a positive list was initially discussed at a WHO workshop in 1993. It was considered that it was not possible at that time to develop a list that did not cause food safety concerns. Further development work was carried out by the Nordic Working Group on Food Toxicology and Risk Assessment under the auspices of the Nordic Council of Ministers (Karenlampi, 1996).

337

The Nordic Working Group proposed the kanamycin resistance gene and glyphosate resistance gene for the list.


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338

Inclusion of a marker gene in a positive list indicates that the marker gene itself, regardless of any promoter and terminal sequences, and its gene product are considered safe for human consumption irrespective of the host plant in which the gene is inserted. Secondary effects are not evaluated as part of the procedure for inclusion because these may differ according to the site of insertion.

339

A marker gene can be included in the list without restrictions or with specific conditions (eg amount of the gene or its product present in the GM plant) for a specific plant family. Before acceptance on a positive list marker genes should be shown to be safe in at least two to three different plant families (Karenlampi, 1996).

United States International Food Biotechnology Council 340 The International Food Biotechnology Council concluded in 1990 that the use of antibiotic resistance marker genes does not pose any risks unless selection pressure occurs at the time of the probably very rarely occurring gene transfer event from the plant genome to gut micro-organisms. Food and Drug Administration (FDA) 341 In 1992 the FDA issued a policy statement on foods derived from GM plant varieties. Both the antibiotic resistance gene and its product, unless removed, are expected to be present in foods derived from such plants. Selectable marker genes that produce enzymes that inactivate clinically useful antibiotics theoretically may reduce the therapeutic efficacy of the antibiotic when taken orally if the enzyme in food inactivates the antibiotic. The FDA stated that it will be important to evaluate such concerns with respect to commercial use of antibiotic resistance genes in food in particular those that will be widely used (US FDA, 1992).

342

In 1994 the FDA amended its food additive regulations to permit the use of the kanamycin resistance gene and its expression product in the development of new varieties of tomato, oilseed rape and cotton. It concluded that if gene transfer did occur from plants to microorganisms there would be no significant increase in antibiotic resistant human pathogens. Some members of the FDA Food Advisory Committee, although convinced that gene transfer from tomato plants to soil micro-organisms was improbable, were concerned about the use of the kanamycin resistance gene in other crops that may be grown on a wide scale. The FDA concluded that gene transfer from crops to micro-organisms, as well as other antibiotic resistance genes, should be evaluated on a case-by-case basis (US FDA, 1994).

343

The FDA carried out consultation with external experts between November 1996 and February 1997 to determine whether circumstances exist under which the FDA should recommend that a given antibiotic resistance gene not be used in food crops and if so, to identify the nature of those circumstances.

344

The kanamycin resistance gene was regarded as acceptable for use by these scientific experts. Some suggested hygromycin and others included the beta-lactamase gene of pUC18 and the tetracycline resistance gene. Any potential transfer was felt unlikely to add to the existing high levels of resistance in any meaningful way. It was suggested that use of genes other than


67


kanamycin and hygromycin might be acceptable on the basis of studies to address potential transfer and post-market surveillance for transfer of the gene in question. Surveillance would give regulatory agencies an opportunity for early intervention to prevent an adverse impact on public health (US FDA, 1998). 345

The potential effects due to ingestion of enzymes encoded by antibiotic resistance genes as food components raised little concern in comparison to potential health effects from gene transfer to micro-organisms.

346

The FDA concluded that the probability of gene transfer from plants to gut micro-organisms (human or animal) and to micro-organisms in the environment is remote. Several barriers operate against such transfer. The rate of such transfer, if any, would be insignificant when compared to transfer between micro-organisms and in most cases would not add to existing levels of resistance in bacterial populations in any meaningful way. Caution should be the rule for marker genes that encode resistance to antibiotics that are the only drug available to treat certain infections (eg vancomycin); they should not be used in GM plants (US FDA, 1998).

347

Developers should evaluate the use of antibiotic resistance genes in crops on a case-by-case basis taking into account information on the therapeutic importance of the antibiotic, frequency of use, route of administration, uniqueness, whether there would be selective pressure for transformation to take place, and the level of resistance to the antibiotic present in bacterial populations. If the gene or its gene product in food or feed could compromise the use of the relevant antibiotic it should not be present in the finished food or feed (US FDA, 1998).

Environmental Protection Agency (EPA) 348 The kanamycin resistance gene nptII and its enzyme neomycin phosphotransferase are considered inert ingredients by the EPA when they are introduced into a plant in order to ensure or confirm the presence of a plant pesticide and are exempted from the requirement of a tolerance in or on all raw agricultural commodities (US EPA, 1994).

World Health Organisation 349

In 1991 a FAO/WHO assessment of biotechnology in food production and processing concluded that the risk of transfer of antibiotic resistance genes from food in the gut to gut micro-organisms can be considered insignificant in comparison with the risk of the microorganisms becoming resistant to antibiotics by other mechanisms.

350

However vectors should be modified so as to minimise the probability of transfer of antibiotic resistance genes to other micro-organisms and genes that encode resistance to clinically useful antibiotics should not be used in micro-organisms intended to be present as living organisms in food (eg yoghurt). Food components obtained from micro-organisms containing antibiotic resistance marker genes should be demonstrated to be free of viable cells and genetic material that could encode antibiotic resistance (WHO, 1991).

351

These earlier recommendations relating to safety assessment of food and food components derived from GM micro-organisms were endorsed in 1996 (FAO and WHO, 1996).


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352

A WHO workshop on the health aspects of marker genes considered the probability of gene transfer from plants to micro-organisms to be extremely small. It concluded that there is no substantial evidence for gene transfer from ingested plant material to gut micro-organisms. If transfer were to occur, the nature of the gene and its product and the conditions in the gastro-intestinal tract will determine whether or not it is a food safety problem. Evaluation should be on a case-by-case basis (WHO, 1993).

353

Joint consultation by FAO and WHO concluded that as the probability of gene transfer is very low data on such gene transfer will only be needed when the nature of the marker gene is such that if transfer were to occur it would give rise to a health concern. In assessing potential health impact the human or animal use of the antibiotic and presence and prevalence of resistance to the same antibiotic in gut micro-organisms should be considered (FAO and WHO, 1996). These conclusions were reiterated recently but the use of alternative strategies to antibiotic resistance marker genes, if shown to be safe, was also encouraged (WHO, 2000).


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Alternative strategies to antibiotic resistance marker genes

7.


354

Questions raised with respect to the use of antibiotic resistance marker genes have led to the development of alternative strategies. Many other strategies for selecting transformed cells now exist and where antibiotic resistance genes are used, strategies exist to remove them from the final GMO.

355

Alternative strategies to antibiotic resistance marker genes must also be acceptable to the public.

Characteristics of a selectable marker 356

Characteristics of a useful selectable marker system include: • a minimum of non-transformed cells or tissue escape the selection; • selection results in a large number of independent transformation events and does not significantly interfere with regeneration; • it works well in many species; and • an assay is available to confirm that the marker is present (ACNFP, 1994).

Choice of a selectable marker 357

Most selectable marker genes confer resistance to an antibiotic, herbicide or other toxic agent.

358

Choice is usually based on: • The effectiveness of the agent in limiting the growth of non-transformed cells. This is determined by the mechanism of toxicity and uptake of the agent by the target tissue. • The efficacy of the selectable marker gene in providing resistance. Efficacy is determined by the mode and site of action of the gene product, the activity of the promoter directing gene expression, efficiency of translation and stability of the gene product. • The availability of vectors (Langridge, 1997).

359

However use of alternative selectable marker genes or subsequent deletion of the antibiotic resistance gene is becoming more common (Donaldson and May, 1999).

Disadvantages of antibiotic resistance marker genes 360

Disadvantages of using antibiotic resistance genes as selectable markers include: • Transformed cells convert the selective agent (ie antibiotic) to a detoxified compound that may still have negative effects on cell proliferation and differentiation.


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• It is difficult to carry out recurrent transformations using the same selectable marker to stack the genes of interest. • Only a limited number of antibiotic resistance genes are available for practical use. • Some plant species are insensitive or tolerant of the selective agents. • Public perception of the risk of antibiotic resistance. Herbicide resistance 361

Herbicide resistance due to detoxification or degradation of a specific broad spectrum herbicide may be relatively independent of the plant species. The resistance gene is therefore useful in genetic modification of a variety of plants. Herbicide resistance can also be used as a marker in the yeast Saccharomyces cerevisiae. Herbicide resistance markers include tolerance to glufosinate, glyphosate, chlorsulfuron, or bromoxynil.

362

Glufosinate inhibits amino acid biosynthesis. Glufosinate tolerance genes include bar (cloned from the soil bacterium Streptomyces hygroscopicus), pat (isolated from Streptomyces viridochromogenes) and synthetic pat. The genes encode the enzyme phosphinothricin acetyltransferase (PAT). Phosphinothricin acetyltransferase detoxifies phosphinothricin which inhibits glutamine synthetase causing rapid accumulation of ammonia and plant cell death. PAT is not an endogenous enzyme in humans. It has no homology to known toxins or allergens and is rapidly degraded in simulated digestion studies (Karenlampi, 1996).

363

Glyphosate prevents synthesis of the aromatic amino acids that are essential for protein synthesis. Glyphosate tolerance genes include epsps from E. coli, Salmonella typhimurium, and Agrobacterium and mutated epsps in plants. The epsps gene encodes the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). All plant, microbial and fungal food sources contain EPSPS protein. EPSPS has no significant homology to known toxins or allergens, is rapidly degraded in simulated digestion studies and displays no enzymatic activity in the stomach. No adverse effects were found in acute mouse gavage studies. Low levels of the protein are expressed in GM plants (Karenlampi, 1996).

364

Chlorsulfuron is a sulfonylurea and interferes with amino acid biosynthesis. Chlorsulfuron tolerance genes are mutated als genes isolated from various plants (eg tobacco, maize, sugar beet). The als gene is present in all plants and encodes the enzyme acetolactate synthase (ALS). The enzyme is not novel in the human diet. Interest in its use in genetic modification decreased with the appearance of spontaneous resistance to chlorsulfuron.

365

Glyphosate and chlorsulfuron markers differ from the glufosinate marker in that the genes conferring tolerance encode proteins normally present in all plants, except that they are mutated (single base pair substitution). The mutated enzymes have retained their normal physiological function in amino acid biosynthesis but have obtained an altered affinity to the herbicide. Consequently they are not considered novel components of food plants. They are considered good candidates for a positive list of marker genes if the enzyme concentrations in the transformed plants are not markedly different from the concentrations in herbicide sensitive plants (Karenlampi, 1996).


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366

Health concerns relate to potential new metabolites or herbicide residues that could occur as a result of the introduced resistance and interaction between the gene product and the herbicide. If resistance is introduced only for herbicide use as a selective agent and there is no field application of the herbicide, direct plant exposure to the herbicide does not occur and metabolites and/or residues of the herbicide do not raise food safety issues. However the use of herbicide resistance genes in plants should be avoided for use only as a selectable marker if the gene will be treated for regulatory purposes as if field application of herbicide will occur (Langridge, 1997).

Metabolic markers 367

A large number of genes other than antibiotic and herbicide resistance genes have been isolated for use as selectable markers. Some have been important markers in insect (eg organophosphate resistance) and fungal (eg benomyl resistance) transformation but are of minimal use for plants. Of those used in plants, many are based on resistance to toxic agents (eg methotrexate) that do not occur in the natural environment.

368

The use of a toxic agent as a selectable marker as an alternative to an antibiotic resistance gene in food plants and animals may not be acceptable among consumers and may arouse similar concerns.

369

Resistance to heavy metal ions has been used in GM micro-organisms. Genes conferring resistance to arsenite and mercuric salts or organomercurials have been used as selectable markers in Tn5- and Tn10-derived transposon vectors (de Lorenzo, 1992).

370

Resistance to copper has been used as a marker in yeasts. Metal resistance, through insertion of a mammalian metallothionein gene can be used as a selectable marker in plants. It has been suggested that crop plants containing a metal resistance marker that encodes for a protein that binds heavy metals might become toxic through the accumulation of metals but this has not been tested experimentally (ACNFP, 1994).

371

Tryptophan decarboxylase (TDC) catalyses the conversion of L-tryptophan into non-toxic tryptamine. The tdc gene has been used as a selectable marker in the transformation of species like Nicotiana tabacum that have no detectable endogenous TDC activity. A possible disadvantage is accumulation of tryptamine in the transformed tissue. Its applicability in other species will depend on endogenous TDC activity and their tolerance to elevated tdc gene-directed tryptamine levels (Goddijn et al, 1993).

372

A recent development is based on the use of selectable marker genes that give the transformed cells a metabolic advantage compared to the non-transformed cells which are starved with a concomitant slow reduction in viability. One method depends on conversion by the E. coli beta-glucuronidase gene (GUS) of the inactive form of the plant hormone cytokinin to a biologically active form that stimulates the transformed cells to regenerate. In this selection system the GUS gene functions as a selectable gene as well as a reporter gene (Okkels et al, 1997). The method uses cytokinin glucuronides as selective agents and is based on the fact that in vitro grown plant tissue cultures are auxotrophic in several aspects. In particular exogenously applied cytokinin significantly enhances regeneration and growth.


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373

Higher transformation frequencies (1.7-2.9 fold higher) are obtained than with kanamycin selection. This could be related to the fact that most cells in the explants die during traditional antibiotic resistance selection. The effect of necrotic tissue during selection is presumably reduced mitotic activity of the transformed cells resulting in less GM shoots emerging from the explants (Joersbo and Okkels, 1996).

374

Another method involves the use of mannose which cannot be metabolised by many plant species as a selective agent. After uptake it is phosphorylated to the sugar mannose-6phosphate that accumulates resulting in severe growth inhibition. The manA gene encodes the enzyme phosphomannose isomerase (PMI) that converts mannose-6-phosphate into fructose-6-phosphate which is easily metabolised. The gene exists in humans, many animals and many species of gut bacteria. It cannot work in all plants (eg soya) as they already have the manA gene (Coghlan, 1999). It has been successfully used to date in wheat, maize and sugar beet.

375

Joersbo et al (1998) obtained transformation frequencies about 10-fold higher compared to kanamycin selection and at least 80-90 percent of the isolated shoots contained the transgene. High selection efficiency means the use of a reporter gene is not required. Rooting of the GM shoots was markedly improved using mannose instead of kanamycin selection. Nontransformed explants and shoots grew very slowly, lost vigour and acquired a light brown colour. The higher transformation frequency may occur because transformed cells are actively encouraged to grow rather than just allowed to survive. As a result the adverse effects of dying cells are to a large extent avoided. The assay for PMI activity is also more convenient than for traditional selectable genes in particular nptII.

376

It is considered that this selection system should replace antibiotic resistance and other markers currently in use (Malik and Saroha, 1999).

377

The xylose isomerase gene (xylA) has been used as an alternative selectable marker. It also favours regeneration and growth of the transformed cells while non-transformed cells are starved but not killed. The gene enables transformed cells to use xylose as a carbohydrate source. Even without optimisation, transformation efficiency is higher than kanamycin selection and fully grown plants can be produced about two weeks earlier than kanamycin selected plants because of vigorous shoot development. It is inexpensive and considered to be applicable to a broad range of species since the plant species (potato, tomato, tobacco) tested produced GM plants in the first transformation experiment (Haldrup et al, 1998).

378

Biosynthesis of the amino acids lysine, threonine and S-aminoethyl L-cysteine is controlled by aspartate kinase and dihydrodipicolinate synthase both of which are subject to feedback inhibition. Bacterial genes for these enzymes are less sensitive to inhibition than their counterparts in plants and have been used for genetic modification of plants. Genetically modified plants containing E. coli genes for their expression can be grown on media containing lysine and threonine, and cysteine respectively (ACNFP, 1994).

379

The isopentenyltransferase gene (ipt) has been used as an alternative marker for Agrobacteriummediated transformation. The gene of interest is linked to the gene from Agrobacterium that encodes isopentenyltransferase, an enzyme involved in cytokinin biosynthesis. Cytokinins


73


stimulate shoot formation from plant cells so only cells containing the introduced DNA form shoots and differentiate into mature plants. An inducible system has been developed to carefully control ipt expression so that it is only turned on when needed for shoot formation and not long enough to cause abnormalities. Future refinements to the approach could avoid introducing the bacterial ipt gene, instead using controlled expression of shoot-inducing genes from plants as a marker (Kunkel et al, 1999). 380

Metabolic markers function by interfering with the metabolism of the host plant. They may reduce the sensitivity of the plant to high concentrations of substances normally found in the plant. There could be changes in some important components of the plant (eg vitamin or natural toxin content) because of the direct interference with the plant’s metabolism. The extent of interference to a metabolic pathway that might occur from the product of the inserted metabolic marker gene can be expected to vary considerably between different plant species. As a result it may not be possible to extrapolate from the results of a safety evaluation of a metabolic marker gene in one plant species to another (WHO, 1993).

Other selectable markers in plants 381

Negative selection systems include root inhibition markers and induced toxicity markers. The concept of negative selection is based on the expression of a marker gene that causes immediate or conditional cell death. It can be used when a particular class of cells needs to be eliminated.

382

The aux2 or tms2 gene derived from Agrobacterium species encode the enzyme amidohydrolase that catalyses conversion of indole acetamide and naphthalene acetamide to indole acetic acid (IAA) and naphthalene acetic acid (NAA) respectively. Modified plants containing these genes overproduce IAA and NAA when grown on a medium containing indole or naphthalene acetamide that inhibits root system formation (Harding, 1999).

383

The bacterial cytosine deaminase gene (codA) encodes the enzyme cytosine deaminase. Cytosine deaminase activity is not usually detectable in higher organisms. Its absence in many plant species indicates wide applicability as a negative selection marker. Cytosine deaminase converts the selective agent 5-fluorocytosine to toxic 5-fluorouracil which kills the GM plants (Kobayashi et al, 1995).

384

When plants are grown in the presence of ammonium instead of nitrate the endogenous nitrate reductase gene is expressed at very low levels and is not sensitive to chlorate, an inhibitor of nitrate reductase. In GM plants containing endogenous and introduced nitrate reductase genes the modified gene is constitutively expressed in the presence of ammonium so that GM plants are killed by chlorate and only non-GM plants survive (Harding and Harris, 1997).

Other selectable markers in mammalian cells 385

Glutamine synthetase catalyses the formation of glutamine from glutamate and ammonia which is essential to the cell for growth. It has been used as a marker for mammalian cells in


74


the presence of the glutamine synthetase inhibitor, methionine sulphoximine (Harding, 1999). 386

Micro-organisms catalyse synthesis of tryptophan from indole and serine. The bacterial trpB gene produces tryptophan synthase and its transfer into mammalian cells allows the positive selection of such cells on medium containing indole (Harding, 1999).

387

The bacterial hisD gene produces histidinol dehydrogenase which catalyses oxidation of histidinol to histidine. When this gene is transferred into mammalian cells it allows the selection of cells on growth medium containing histidinol only. This is a double selection system since histidinol is toxic and histidinol dehydrogenase detoxifies it while providing histidine for growth (Harding, 1999).

Other markers in yeast and micro-organisms 388

L-canavanine is an amino acid analogue that can be included in culture media to select for transformed L-canavanine resistant cells. The marker has been used in S. cerevisiae (ACNFP, 1994).

389

Complementation systems involving auxotrophic markers have been used widely for the yeast S. cerevisiae and have been described for E. coli (ACNFP, 1994). The success of transformation in yeast is largely due to the ease of selection for transformants. As laboratory strains of yeast are generally haploid, a mutation or deletion of a gene in an important biosynthetic pathway can be complemented with the wild type gene in a transformation vector. Since a large number of such auxotrophic mutants are available and many of the wild type genes have been cloned this provides a ready series of selectable markers. A similar approach is possible in diploid species but the target for the transformation must be made homozygous for the mutation. Problems with the method are that homozygous auxotrophic mutants and the cloned wild type gene must be available and it is restricted to these defined genotypes (Langridge, 1997).

390

Sugar catabolism markers that use the presence of enzymes involved in breakdown of specific sugars have been used to identify transformed bacteria.

391

Alternative markers for GM micro-organisms include resistance to the antimicrobial, nisin and resistance to bacteriophage. Nisin is found naturally in some lactic acid bacteria and is used in food preservation. Resistance does not involve enzyme production; its mechanism is unknown but is thought to involve either tolerance or receptor resistance. If the nisin resistance marker were acquired by a spoilage micro-organism this could result in spoilage of unprocessed food as the micro-organism would continue to grow in the presence of nisin. Resistance to bacteriophage is a potential selectable marker for lactic acid bacteria (ACNFP, 1994).


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Alternatives to the selection of antibiotic resistance genes in bacteria 392

Use of antibiotic resistance genes is a convenient but not an absolute requirement for cloning genes in E. coli. Selection vectors are now available based on the control of cell death. The gene sequences for biosynthesis of amino acids, purines and pyrimidines are routinely used for cloning genes in yeast and fungi and similar markers can be used for cloning genes in E. coli. Such markers have appeal because the construction of the recipient host by homologous recombination with deletion of selectable marker sequences is possible. The marker gene can also be cleaved out of the recombinant plasmid before it is introduced into the plant tissue. Cotransformation with a plasmid that can be used to select the transformed cells and later eliminated is also a possibility (Malik and Saroha, 1999).

393

Specialised host-vector systems in which an essential gene that has been deleted from the host chromosome is supplied by a recombinant plasmid have been developed. As a result direct selection of recombinant clones is possible without addition of a selective agent. It has been successfully tested in construction of live vaccine candidates. Broad host range plasmids carrying the thyA+/thymidylate synthase autoselective marker have been constructed which use a thyA-- mutant strain as the host (de Lorenzo, 1992).

394

Ideally if a gene is to have broad application as a marker it should be expressed in a variety of organisms. The thymidylate synthase gene (thyA) is from a species of bacteria used routinely for the manufacture of cheese. Mutants have been isolated from many micro-organisms. They lack thymidylate synthase activity and rely on an alternate pathway of DNA synthesis from exogenous thymidine or thymine.

395

In a thyA mutant background the thyA gene can become a positive marker for strain construction. Plasmids marked with the thyA gene are likely to be stably maintained in thyA mutant strains, in media or environments lacking or limiting thymine or thymidine. It is unlikely that natural environments have sufficient thymidine or thymine to interfere with the effectiveness of the thyA gene marker system although further experimentation is required (Ross et al, 1990).

Elimination of the selectable marker 396

Except for herbicide resistance, selectable marker genes are not aimed at any change or improvement of the agronomic or other characteristics of the organism involved. Elimination of the gene is therefore an alternative strategy.

No selectable marker or reporter gene 397

Direct introduction of DNA into a cell with the potential to develop into a whole organism is the best method of genetic modification for the release of organisms into the environment. Micro-injection has been applied to a wide range of higher organisms in particular animals, including fish, and some plant species, and avoids the use of markers (Harding, 1999). Direct DNA transfer via micro-injection of DNA into cells of microspore-derived embryoids may improve the efficiency of gene transfer to acceptable values. Cell finder systems in which a


76


computer-controlled microscope allows easy positioning and relocation of cultured cells and protoplasts, in combination with improved gene delivery techniques, may also develop into a system that allows the identification and isolation of GM plants only carrying the desired gene (Metz and Nap, 1997). 398

Currently it is not practical to avoid the use of marker genes. Foreign DNA can be readily identified by PCR based assay systems and methods are available that do not require direct isolation of DNA so large numbers of cells can be screened rapidly. However transformation efficiency is still very low. A single GM plant is also rarely enough; several GM plants are usually sought for each construct due to variation in transgene expression levels and stability (Langridge, 1997).

Reporter gene only 399

When marker genes are used for the rapid analysis of promoter activity they are called reporter genes. Another way to eliminate selectable marker genes is to use a reporter gene to monitor transformation. For plant cells the use of reporter genes to identify or tag GM cells is feasible only when transformation efficiency is high. Although the currently most frequently used reporter gene, the GUS gene requires assays that destroy the GM material, reporter genes that encode firefly luciferase and the jellyfish Aequorea victoria green fluorescent protein can be assayed non-destructively. These genes allow the identification of transformed cells through a change in their colour. Non-destructive assay raises the possibility of monitoring transformation during the development of the GM shoot and selection of the shoot in a very early stage (Ghorbel et al, 1999).

400

Green fluorescent protein has been expressed in a wide variety of organisms (eg E. coli, yeast, Drosophilia, mammals, worms and plants) and only needs ultraviolet or blue light to fluoresce. Detection of other commonly used reporter genes requires enzymes, cofactors or exogenous substrates. Expression of GFP is cell autonomous and independent of cell type and location (Sheen at al, 1995).

401

It is possible that the frequency of recovery of transformants will be increased by the use of colour markers relative to selection based on a toxic agent. In selecting for transformed cells in the presence of a toxic agent it is unclear whether or not transformed but poorly expressing GM cells are being killed or whether the regeneration rates are being decreased by having the GM cells surrounded by cells that are dead or dying (Langridge, 1997).

402

Considerable effort is being expended on developing and testing transformation procedures based on colour markers.

Inactivation of the selectable marker gene 403

Limiting the expression of the selectable marker gene to the stages at which selection for transformation is applied will result in GM plants in which the transgene is present but not the transgene encoded protein. It has been shown that the wound-inducible promoter AoPR1 isolated from asparagus, when fused to the kanamycin resistance gene, allows selection during transformation but results in very low levels of the transgene product in the mature plant (Metz and Nap, 1997).


77


404

Gene silencing may possibly be developed into a method to obtain plants without selectable marker gene activity (Metz and Nap, 1997). Schmulling and Rohrig (1995) have demonstrated in GM tobacco hybrids that single genes could be selectively inactivated on T-DNAs harbouring several genes. They concluded that it is likely that several factors determine the probability of interaction between different loci and therefore influence the differences in the kinetics of inactivation and restoration of gene expression (eg accessibility of the loci for pairing and relative positions of the inserts in the genome).

Removal of the selectable marker gene 405

Removal of the selectable marker gene allows the same marker to be used repeatedly in subsequent transformations into the same host. As successive rounds of transformation with additional genes of interest become prevalent this will be an important attribute given the limited availability of selectable markers. Removal also minimises the amount of foreign DNA to that which is actually involved in conferring the desired trait(s) and could have positive effects on consumer acceptance (Ow and Medberry, 1995).

406

When using Agrobacterium T-DNA transfer as the means of transformation the selectable marker gene, being on the same T-DNA as the desired gene, is linked to the desired gene. When unlinked the gene can be segregated from the selectable marker gene and plants carrying only the desired gene can be identified with routine procedures.

407

Several strategies have been used to unlink the selectable marker gene from the desired gene and generate marker-free GM plants. They include cotransformation of two T-DNA molecules (Komari et al 1996), site-specific recombination (Dale and Ow, 1991) and transposition-mediated repositioning of the marker gene (Goldsbrough et al, 1993). These strategies require sexual crossing to eliminate the marker genes and hence their applicability to plant species that have a long generation time is limited. However some recently developed systems can generate marker-free GM plants without the need to sexually cross plants eg the multi-autotransformation (MAT) vector system (Ebinuma et al, 1997).

Cotransformation 408 The cotransformation systems appear to be the simplest of the strategies to eliminate marker genes and have been widely used in direct transformation methods. In these systems the marker gene and desired gene are introduced into the plant genome on separate vectors. Following several generations of crossing the marker gene will be eliminated in some progeny through genetic segregration.

409

Another advantage of cotransformation systems is that the construction of the separate molecules for the marker gene and desired gene is less tedious than creation of linked DNA fragments.

410

Komari et al (1996) obtained GM tobacco and rice plants free from selectable markers by a relatively simple procedure consisting of Agrobacterium-mediated cotransformation and segregation of the progeny. Since the vector system functioned efficiently both in a dicotyledon, tobacco, and a monocotyledon, rice, this system is potentially useful for a wide range of plant species.


78


411

The feasibility of cotransformation is disputed but it has been suggested that the method would be suitable for transformations using Agrobacterium and cloned gene transfer strategies. Frequencies of cotransformation are low and not potentially useful for species that are difficult to transform (ACNFP, 1994).

412

Biolistics with independent DNA molecules may also result in unlinked cotransformation (Metz and Nap, 1997).

Site-specific recombination 413 Site-specific recombination is the enzyme-mediated cleavage and ligation of two defined deoxynucleotide sequences. Once transformants with the desired gene are obtained the selectable marker gene is no longer necessary. Removal of such unnecessary DNA has been demonstrated using the Cre/lox recombination system (Dale and Ow, 1991).

414

Using the Cre/lox recombination system a plant is transformed with the desired gene using a selectable marker gene flanked by lox recombination sites. The cre gene accompanied by a second marker gene is introduced through sexual hybridisation or a second transformation round. The initial marker is excised from the genome through the action of cre recombinase (bacterial recombination enzyme) which acts at the lox sites. After its removal plants are left to flower and set seed and the progeny are screened for segregation of the cre gene which is linked to the remaining marker gene. Plants without the cre and marker genes are selected and propagated.

415

Gleave et al (1999) have developed a plant transformation vector incorporating the Cre/lox site-specific recombination system to facilitate the elimination of marker genes from GM plants and the cytosine deaminase gene (codA). Transient expression of cre recombinase is used to mediate excision of lox flanked marker genes from GM plants and the codA gene is used to select plants that have undergone Cre-mediated recombination. Using this approach marker-free plants can be produced without sexual crossing. It should be applicable to many established perennial horticultural cultivars requiring vegetative propagation to maintain their elite genome.

416

Several DNA site-specific recombination systems have been shown to function in higher eukaryotic cells. Strategic placement of the recognition sites into the plant genome has permitted the deletion, inversion, integration and translocation of host and introduced DNA fragments. Recombinase-based strategies afford precise and predictable engineering of the plant genome (Ow and Medberry, 1995). The approach is also relevant to genetic modification of animals and cell lines. In the case of cell lines, where loss of the cre locus cannot be achieved by sexual segregation, transient expression of the cre gene or direct introduction of purified cre protein could be used (Dale and Ow, 1991).

417

It can be applied to a wide range of plants but is not feasible in vegetatively propagated crops with low fertility and seed selection might scramble elite genomes in clonally propagated plants (Harding, 1999).

418

A lot of progress has been made since the first reports of site-specific recombination in plants. However recombinase-based technology is still an academic exercise. It has yet to be


79


seen if and when it will have an impact on crop improvement (Ow and Medberry, 1995). Two other recombination systems (FLP-frt and R-rs) are also being evaluated in plants (Metz and Nap, 1997). 419

Zubko et al (2000) have developed a system to remove selectable marker genes from tobacco based on intrachromosomal homologous recombination between two homologous sequences using a vector containing nptII and attachment regions from bacteriophage λ. It does not require removal of recombinase by genetic segregation and is therefore less time consuming.

Transposition-mediated repositioning 420 Transposition-mediated repositioning requires the introduction of the selectable marker gene and the desired gene on a vector containing a transposase function. The selectable marker gene moves away from the gene or vice versa to become unlinked from the gene (eg transformation system using the Ac/Ds maize transposable element) (Metz and Nap, 1997). Large numbers of progeny plants are needed for selection and the construction of the vector has to allow transposition to occur at a sufficient distance from the selectable marker gene to ensure that the selectable marker gene and desired gene are separated during recombination (ACNFP, 1994).

421

Removal of the selectable marker gene has also been achieved by linking the marker to a negative selectable marker eg codA gene; human herpes simplex virus thymidine kinase type 1 gene (HSVtk) on the same DNA molecule. Plant cells expressing cytosine deaminase will convert 5-fluorocytosine to toxic 5-fluorouracil. The HSVtk encoded enzyme inhibits growth in the presence of the antiviral drug, ganciclovir. All plants carrying the antibiotic resistance selectable marker will also carry the negative selectable marker and will be killed by treatment with 5-fluorocytosine or ganciclovir. Only plants without the selectable marker will survive and about half will carry the desired gene. These can be identified by PCR assays (Langridge, 1997).

422

Ebinuma et al (1997) have developed a plant vector system for repeated transformation (called multi-autotransformation (MAT)) in which a chimaeric ipt gene inserted into a transposable element Ac is used as a selectable marker. It is easy to detect visually GM plants that carry a functional ipt gene due to shoot formation. Chimeric ipt genes are not commonly used as markers because the resulting GM plants lose apical dominance and are unable to root due to overproduction of cytokinins. Hence Ac was used to remove the ipt gene after transformation although the frequency of marker-free GM plants obtained was relatively low.

423

The vector system may provide an alternative approach to regenerate some plant species that have been difficult to transform and provides a way of bypassing the problem of long generation times (eg tree species). The system allows removal of a marker gene from a vegetatively propagated crop without crossing. Other transformation systems for eliminating marker genes cannot be applied to those crops because they need sexual crosses to produce marker-free plants and to be able to carry out successive transformation.

424

Strategies to obtain GM plants without a selectable marker gene have been successful in some cases. However these approaches are far from routine or from being generally applicable and are more time consuming. These techniques could be optimised further for


80


more routine GM plant production. Approaches using protoplasts are likely to suffer from disadvantages compared to Agrobacterium-mediated plant transformation due to somaclonal variation. Approaches that are based on segregating away the unlinked selectable marker gene require plants that are relatively homozygous and that can be easily backcrossed. These approaches will be essentially impossible for some plants (eg potato). Removal of marker genes from micro-organisms 425 Removal methods from GM micro-organisms involve use of enzymic excision or of homologous recombination.

426

During transformation, target sites are inserted to flank the selectable marker gene. Selected GM micro-organisms are then further modified to include sequences which code for enzymes active at the target sites and the action of these enzymes excises the selectable marker gene.

427

The delivery vector which is unable to replicate in the recipient organism carries the desired gene flanked by DNA homologous to that on the recipient chromosome and antibiotic resistance and beta-galactosidase (lacZ) marker genes. Crossover of homologous DNA integrates the vector into the recipient chromosome, indicated by antibiotic resistance. The vector DNA, including the markers, then separates from the recipient chromosome and is lost, indicated by the absence of lacZ activity. The desired gene remains in the recipient chromosome.

428

A site-specific recombination vector has been developed that selectively removes the antibiotic resistance marker gene from Bacillus thuringiensis (Bt) after introduction of the plasmid into the host strain (Sanchis et al, 1997).

Modulation of gene expression 429

Antisense technology could be applied to marker genes. When an antisense gene is introduced into a cell it encodes antisense RNA that is complementary to the messenger RNA of the original marker gene and blocks its expression.

430

Catalytic RNA such as ribozymes cleaves either itself or other RNA molecules. It has created new opportunities to repress the expression of selectable marker genes (Harding, 1999). Expression of a ribozyme gene directed towards the target mRNA of the npt marker gene in plants resulted in a reduction of the gene product expression (Steinecke et al, 1992).

431

Genetically modified plants containing selectable markers could be further modified with genes that encode antibodies against the gene product (Hiatt et al, 1989).

Intron-containing antibiotic resistance genes 432

Most of the antibiotics used for selection of transformed plant tissues can also inhibit Agrobacterium growth. This inhibition is usually not effective against Agrobacterium during the plant transformation process because the antibiotic resistance gene used for plant selection is expressed in Agrobacterium.


81


433

Introns have been inserted into the hygromycin resistance gene (hph) coding region to abolish its expression in Agrobacterium and hence minimise the problem of Agrobacterium overgrowth during plant transformation. Control of overgrowth reduces the amount of labour and increases the chance of success of each experiment. Introns also enhanced the expression level of the hph gene and as a result either maintained or enhanced the selection efficiency of the hph gene during transformation. Better quality transgene mRNA and GM lines with low copy numbers of the transgene were also produced.

434

An advantage of using an intron-disrupted antibiotic resistance gene as a selectable marker is a reduced probability of antibiotic resistance being acquired by gut bacteria following consumption of GM material by humans or animals (Wang et al, 1997).


82

Conclusion

8.

Conclusion

435

People already consume naturally occurring micro-organisms on or in food or water that contain antibiotic resistance genes and also acquire antibiotic resistant micro-organisms as a result of antibiotic treatment. These are the major sources of antibiotic resistance in humans and relate to decades of widespread antibiotic use in humans and animals.

436

The scientific literature on the use of antibiotic resistance genes in GMOs is characterised by many opinions and relatively few data. Many different scientific disciplinary approaches have assessed the uncertainty and the available evidence. Opinion is polarised between scientists who are concerned about what might happen based on extrapolations from laboratory experiments and those who maintain the weight of evidence for successful gene transfer is not sufficiently convincing for there to be any concern. However often the overall importance of a value to be protected such as human health is more a matter of science policy, to be determined in the New Zealand context by the ERMA, rather than a scientific issue.

437

Scientific concern about the use of antibiotic resistance genes in GMOs relates more to potential health effects from gene transfer to micro-organisms than to potential health effects due to ingestion of the antibiotic resistance gene or its gene product or transfer to gut epithelial cells.

438

As there is no evidence that antibiotic resistance marker genes have transferred from GM plants to pathogenic micro-organisms, and experimental evidence suggests the probability of such an event is remote, scientists generally consider that the risk of antibiotic resistance arising from the presence of antibiotic resistance genes in GMOs is extremely low. Even if the probability of horizontal gene transfer is extremely low this should not however be confounded with the potential outcome. Rare transfer events can be amplified rapidly under selective pressure. The health impact would be significant if a gene conferring resistance to a clinically important antibiotic was transferred and expressed in a pathogenic micro-organism normally treated with that antibiotic. From what is known about mechanisms of horizontal gene transfer between organisms and the survival of intact DNA following processing and digestion it is concluded that the risk is highest for ingested viable GM micro-organisms, in particular if the antibiotic resistance gene confers resistance to a clinically important antibiotic, and lowest for highly processed GM food.

439

However there are gaps in scientific knowledge in this area. Assessment of the potential for transfer of antibiotic resistance marker genes from GM plants or other eukaryotes into micro-organisms needs to be examined more closely. Studies are needed that incorporate selective pressure during the exposure of competent bacteria with DNA. Data are also limited about the antibiotic resistance genes with the exception of the kanamycin resistance gene.

440

Relatively little consideration has been given to date to the potential cumulative consequences of a rare event in a scenario of widespread cultivation of GM plants and ingestion of raw and unprocessed GM plant material by millions of people and animals.


83

Conclusion

441

Widespread disagreement exists on what constitutes a tolerable risk when it comes to antibiotic resistance. There is a dichotomy of opinion between the view that any further increase, irrespective of how small, in the prevalence of antibiotic resistant micro-organisms through the transfer of antibiotic resistance genes from GMOs would be undesirable and the view that the consequence would not be meaningful due to existing levels of resistance in the human gut and environment.

442

The concept of the precautionary approach as a matter to be taken into account under the HSNO Act 1996, though not defined, takes into account scientific uncertainty and conflict. It also allows for consideration of the social construct of tolerable or acceptable risk.

443

Risk assessment reports have argued that there are no scientific, health or safety reasons to restrict the use of the nptII gene and even if methods for removal are available and prove generally applicable, removal of the nptII gene should not be required (Appendix I). These conclusions have also been broadened by some to there being no scientific reason to prohibit or limit the use of antibiotic resistance marker genes, nor to encourage or require their removal from GM plants. The arguments put forward to justify the inclusion of antibiotic resistance marker genes do not necessarily apply to all pathogenic micro-organisms and to all geographical locations. In this respect it is important to consider the antibiotic involved: how it is administered, its uses, the potential for cross-resistance to other antibiotics, the prevalence of resistance, and the existence of alternative therapeutic agents.

444

The commonly used kanamycin resistance marker gene confers resistance against kanamycin and neomycin. These antibiotics have limited clinical use because of their side effects and are not often used orally. Although there are no New Zealand data on the prevalence of kanamycin resistance it is unlikely to differ markedly from that found in other developed countries. Kanamycin is however used as a reserve drug in the treatment of tuberculosis.

445

The ampicillin resistance gene used in bacterial transformation is often not present intact in the final GMO. If it is present the probability of transfer from the GMO to pathogenic micro-organisms and gene expression needs to be considered in a New Zealand context of high use of beta-lactams, the clinically important group of antibiotics to which ampicillin belongs.

446

In the environment if transfer from the plant genome to soil micro-organisms did occur in most cases there would be no selective pressure. Exceptions are streptomycin use in horticulture or when manure is used as fertiliser following in-feed antibiotic use for animal growth promotion and prophylaxis.

447

There appears to be an emerging international consensus among regulatory agencies to evaluate each GMO containing antibiotic resistance marker genes on a case-by-case basis and a move to advocating restriction of introduced genes in the final GMO to those genes needed to confer the desired trait(s).

448

Irrespective of the scientific conclusions removal of the antibiotic resistance gene from the final GM plant or use of alternative strategies is now being recommended whenever feasible. It is consumer acceptance of a product that governs its market performance. Elimination of antibiotic resistance marker genes could have positive effects on consumer acceptance by


84

Conclusion

alleviating perceived risks. In addition antibiotic resistance marker genes serve no useful purpose in the final genetically modified organism. 449

Post-market surveillance for transfer of the antibiotic resistance gene has been suggested as a mechanism to safeguard against occurrence of antibiotic resistance from the use of marker genes other than hygromycin (which is not used in human medicine) and kanamycin. It would provide regulatory agencies an opportunity for early intervention to prevent an adverse impact on public health.

450

The potential impact of the use of antibiotic resistance genes in GMOs on the prevalence of antibiotic resistance is far less significant than the impact of the current use of antibiotics in humans and animals in New Zealand. However antibiotic resistance is receiving increasing scrutiny nationally and internationally, and there are an increasing number of strategies being implemented with the aim of curbing all antibiotic use. In addition alternative strategies to antibiotic resistance marker genes such as gene removal or other selectable markers are increasing, some of which are looking feasible in terms of safety and practicability.


85

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McConnell MA, Mercer AA, Tannock GW. Transfer of plasmid pAMβ1 between members of the normal microflora inhabiting the murine digestive tract and modification of the plasmid in a Lactobacillus reuteri host. Microbial Ecol Health Dis 1991; 4: 343-55. Mercer DK, Scott KP, Bruce-Johnson WA, Glover LA, et al. Fate of free DNA and transformation of the oral bacterium Streptococcus gordonii DL1 by plasmid DNA in human saliva. Appl Environ Microbiol 1999; 65(1): 6-10. Metcalfe DD, Astwood JD, Townsend R, Sampson HA, et al. Assessment of the allergenic potential of foods derived from genetically engineered crop plants. Crit Rev Food Sci Nutr 1996; 36(S): S16586. Metz PL, Nap JP. A transgene-centred approach to the biosafety of transgenic plants: overview of selection and reporter genes. Acta Bot Neerl 1997; 46(1): 25-50. Miller RV. Bacterial gene swapping in nature. Sci Am 1998; 278: 66-71. Minekus M, Marteau P, Havenaar R, Huis in’t Veld JHJ. A multicompartmental dynamic computercontrolled model simulating the stomach and small intestine. Alta-Altern Lab Anim 1995; 23(2): 197209. Nap JP, Bijvoet J, Stiekema WJ. Biosafety of kanamycin-resistant transgenic plants. Transgenic Res 1992; 1: 239-49. Nielsen KN, Gebhard F, Smalla K, Bones AN, et al. Evaluation of possible horizontal gene transfer from transgenic plants to the soil bacterium Acinetobacter calcoaceticus BD413. Theor Appl Genet 1997; 95: 815-21. Nielsen KM, Bones AM, Smalla K, van Elsas JD. Horizontal gene transfer from transgenic plants to terrestrial bacteria – a rare event? FEMS Microbiol Rev 1998; 22: 79-103. Ochman H, Lawrence JG, Groisman EA. Lateral gene transfer and the nature of bacterial innovation. Nature 2000; 405: 299-304. Okkels FT, Ward JL, Joersbo M. Synthesis of cytokinin glucuronides for the selection of transgenic plant cells. Phytochem 1997; 46: 801-4. Organisation for Economic Cooperation and Development. Safety evaluation of foods derived by modern biotechnology: concepts and principles. Paris: Organisation for Economic Cooperation and Development, 1993. Organisation for Economic Cooperation and Development. Report of the Task Force for the Safety of Novel Foods and Feeds. Paris: Organisation for Economic Cooperation and Development, May 2000a.


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95

Glossary

10.

Glossary

Auxotrophic markers Bacteriophage Cytokinin DNA Eukaryote Gene Horizontal gene transfer Intron Mismatch-repair system Phytosphere Pleiotrophic effects Polymerase chain reaction Probiotics Prokaryote Promoter Replicon Transposon

Zoonosis

Allow cells to synthesise an essential component, usually an amino acid, a vitamin or other minor nutrient in media that lack that component. A virus that infects bacteria. Plant growth hormone. Deoxyribonucleic acid, which is present in all living cells and contains the information for cell structure, organisation and function. An organism that contains a nucleus like plants and animals. An ordered sequence of nucleotide bases comprising a segment of DNA. Non-sexual or parasexual transfer of genetic material between genomes of organisms of the same or different species. Regions within genes that do not code for protein sequences. Comprises enzymes that recognise and process mispaired and nonpaired bases. Plant environment ie leaves and roots. Multiple effects resulting from a single genetic change. Artificial amplification of a DNA sequence by repeated cycles of replication and strand separation. Products that are consumed intentionally to alter the number and type of gut bacteria. An organism without a nucleus ie bacteria. DNA sequence that receives specialised proteins that bind and switch on a gene. Length of DNA that behaves as an autonomous unit during DNA replication. Mobile segment of DNA that has the capacity to move from one site in the genome to another. Transposons vary in size and often contain antibiotic resistance genes as well as genes encoding for functions concerned with their mobility. Disease of animals that may be transmitted to humans.


96

Appendices

11.

Appendices

Appendix I: Evaluation of the kanamycin resistance gene 451

Detailed evaluations have been published on the kanamycin resistance gene (nptII) and its product neomycin phosphotransferase (NPTII) that indicate that NPTII produced in GM plants presents no discernible food or feed safety or environmental concerns (Nap et al, 1992; Flavell et al, 1992; Fuchs et al, 1993; Redenbaugh et al, 1994; US FDA, 1994).

452

A review on the biosafety of kanamycin resistant GM plants concluded that use of the kanamycin resistance gene is an excellent choice because of the high substrate specificity of the enzyme encoded. Physico-chemical properties of the antibiotic exclude the existence of selective conditions in the environment and hence the expression of nptII in GMOs will not give the organism any selective advantage outside the laboratory because of this gene compared to the non-GM parent organism (Nap et al, 1992).

453

Nap et al (1992) also concluded that even if methods are available and become generally applicable, removal of the nptII gene from the final GMO should not be required.

454

NPTII does not contain any properties that would distinguish it toxicologically from any other phosphorylating enzymes that historically have been part of the food supply without adverse consequences.

455

The kanamycin resistance gene has been used in clinical studies reported to the National Institutes of Health involving human gene therapy. They represent a human in vivo safety evaluation of the marker gene. No adverse effects of the gene or gene product were observed (Redenbaugh et al, 1994). However this research provides little information concerning the safety of the gene and its product in food.

Impact of the gene product NPTII 456

The fate of the protein during digestion was assessed using a simulated in vitro mammalian digestion model. No protein was detected at 10 seconds in gastric fluid and 50 percent degradation had occurred after two to five minutes in intestinal fluid. Enzymatic activity was lost by two minutes in gastric fluid and 15 minutes in intestinal fluid. If analysis had been carried out at earlier time points loss of functional activity would have been detected sooner (Fuchs et al, 1993). These data confirmed findings submitted to the FDA by Calgene Inc (developer of the Flavr Savr tomato) and suggested consumption should not pose any significant allergenic concerns.

457

Solid food empties from the stomach by about 50 percent in two hours while liquid empties by 50 percent in about 25 minutes. Intestinal transit times of NPTII were measured using isotopically labelled chromate which is not absorbed. It was first detected in the faeces at four to 10 hours and last at 68 to 165 hours. Hence there is minimal, if any, potential for NPTII to reach the intestinal mucosa to trigger an IgE-mediated response. The protein also has no homology to any reported allergen (Fuchs et al, 1993).


97

Appendices

458

Since proteins that are toxic produce toxic effects following acute exposure an acute toxicological study was carried out. An acute mouse gavage study confirmed that the protein caused no adverse effects when administered by gavage at a cumulative target dosage of up to 5,000 mg/kg of body weight. This dosage correlates to at least a million-fold safety factor relative to the average daily consumption of potato or tomato assuming that all the potatoes or tomatoes consumed contained the protein. In other words a 5,000 mg/kg body weight dose is equivalent to an average human consuming in one day more than one million tomatoes or potatoes expressing the level of NPTII previously reported for these GM crops. Whole food feeding studies also showed no adverse effects. Rats were fed potatoes or tomatoes at a level equivalent to an average human consuming 40 raw potatoes or 100 tomatoes per day for 28 days with no adverse effects (Fuchs et al, 1993).

459

Human exposure to the protein can also be assumed to occur as a result of the background of kanamycin resistant micro-organisms in the environment and the human gut.

460

Humans continually ingest kanamycin resistant micro-organisms. The diet, particularly raw vegetables is the major source. It has been conservatively estimated that a human ingests 1.2 x 106 kanamycin resistant micro-organisms daily (Flavell et al, 1992).

461

With respect to the possibility of increased intestinal absorption of proteins in neonates and people with certain conditions (eg ulcers) the FDA concluded there is no reason to expect that absorption of intact or partially digested NPTII protein would present a safety concern different from absorption of any other protein in the diet.

462

Phosphorylation of kanamycin or neomycin by NPTII requires the cofactor ATP. ATP is unstable in the low pH of the digestive system and endogenous concentrations in the stomach are below that required for catalytic activity. It is susceptible to inactivation by heat and by enzymes (eg intestinal alkaline phosphatases). The primary source of ATP in the gastro-intestinal tract is uncooked fruits and vegetables.

463

Dietary exposure of NPTII was estimated by Calgene Inc as very low - 480 µg per person per day or 0.16 parts per million in the diet based on a 100 percent market share for tomatoes containing NPTII. The exposure estimate was based on several conservative assumptions.

464

High temperature treatment denatures proteins and inactivates enzymes and therefore processed products that contain tomatoes with the kanamycin resistance gene are unlikely to contain any enzymatically active NPTII. Purified oils essentially do not contain protein. Intact DNA including the kanamycin resistance gene are not expected to survive production of oils and animal feeds from cottonseed and rapeseed because of release of degradative enzymes normally present within the cell from mechanical grinding and enzyme inactivation from high temperatures and solvent extraction (US FDA, 1994).

Impact of the DNA in food 465

Most of the DNA remaining after digestion would be smaller than the kanamycin resistance gene which is about 1,000 base pairs long.


98

Appendices

466

Calgene Inc calculated that for consumption of fresh GM tomatoes containing nptII at the 90th percentile the transformation frequency of intestinal micro-organisms will be about 3 x 10-15 transformants per day.4 This is more than five orders of magnitude less than the frequency of mutation to kanamycin resistance per bacterial replication (ie 10-9). For every 300,000 bacteria that mutate to kanamycin resistance per replication (generally a matter of hours) there would be at most under worst case conditions one kanamycin resistant bacterium per day added to that number due to gene transfer. The number of kanamycin resistant micro-organisms that would result from potential gene transfer in the human gastrointestinal tract is therefore negligible with respect to the number of intrinsically resistant micro-organisms already present (Nap et al, 1992).

Impact on the efficacy of antibiotics 467

To be of clinical significance the nptII gene would have to be transferred to and expressed in a pathogenic micro-organism that is treated with kanamycin or neomycin.

468

Calgene Inc reported that studies that simulated abnormal gastric conditions (eg patients treated with antacids) showed that NPTII is not degraded in neutralised (pH 7) simulated gastric fluid and therefore may remain active. In this situation the concentration of ATP which the enzyme requires to inactivate kanamycin and neomycin would be limiting.

469

Using a worst case scenario Calgene Inc concluded that compromised oral antibiotic efficacy due to ingestion of GM food containing NPTII would be extremely unlikely. The worst case scenario was based on a number of assumptions several of which are extremely unlikely to occur. Assumptions made were: • 95th percentile consumption at one sitting of fruits or vegetables with high ATP content; • stoichiometric reaction of 100 percent of the ATP in ingested food with orally administered neomycin (highly unlikely); • administration of neomycin simultaneously with consumption of GM food containing NPTII and with other fruits or vegetables rich in ATP; • presence of intact functional NPTII which requires a buffered stomach environment (pH 7); and • stability of ATP in the stomach.

470

Oral kanamycin or neomycin is administered only for hepatic encephalopathy and preoperative bowel preparation. During preoperative preparation for bowel surgery it is highly unlikely that patients would be consuming any solid foods. For patients with hepatic encephalopathy who consume fresh fruit and vegetables Calgene Inc calculated that 1.5 percent of a 1 g dose of antibiotic would be inactivated by consuming NPTII as a component of fresh tomatoes (Redenbaugh et al, 1994). This conclusion was supported by data from an in vitro study that showed no significant inactivation of kanamycin when tomato extract containing NPTII and kanamycin was incubated over a four hour period.

A model showed that 3 mg of tomato genomic DNA (from consumption of 250 g tomatoes) in 1500 ml (volume of fluid in the small intestine) would generate 0.0024 transformants, an increase of 2.4 x 10-13 percent of the transformable intestinal microflora or 2.4 x 10-15 percent of the total intestinal microflora (Nap et al, 1992).

4


99

Appendices

471

None of the bacterial species known to be present in the gastro-intestinal tract has been found capable of acquiring foreign DNA by natural transformation. Calgene Inc noted that although they developed their transformation model for certain Streptococcus species they were not aware of any information indicating that Streptococcus found in the gastro-intestinal tract can be naturally transformed.

472

E. coli contains DNA segments homologous with part of the kanamycin resistance construct because the construct contains part of an E. coli gene. Although E. coli constitutes one of the predominant species of aerobic gastro-intestinal tract bacteria it is not transformation competent under normal gastro-intestinal conditions.

473

The FDA concluded that the therapeutic efficacy of neomycin in animal feed would not be affected since Calgene Inc found no significant inactivation of neomycin in feed manufactured using GM cottonseed or rapeseed during an eight week storage period.

474

In the event that DNA was not completely degraded by processing during feed production any remaining DNA would be degraded by digestion. Studies have shown that nucleic acids entering the rumen are rapidly degraded (McAllan, 1980; 1982). Also many rumen bacterial strains have nuclease activity which degrades DNA and provides another barrier to transformation (Flint and Thomson, 1990).

475

The probability of generating a kanamycin resistant organism in cattle as a result of gene transfer from GM feed was concluded as being equally negligible (Nap et al, 1992).

Impact on the environment 476

It is unlikely that as a result of the increase in resistance genes that the gene product could reach environmental concentrations that may be toxic to plants, soil micro-organisms or wildlife because of a lack of persistence and low toxicity of most proteins in the environment and narrow substrate specificity of most enzymes including NPTII. Generally natural organic materials are rapidly degraded in the soil (Redenbaugh et al, 1994).

477

Kanamycin resistant micro-organisms represent a small proportion of the total soil microflora but are present at detectable background levels. The probability of a bacterium obtaining the nptII gene from plant DNA compared to from another bacterium is extremely low.

478

Calgene Inc calculated that at worst kanamycin resistant transformants resulting from plant DNA left in the field would represent no more than one micro-organism in 10 million existing kanamycin resistant soil micro-organisms. This is the estimated number of kanamycin resistant soil micro-organisms present in one hectare (Redenbaugh et al, 1994).

479

It was concluded that even if horizontal gene transfer occurred release of NPTII into the environment would not be of concern since calculations using the most favourable assumptions and probabilities do not show significant changes in the potential numbers of micro-organisms containing the resistance gene or potential impact on the level of NPTII in the environment (Redenbaugh et al, 1994).


100

Appendices

Appendix II: Genetically modified insect resistant maize 480

Considerable controversy has been associated with the inclusion of the ampicillin resistance gene (bla) that encodes resistance to ampicillin in insect resistant maize developed by CibaGeigy (now Novartis) and Dekalb for animal feeds.

481

This controversy centres on the scenario that as a result of feeding unprocessed GM maize containing the bla gene to animals, the gene may be transferred to bacteria in the animals’ guts and ampicillin resistant bacteria may subsequently be transmitted to humans via the food chain.

482

Genetically modified maize varieties have been produced by transformation with directly introduced linearised plasmid DNA containing the desired gene. An antibiotic resistance gene is usually present as a result of the use of antibiotic resistance to select recombinant bacteria in the laboratory.

483

The Novartis and Dekalb maize contains an intact bla gene with a promoter and an origin of replication (ori) derived from the pUC18 cloning vector. The pUC ori generates over 600 copies per cell (compared to the four to 18 copies per cell of the ColE1 ori vector found in nature).

484

Little information is available on the state of the pUC18 DNA that is integrated in the Novartis or Dekalb maize genome. It is not certain that the bla gene and the region of the plasmid necessary for plasmid replication are intact. There is no selection for maintenance of an intact bla gene or pUC18 ori in a plant cell because pUC18 does not replicate in an eukaryotic host and the bla gene does not have an eukaryotic promoter. Maize is naturally resistant to penicillins so there is no selective advantage even if the gene were to be expressed. As a result of lack of selection parts of pUC18 could have undergone deletions or other modifications during or after integration (Salyers, 1999).

485

The smallest contiguous DNA fragment from pUC18 that could contain the bla gene is about 900 base pairs. The fragment size necessary to contain both the gene and the origin of replication is about 1600 base pairs. It has been found experimentally that during DNA isolation within one hour of tissue disruption all DNA is degraded to fragments of less than 500 base pairs, the majority to less than 150 base pairs. It is therefore unlikely that DNA fragments of sufficient size would survive plant nuclease activity (Malik and Saroha, 1999).

486

Although DNA can be partially protected from digestion when associated with particulate matter (eg soil, plant) it would then also be unavailable for binding to proteins on the bacterial cell surface, a prerequisite for DNA uptake into the bacterial cell. Multiple copies of the intact DNA fragment would need to aggregate at the same binding site on the bacterial cell surface for uptake to occur. This is considered highly improbable. There is also a very large quantity of other DNA fragments competing for the binding sites that further reduces the opportunity for successful bacterial transformation (Malik and Saroha, 1999).

487

If pUC18 and the bla gene are intact and unaltered, and one or more tandem duplications of the plasmid occurs less than one in 10,000 cells would contain the circular form of the plasmid as a result of homologous recombination (Salyers, 1999).


101

Appendices

488

It is highly unlikely but not impossible that the bla gene could be incorporated as a single copy via illegitimate recombination into the bacterial chromosome of E. coli or its relatives. The frequency of illegitimate recombination is less than that of homologous recombination (Salyers, 1999).

489

The bla gene is expressed in E. coli or its close relatives such as Salmonella species but not in ruminal or intestinal anaerobes. A strong selective pressure would be necessary to maintain the gene in a gut bacterium in the competitive environment of the rumen or intestine (Salyers, 1999).

490

In 1996 the ACNFP recommended approval be declined for unprocessed insect resistant maize for animal feeds. Although transfer of the bla gene to ruminal or intestinal bacteria would be a very rare event there was concern that long term high level consumption by animals, in particular ruminants, might allow such an event to occur and as a result of the presence of a promoter and the pUC ori on the gene such transfer would have serious consequences for therapy with beta-lactam antibiotics (US FDA, 1998).

491

Initially the European Union (EU) did not approve the GM maize. Subsequently the European Commission Scientific Committee for Food concluded that the risk of bacterial transformation from GM maize is extremely low. If transfer did occur it would have no detectable additional effect as the bla gene is already widespread in nature including the human and animal gastro-intestinal tracts. The European Commission Scientific Committee for Animal Nutrition also concluded that the probability of transfer of a functional bla gene into bacteria is virtually zero, and if it did occur it would be clinically insignificant.

492

A group of scientists at a conference in September 1996 sponsored by Tufts University and the Foundation for Nutritional Advancement also concluded that use of the bla gene constitutes an insignificant to near zero risk of causing ampicillin resistance in animals or humans. This is because the probability of DNA survival in fragments large enough to be taken up by bacteria is very low, the probability of bacteria taking up or incorporating DNA into the bacterial genome is virtually zero, if it was incorporated there is a low probability that it would be expressed, and the clinical significance is virtually zero because ampicillin resistance is widespread and can be overcome by antibiotics other than ampicillin (US FDA, 1998).

493

The majority scientific opinion was that the presence of the bla gene in the maize genome posed no significant antibiotic resistance risk even if large amounts were ingested regularly by animals over prolonged periods. As a result in December 1996 the EU authorised the sale and cultivation of insect resistant maize.

494

This decision has been challenged by some countries on grounds including health (eg France, Austria, Luxembourg, Italy). French regulatory authorities claimed that the proportion of ampicillin resistant gut bacteria was much lower than that stated by proponents of GM maize (Salyers, 1999). More recently Germany halted the approval process for commercial cultivation citing the need for further research with respect to the transfer of antibiotic resistance (Abbott, 2000).


102

Appendices

495

The difference of opinion arises mainly over the question of whether extremely low but nonzero risks of increased antibiotic resistance are acceptable.

496

Preliminary results from a study by Heritage and coworkers of gene transfer from GM maize to chicken gut bacteria have found no evidence of transfer of the bla gene to normal flora. However this study has yet to be completed and published in the peer reviewed scientific literature (Coghlan, 2000).


103

Appendices

Appendix III: Summary of submissions The following submissions were received as a result of the draft report’s availability for public comment and were considered in the preparation of the final version. Submission Wendy Johnson, Friends of the Earth (NZ)

Jim Waters, Ministry of Health

Comments Issues were raised that are outside the Terms of Reference of this report: • Extension of the report to include all possible scenarios of ingestion of genetically engineered DNA • Intestinal permeability - its association with certain diseases, causes, and social and public health costs • Approach of the ERMA to applications involving GM foods •

The unique narrow specificity of certain aminoglycosides means that in spite of their side effects they continue to be important antibiotics in an era of complex medical care with greater risk of complications from infection due to resistant micro-organisms.

•

Development of resistance to antibiotics needed for the control of meningococcal disease and to the aminoglycosides needs to be prevented in New Zealand.

•

The Ministry supports reducing unnecessary antibiotic use in the interest of reducing selection pressure around antibiotic resistance. A side benefit of this is a reduction in selection of antibiotic resistance marker genes but the main focus is other sources of resistance.

•

Issues were raised that relate to the Authority’s decision-making process and are outside the Terms of Reference of this report.


104