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Antibiotic resistance among bacteria isolated from seawater and penguin fecal samples collected near Palmer Station, Antarctica1 Robert V. Miller, Katharine Gammon, and Martin J. Day
Abstract: Antibiotic resistance in aquatic bacteria has increased steadily as a consequence of the widespread use of antibiotics, but practice and international treaty should have limited antibiotic contamination in Antarctica. We estimated antibiotic resistance in microorganisms isolated from the Antarctic marine waters and a penguin rookery, for 2 reasons: (i) as a measure of human impact and (ii) as a potential ‘‘snapshot’’ of the preantibiotic world. Samples were taken at 4 established sampling sites near Palmer Station, which is situated at the southern end of the Palmer Archipelago (64810’S, 61850’W). Sites were chosen to provide different potentials for human contamination. Forty 50 mL samples of seawater were collected and colony-forming units (CFU)/mL were determined at 6 and 20 8C. For this study, presumed psychrophiles (growth at 6 8C) were assumed to be native to Antarctic waters, whereas presumed mesophiles (growth at 20 8C but not at 6 8C) were taken to represent introduced organisms. The 20–6 8C CFU/mL ratio was used as a measure of the relative impact to the ecosystem of presumably introduced organisms. This ratio was highest at the site nearest to Palmer Station and decreased with distance from it, suggesting that human presence has impacted the natural microbial flora of the site. The frequency of resistance to 5 common antibiotics was determined in each group of isolates. Overall drug resistance was higher among the presumed mesophiles than the presumed psychrophiles and increased with proximity to Palmer Station, with the presumed mesophiles showing higher frequencies of single and multiple drug resistance than the psychrophile population. The frequency of multidrug resistance followed the same pattern. It appears that multidrug resistance is low among native Antarctic bacteria but is increased by human habitation. Key words: natural resistance to antiobiotics, Antarctic, plasmids, Palmer Station, penguins. Re´sume´ : La re´sistance aux antibiotiques des bacte´ries aquatiques a augmente´ progressivement a` cause de l’utilisation re´pandue des antibiotiques, mais un meilleur usage et la signature d’un traite´ international devraient avoir limite´ la contamination de l’Antarctique par les antibiotiques. Nous avons estime´ la re´sistance aux antibiotiques des eaux Antarctiques et d’une colonie de pingouins pour deux raisons : (i) comme mesure de l’impact humain et (ii) comme « instantane´ » du monde pre´-antibiotique. Des e´chantillons ont e´te´ recueillis a` partir de 4 sites e´tablis a` proximite´ de la Station Palmer situe´e a` l’extre´mite´ sud de l’Archipel Palmer (648 10’ S, 618 10’ W). Ces sites ont e´te´ choisis pour repre´senter diffe´rents potentiels de contamination humaine. Quarante e´chantillons de 50 mL d’eau de mer ont e´te´ recueillis et le nombre d’unite´s de formation de colonies UFC/mL a e´te´ de´termine´ a` 6 et 20 8C. Lors de cette e´tude, on a suppose´ que les psychrophiles pre´sume´s (croissance a` 6 8C) e´taient indige`nes des eaux Antarctiques alors que les me´sophiles pre´sume´s (croissance a` 20 8C mais pas a` 6 8C) repre´sentaient des organismes introduits. Le ratio UFC/mL a` 20 8C / UFC/mL a` 6 8C a e´te´ utilise´ pour mesurer l’impact relatif sur l’e´cosyste`me des organismes vraisemblablement introduits. Le ratio le plus e´leve´ a e´te´ observe´ au site le plus pre`s de la station Palmer et diminuait a` mesure que l’on s’en e´loignait, ce qui sugge`re que la pre´sence de l’humain ait un impact sur la flore microbienne naturelle du site. La fre´quence de la re´sistance envers 5 antibiotiques communs a e´te´ de´termine´e chez chacun des groupes d’isolats. La re´sistance globale e´tait plus e´leve´e parmi les me´sophiles pre´sume´s que chez les psychrophiles pre´sume´s et augmentait en fonction de la proximite´ de la station Palmer, les me´sophiles pre´sume´s faisant preuve d’une fre´quence de re´sistance simple ou multiple plus e´leve´e que la population de psychrophiles. La fre´quence de multire´sistance aux me´dicaments suivait le meˆme patron. Il semble que la multire´sistance aux me´dicaments soit faible chez les bacte´ries antarctiques indige`nes mais qu’elle soit augmente´e par l’habitat humain. Mots-cle´s : re´sistance naturelle aux antibiotiques, Antarctique, plasmides, Station Palmer, pingouins. [Traduit par la Re´daction]
Received 15 September 2008. Accepted 10 October 2008. Published on the NRC Research Press Web site at cjm.nrc.ca on 29 January 2009. R.V. Miller.2 Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA. K. Gammon3 and M.J. Day. School of Biosciences, Cardiff University, Cardiff, CF10 3TL, Wales, UK. 1This
article is one of a selection of papers in the Special Issue on Polar and Alpine Microbiology. author (e-mail:
[email protected]). 3Present address: Biotechnology Team, Detection Department, DSTL Porton Down, Salisbury, SP4 0JQ, UK. 2Corresponding
Can. J. Microbiol. 55: 37–45 (2009)
doi:10.1139/W08-119
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Introduction As recently as 1984, Kobori et al. (1984) were able to state that ‘‘although physically an extreme environment, the Southern Ocean regions have been little influenced by human activities.’’ Perhaps one of the most sensitive ways of determining human impact on this environment is to assess the potential resistance of the native bacterioplankton to antibiotic resistance. The incidence of antibiotic-resistant bacteria in aquatic environments throughout the world has increased steadily as a consequence of the widespread use of antibiotics by humans (Baya et al. 1986). Still, practice and international treaty have limited the exposure of the Southern Ocean surrounding Antarctica to antibiotics. Direct release of antimicrobials or human-associated bacteria is strictly limited, and all wastes must be quarantined and removed (Bonner 1984). Various authors, including Stuart Levy (2002), have suggested that a more frugal use of antibiotics in medicine, veterinary medicine, animal husbandry, and crop agriculture will lead to a reversal of the current trend to increased incidence of multiantibiotic resistance microorganisms in the environment and, hence, to a reversal in the ever-increasing crises in infectious disease therapy that this increased incidence has produced. Without some indication of the nature of antibiotic resistance among bacterial communities in the preantibiotic age, the potential benefit of this return is difficult to assess. Yet, there are few environments that have not been inundated with antibiotic use (Baya et al. 1986). For instance, a recent study by Skurnik et al. (2006) determined that the frequency of antimicrobial resistance and integrons in animal fecal Escherichia coli was anthropogenic in origin. Antarctica is potentially one of the few, it not the only, environment where antibiotic use by humans has been limited, and it offers the best chance to assess preantibiotic era levels of resistance among natural populations of bacteria. Hence, a study of the frequency of antibiotic resistance in the Antarctic is potentially interesting for 2 reasons: (i) as a measure of the impact of human intervention and (ii) as a potential ‘‘snapshot’’ of the preantibiotic environment.
Materials and methods Sampling site Seawater samples were taken from 4 sampling stations near Palmer Station, Antarctica (Fig. 1). Palmer Station (United States) is at the southern end of Anvers Island, the southernmost major island of the Palmer Archipelago. This archipelago is separated from the Antarctic Peninsula by the Gerlache Strait and is situated approximately 64810’S, 61850’W. The sites are well established and were chosen to provide sample areas with the potential for differing levels of human contamination. Station X is at the pump house at Palmer Station where seawater is drawn for various uses around the station. No return of water to the sea is made at this site. Station A is opposite Palmer Station on the far side of a small peninsula on Anvers Island. It is in Arthur Harbor, approximately 0.2 mi. (1 mi. = 1.609 km) overland from the station. Station T is situated near Torgersen Island that supports a large penguin colony. The station is approximately 1 nautical mi. from Palmer Station. It is located in the runoff from the penguin colony and hence is ‘‘naturally’’
Can. J. Microbiol. Vol. 55, 2009
contaminated with penguin feces. Seawater was sampled from Station T and penguin fecal material was collected from the colony on Torgersen Island. Station E is a deepwater station located approximately 2 nautical mi. seaward of Palmer Station in Bisco Bay. Sampling Forty 50 mL samples were collected at each station from surface waters or as fecal material. Samples were taken during December 2000 and were transported frozen by air to Cardiff University, Cardiff, Wales, where they were immediately stored. Individual samples were thawed and 1 mL and 100 mL portions were taken from each sample and plated on R2A medium (0.05% yeast extract, 0.05% protease peptone, 0.05% casamino acids, 0.05% soluble starch, 0.05% dextrose, and 0.03% sodium pyruvate) containing 3% sea salt (Instant Ocean synthetic sea salt). Similar sets of plates were incubated at 6 and 20 8C. Plates were incubated for 2 weeks and the concentration of colony-forming units (CFU) was estimated at each temperature. The ratio of CFU/mL at 6 8C (presumed psychrophiles that are defined as being able to grow below 8 8C; Ingraham and Stokes 1959) to that at 20 8C (presumed psychrophiles + presumed mesophiles) was determined for each sample. Plates were observed daily for the 2 week period. No increase in colonies was noted after the initial 7 days of incubation. All colonies were selected for further analysis from samples with low CFU/mL. For samples with high counts, colonies were selected on morphology so that all representative colony types were among those selected for further analysis. All 6 8C isolates were tested for growth at 20 8C. Approximately 60% grew at 20 8C, whereas the remainder did not. Thus, they represent a mixture of true psychrophiles and psychrotolerant organisms, and we have chosen to designate them as ‘‘presumed psychrophiles’’ throughout this communication to indicate this possibility. Isolation from penguin feces Samples of penguin fecal material were taken from the colony on Torgersen Island (Fig. 1) adjacent to sampling site T. Samples were homogenized in 10 mL of 3% sea salt and then analyzed in a manner similar to the water samples (see Sampling section). Antibiotic and mercury concentrations Antibiotics used in this study included tetracycline (Tet, either 25 or 100 mg/mL of medium), kanamycin (Kan, 500 mg/mL), ampicillin (Amp, 1000 mg/mL), naladixic acid (Nal, 500 mg/mL), and streptomycin (Str, 500 mg/mL). Mercury (HgCl2) was used at a concentration of 27 mg /mL. Antibiotics and Hg were added to the medium singly and plates were incubated at 6 or 20 8C as appropriate. Growth was assessed daily for up to 1 week. Ultraviolet sensitivity Resistance to UV light was assessed by the method of Wilson et al. (2004). Growth was compared with the positive control Pseudomonas putida strain pQM1 (Bale et al. 1987). Organisms were scored as UVR if they were equal to or more resistant than the P. putida strain. Published by NRC Research Press
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Fig. 1. Sampling sites in and around Palmer Research Station, Antarctica. Sampling points A, E, T, and X are indicated. This is an adaption of the Palmer Station Safe Boating Map provided to us by the United States Arctic Programs. The original is available at http://www.usap. gov/USAPgov/sciencesupport/GIS/images/BoatingMap.gif.
DNA and plasmid isolation DNA isolation was by the method of Powell et al. (1993). Plasmid DNA was isolated using a Promega plasmid isolation kit or by the method of Birnboim and Doly (1979). PCR analysis PCR and sequencing of genomic DNA were carried out by the method of Marchesi et al. (1998). For 16S rRNA gene sequencing, primers 63f and 1387r were used.
Results Frequency of presumed mesophiles as a measure of anthropogenic activity The ratio of CFU/mL at 6 8C to that at 20 8C was determined for each sample (Fig. 2). For the purposes of this study, presumed psychrophiles were assumed to be native to seawater, whereas presumed mesophiles were presumed to represent organisms introduced from other environments. The ratio of the CFU/mL on the 20 8C-incubated plates to the CFU/mL on 6 8C-incubated plates was taken as a meas-
ure of the relative impact of non-native organisms on the sampling site. The ratio of CFU/mL enumerated at 20 and 6 8C was highest for Station X (the pump house at Palmer Station), followed by Station T (off the Torgersen Island penguin colony), Station A (in Arthur Harbor), and finally Station E (deep water) that showed the lowest ratio. Thus, we concluded that the contamination of the sites with nonnative presumed mesophiles was greatest at Station X and lowest at Station E. Low-level antibiotic resistance in samples Preliminary evaluation of antibiotic resistance was carried out by replicating plates using R2A medium containing 25 mg Tet/mL of medium. Data from these experiments showed that on average 1% of the CFU from Station E was resistant. This number rose as the level of organisms capable of growth at 20 8C rose, with the highest percentage (14%) observed at Station X (Table 1). These data suggested that antibiotic resistance was low in the Antarctic Ocean but that intervention by vertebrate contaminators (penguins or human) increased the frequency of TetR. Published by NRC Research Press
40 Fig. 2. Ratio of presumed psychrophiles to presumed mesophiles at each of the sampling sites. The CFU/mL that grew at 6 and 20 8C were determined as described in the Materials and methods section. Each symbol represents the result from a single seawater sample. The symbols indicate the sampling site as shown in Fig. 1.
Can. J. Microbiol. Vol. 55, 2009 Table 1. Frequency of low-level tetracycline (Tet) resistance in marine isolates from the Southern Sea. Average CFU/mL*
Station E A T X
6 8C 35±20 96±58 486±160 6±3
20 8C 2±1 44±40 1154±340 123±64
No. of colonies tested 43 61 242 240
Percent tetracycline resistant 1 4 13 14
Note: Isolates were screened for resistance to Tet at 25 mg/mL, as described in the Materials and methods section. In this preliminary study, presumed psychrophiles and presumed mesophiles were not distinguished. *Mean ± standard error of the mean.
High-level, multiple drug resistance Although any antibiotic resistance phenotype is important in the consideration of the continued use of antibiotics, medical difficulties most often arise when organisms from clinical infections are resistant to several drugs at the same time (Levy 2002). Selection of resistance owing to simple spontaneous mutations by antibiotic exposure is most likely to produce a phenotype showing resistance to a single antibiotic. This is true because the mutation maps to a single locus, which is usually a structural gene for some component of the antibiotic’s target organelle. The changes in interaction between the antibiotic and target caused by these mutations are usually highly specific and therefore limited to affecting the action of a single antibiotic. On the other hand, phenotypes associated with selection for plasmids or transposons that contain antibiotic-resistance loci often demonstrate multiple drug resistance. The molecular mechanisms leading to these drug-resistant phenotypes are often from the introduction of antibiotic-degrading enzymes or antiporter systems into the cell (Levy 2002). To assess the frequency of multiple drug resistance in our Antarctic isolates, we tested them using 5 drugs that affect different features of bacterial cytology and physiology (Mann and Crabbe 1996). Tetracycline affects protein synthesis by inhibiting aminoacyl-tRNA binding to the ribosome. Because of the possibility that a high-salts medium could reduce the efficacy of Tet, we retested our isolates at the higher concentration. We also chose streptomycin that inhibits protein synthesis by binding to the 30S ribosomal subunit, inhibiting both initiation and elongation of the growing peptide. Kanamycin also binds to the ribosome and inhibits translocation. Ampicillin is a b-lactam and affects the cross-linking of the peptidoglycan during bacterial cellwall synthesis. Finally, naladixic acid affects bacterial DNA synthesis by covalently linking DNA gyrase to the bacterial DNA. To date, all documented resistance to Nal is chromo-
somal in origin and involves the alteration of gyrase structure to reduce binding of the antibiotic to the protein. Plasmid-associated resistance has not been identified (Mann and Crabbe 1996). The frequency of antibiotic resistance to each of these 5 common antibiotics was determined in each group of isolates (presumed psychrophiles and presumed mesophiles) at each station. The frequency of organisms resistant to 2 or more of the 5 antibiotics was also determined for each group and station (Fig. 3). The frequency of drug resistance was higher among the presumed mesophiles than the presumed psychrophiles and increased with proximity to Palmer Station (Fig. 3). The frequency of multidrug resistance followed the same pattern. Not only was the frequency greater near Palmer Station but the maximum number of drugs to which a single isolate was resistant also increased with proximity to the experimental station. The various combinations of resistant phenotypes in each population are summarized in Table 2. Plasmid content of multiple-resistant isolates An examination of isolates for plasmids that might carry drug resistance showed that the frequency of plasmids was no greater in isolates carrying antibiotic resistance (single or multiple) than it was in isolates not displaying resistance to any of these antibiotics (Fig. 4). These plasmids ranged in molecular size from approximately 30 to 300 kb. In all cases, the incidence of plasmids was very low, with most isolates not containing detectable extrachomosomal DNA. Identification of organisms isolated (16S RNA gene sequence analysis) rRNA gene amplification was conducted on selected organisms using universal primers 63f and 1387r (Marchesi et al. 1998). A proportion of colonies representing different morphologies was subjected to 16S RNA gene analysis. The majority of isolates fell into 3 generic phylotypes: Alteromonas, Pseudoalteromonas, and Bacillus. These are common genera occurring in marine environments (Belliveau et al. 1991; Jensen et al. 1980). Frequency of UVR and Mercury (HgR) resistance in isolates De Souza et al. (2006, 2007) explored metal resistance in Antarctic bacteria. They observed high levels of metal rePublished by NRC Research Press
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Fig. 3. Fraction of isolates showing no, single, and multiple drug resistance, from the 4 sampling sites shown in Fig. 1. Isolates from each sampling site and incubation temperature were scored for resistance to each of 5 antibiotics as described in the Materials and methods section.
sistance including resistance to HgCl2. Surprisingly, these authors found a negative correlation between antibiotic resistance and metal resistance. Booth et al. (2001a, 2001b) demonstrated that solar UV exposure had a negative effect on Antarctic bacteria and may select for more UV-resistant organisms. We explored the frequency of resistance to HgCl2 and UV radiation among our isolates (Table 3). The frequencies of resistance to both Hg and UV were low in all populations. Both were higher among the presumed mes-
ophiles and followed spatial distributions similar to the antibiotics. We did not observe any correlation between antibiotic resistance and these physical effectors. Antibiotic resistance in fecal isolates from the Torgersen Island penguin colony We were interested in the fact that antibiotic resistance was high at Station T. This station lies off of Torgersen Island and receives runoff from a large penguin colony that Published by NRC Research Press
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Can. J. Microbiol. Vol. 55, 2009 Table 2. Phenotype classes found in each group of isolates. Phenotypic classes observed* Presumed psychrophiles
Station E
No. of isolates tested 12
A
Presumed mesophiles
Phenotype Tet
No. of isolates with phenotype 1
No. of isolates tested 31
21
None
NA{
40
T
97
Tet
10
114
X
85
Tet
2
202
Phenotype Tet Nal Tet, Nal Kan, Nal Kan Nal Tet, Nal Tet Kan Amp Tet, Str Tet, Kan Tet, Amp Tet Kan Nal Tet, Nal Tet, Kan Tet, Amp Tet, Kan, Nal
No. of isolates with phenotype 5 1 1 1 1 1 3 47 5 9 1 3 4 8 6 1 2 4 12 2
Note: The level of resistance to high concentrations of the 5 antibiotics was tested as described in the Materials and methods section. The various phenotypic classes (combinations of resistance markers) in each group of isolates are presented. *Abbreviations: Tet, tetracycline; Nal, nalidixic acid; Kan, kanamycin; Amp, ampicillin; and Str, streptomycin. { Not applicable.
Fig. 4. Number of isolates containing and not containing observable plasmid DNA.
inhabits the island. To see if there was a correlation between the high frequency of resistance in, especially, the presumed mesophiles from Station T and isolates of penguin origin,
we isolated organisms from penguin feces and tested them for antibiotic resistance (Fig. 5). Antibiotic resistance in isolates from penguin feces was high and quite varied (Table 4). Published by NRC Research Press
Miller et al.
43 Table 3. Frequency of mercury (HgCl2) and ultraviolet subregion C (UV-C) resistance in the various groups of isolates. Percent resistance Station E A T X
Growth temperature group Presumed psychrophiles Presumed mesophiles Presumed psychrophiles Presumed mesophiles Presumed psychrophiles Presumed mesophiles Presumed psychrophiles Presumed mesophiles
No. of isolates tested 12 31 21 41 122 144 43 202
Hg resistance 0 0 0 0 0 7 2 3
UV-C resistance 0 0 0 16 3 5 0 5
Note: UV-C: approximately 240–280 nm light (Booth et al. 2001a). Resistance to HgCl2 and UV-C irradiation was determined for each group of isolates as described in the Materials and methods section.
Fig. 5. Fraction of isolated bacteria from penguin feces showing no, single, and multiple drug resistance.
Discussion The study of antibiotic resistance in the Antarctic is important for 2 reasons. First, it can act as a dosimeter of anthropogenic alteration in the microbial ecology of Antarctica and by extension of the ecosystem in general. Second, it can provide us with the best snapshot of the levels and characteristics of antibiotic resistance in an environment that has been little influenced by the misuse of these powerful tools in the fight against infectious disease. In general, the results of our study confirm our hypothesis that in a natural, pristine environment, multiple antibiotic resistance will be low, and what resistance that is observed will be chromosomal in genetic origin. It is likely that those organisms that show single-drug resistance do so as a result of spontaneous mutation in a structural gene for the antibiotic’s target. In the absence of the antibiotic, these mutations often result in reduced fitness, and the organisms containing them will be eliminated from the natural community in a few generations (Replicon et al. 1995; Levy 2002). This then is the picture drawn by many researchers of the frequencies and profiles of antibiotic resistances in natural bacterial populations before the commercial and medical uses of antibiotics. Many investigators, including Levy (2002), pre-
dict that this is also the state to which these populations would return if antibiotics were used in a more prudent manner. Our findings support this prediction. The data presented here suggest the negative effects of human habitation on local pristine environments in the Antarctic. Although practices at Palmer Station are designed to reduce impact on the environment to a minimum, our data show that measurable changes have occurred (are still produced) in 2 ways. First, the frequency of presumed mesophiles near Palmer Station is greater and increases with proximity to the station. Second, the frequency of antibiotic resistance increases with proximity to this research facility, with multidrug resistance also increasing. Each of these parameters suggests that human occupation has altered the bacterioplankton population and provides a warning that, despite the precautions taken, no ecosystem is immune to alteration because of human habitation. It should be noted that these characteristics of higher presumed mesophiles frequencies and certainly antibiotic resistance are also true of Station T, which is located off the penguin colony of Torgersen Island. The presence of these markers of potential human influence was noted both in the organisms isolated from Station T waters and from those isolated from the penguin feces samples. Similar effects of Published by NRC Research Press
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Can. J. Microbiol. Vol. 55, 2009 Table 4. Phenotype classes found in each group of isolates from penguin feces. Phenotypic classes observed* Presumed psychrophiles No. of Phenotype isolates tested 250 Tet Kan Nal Amp Tet, Kan Tet, Nal Tet, Amp Tet, Kan Tet, Kan, Nal Tet, Nal, Str Kan, Nal, Amp Nal, Amp, Str Tet, Kan, Amp, Str Kan, Nal, Amp, Str Tet, Kan, Nal, Amp, Str
No. of isolates with phenotype 12 5 22 14 1 1 2 1 1 1 1 2 2 1 5
Presumed mesophiles No. of isolates Phenotype tested 277
No. of isolates with phenotype
Tet Kan Nal Str Tet, Kan Tet, Nal Tet, Amp Amp, Str Tet, Kan, Nal, Amp Kan, Nal, Amp, Str Tet, Kan, Nal, Amp, Str
25 5 3 24 1 22 1 10 1 2 8
Note: The level of resistance to high concentrations of the 5 antibiotics was tested as described in the Materials and methods section. The various phenotypic classes (combinations of resistance markers) in each group of isolates are presented. *Abbreviations: Tet, tetracycline; Nal, nalidixic acid; Kan, kanamycin; Amp, ampicillin; and Str, streptomycin.
human vicinity on antimicrobial resistance have been noted in animal fecal E. coli (Skurnik et al. 2006). Further investigation of antibiotic resistance in penguin fecal matter will be necessary to determine if this increase is due to interaction with the human population of Palmer Station or is a natural characteristic of penguin-associated microflora. In either case, it is clear that the presence of these birds on Torgersen Island has influenced the antimicrobial resistance characteristics of the marine bacterial population in close proximity to the island. The fact that these do not spread too widely also indicates that the environment has a considerable capacity to remove high densities of microbial populations. Our data suggest that human habitation can alter the antibiotic profile of natural bacterial populations even in environments where efforts to minimize that impact have been taken. They also indicate that the frequency of antibiotic resistance in environments, even where use of antimicrobials by human has been at a minimum, shows an increase, albeit at low levels of resistance, and that this resistance is primarily exhibited to only a single antibiotic. These observations suggest that a more prudent use of antibiotics could reduce the frequency of resistance in the environment and by extension have a positive impact on the treatment of human and animal diseases by reducing the increased levels of microbial antibiotics resistance that the last decade has seen (Levy 2002).
Acknowledgements This research was supported by grants from the National Science Foundation (MCB-0132097), and by a Research Travel Grant from the Burroughs Wellcome Fund to RVM and a grant from The National Environmental Research Council of the United Kingdom (NER/B/S/2000/00821) to MJD.
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