Antimicrobial Resistance in Wildlife - Wiley Online Library

Zoonoses and Public Health

ORIGINAL ARTICLE

Antimicrobial Resistance in Wildlife: Implications for Public Health D. Carroll1, J. Wang2, S. Fanning2,3 and B. J. McMahon1 1 2 3

UCD School of Agriculture & Food Science, University College Dublin, Dublin, Ireland UCD Centre for Food Safety, UCD School of Public Health, Physiotherapy & Population Science, University College Dublin, Dublin, Ireland Institute for Global Food Security, School of Biological Sciences, Queen’s University Belfast, Belfast, Northern Ireland

Impacts

• Antimicrobial-resistant (AMR) bacteria in natural environments are a • •

major concern with serious implications for human and animal health. Bacteria with antimicrobial-resistant genes were found in a number of wildlife species found in different habitats indicating possible widespread prevalence. The presence of AMR in wildlife and the environment has implications for public health and merits further investigation.

Keywords: Wild birds; deer; Escherichia coli; antibiotic resistance; plasmids Correspondence: B. J. McMahon. UCD School of Agriculture & Food Science, University College Dublin, Belfield, Dublin 4, Ireland. Tel.: 353 17167739; Fax: 353 17161102; E-mail: barry. [email protected] Received for publication August 6, 2014 doi: 10.1111/zph.12182

Summary The emergence and spread of antimicrobial-resistant (AMR) bacteria in natural environments is a major concern with serious implications for human and animal health. The aim of this study was to determine the prevalence of AMR Escherichia coli (E. coli) in wild birds and mammalian species. Thirty faecal samples were collected from each of the following wildlife species: herring gulls (Larus argentatus), black-headed gulls (Larus ridibundus), lesser black-back gulls (Larus fuscus), hybrid deer species (Cervus elaphus x Cervus nippon) and twenty-six from starlings (Sturnus vulgaris). A total of 115 E. coli isolates were isolated from 81 of 146 samples. Confirmed E. coli isolates were tested for their susceptibility to seven antimicrobial agents by disc diffusion. In total, 5.4% (8/146) of samples exhibited multidrug-resistant phenotypes. The phylogenetic group and AMR-encoding genes of all multidrug resistance isolates were determined by PCR. Tetracycline-, ampicillin- and streptomycin-resistant isolates were the most common resistant phenotypes. The following genes were identified in E. coli: blaTEM, strA, tet(A) and tet(B). Plasmids were identified in all samples that exhibited multidrug-resistant phenotypes. This study indicates that wild birds and mammals may function as important host reservoirs and potential vectors for the spread of resistant bacteria and genetic determinants of AMR.

Antibiotic use in the non-medical arenas of agriculture, aquaculture and intensive farming can often be up to fourtimes the amount used in human medicine (Schroeder et al., 2004; Simoes et al., 2010; Smith et al., 2014). There is little separation of the types of antibiotics used in human and animal medicine (Laxminarayan et al., 2013). In agriculture, manure and biological solids applied to land might contain both antimicrobials and resistant bacteria. Therefore, run-off from fertilized land or directly from sewage can lead to contamination of surface water and

consequently spread to human beings and animals through possible contact with soil, irrigation of crops, contact with water or with wildlife (Laxminarayan et al., 2013). Wastewater from treatment plants, hospitals and intensive farming practices are important sources of pathogenic and antimicrobial-resistant Escherichia coli being released into the environment (Baquero et al., 2008). It is also estimated that E. coli spends approximately half its life cycle in the external environment, and therefore, environments (soil, surface or ground water) contaminated with these antibiotic-resistant organisms and genes may constitute a reservoir for their dissemination (Gordon, 2001).

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© 2015 Blackwell Verlag GmbH Zoonoses and Public Health, 2015, 62, 534–542

Introduction

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Antimicrobial Resistance in Wildlife

Wild animals do not naturally come into contact with antimicrobials; however, they may be infected with antibiotic-resistant bacteria via the acquisition of resistant bacteria from human sources, agricultural facilities and associated contaminated environments (Dolejska et al., 2007). Once wild animals acquire resistant bacteria, they can serve as reservoirs, vectors and bioindicators of resistant bacterial pathogens and genetic determinants of AMR in the environment (Dolejska et al., 2007; Literak et al., 2007). Prevalence level of resistant bacteria is linked with human activity. This is evident from data by Bonnedahl et al. (2009) and Alroy and Ellis (2011), as antimicrobialresistant profiles of E. coli from herring gulls (Larus argentatus) and yellow-legged gulls (Larus michahellis) mirrored those from human wastewater in the United States and those of clinical human isolates in France (Bonnedahl et al., 2009). In most of these studies, the bird populations have relatively frequent interactions with habitat influenced by human activities, although Sjolund et al. (2008) demonstrated AMR E. coli originating from Arctic birds in a region of the world where human influence on the ecology of AMR was expected to be minimal (Sjolund et al., 2008). Ireland supports a large population of breeding seabirds, passerines and mammalian species such as deer that frequently interact with human-associated environments. According to the Irish Department of Agriculture, Food and the Marine in Ireland, 62% (4.6 million hectares) of the total land surface area (6.9 million ha) is devoted to agriculture (http://www.agriculture.gov.ie/publications/2012/), and given that Ireland has approximately 7524 km of coastline, the potential for the acquisition and transfer of antimicrobial-resistant genes between different wildlife species and environments cannot be underestimated. Wastewater treatment plants and activated sludge have been associated with the development of AMR (Merlin et al., 2011). In Ireland, most wastewater is discharged out to sea after treatment, and therefore, gulls breeding and feeding along the coast are likely to contact this wastewater (Smith et al., 2014). Additionally, in Ireland, the agricultural application of animal manure (common practice), with its high concentration of microbial biomass, is a significant route for the introduction of resistant bacteria into the terrestrial environment, and therefore, both birds and mammalian species such as deer may acquire resistant bacteria as a result of their feeding habits on pastureland. When AMR bacteria colonize wild animals, they in turn become a new environmental reservoir of antibiotic resistance and, especially in migratory birds, they serve as vectors that disperse these bacteria to new localities (Bonnedahl et al., 2009). This may pose a hazard to human and animal health by transmission of resistant strains to waterways, raw food products in fields and human-associated environments via faecal contamination (Waldenstrom et al., 2002; Cole et al., 2005).

For the isolation of E. coli, MacConkey agar No.1 was used. Samples were resuspended by vortex prior to being


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To examine the prevalence of AMR in wildlife, a number of species were selected on the basis of their spatial variation with regard to proximity to human-associated environments and their ability to act as vectors in the dispersal of resistance genes. The aims of this study were to investigate the prevalence of AMR E. coli isolates from wild birds and terrestrial mammals in the Republic of Ireland. The phylogenetic grouping of resistant E. coli isolates was determined, and AMR-encoding genes were characterized by PCR. These data are discussed in the context of public health. Materials and Methods Sample collection Sampling was carried out between the 18 May 2013 and the 28 June 2013 from seven different sites in Ireland. Bird faecal samples were collected from the following sites, Howth Harbour Co. Dublin (coordinates 53°390 05.40 North, 6°070 09.52 West), Goats Island Co. Tipperary (coordinates 52°590 5.48 North, 8°290 59.57 West), Greater Saltee Island, (coordinates 52°120 64.39 North, 6°510 70.10 West), Lough Boora Co.Offaly (coordinates 53°210 12.36 North, 7°750 43.41 West) and Edenderry Co.Offaly (coordinates 53°340 28.42 North, 7°050 24.59 West), see Fig. 1. This research was undertaken in collaboration with the Department of Arts, Heritage and the Gaeltachta in which a licence (No. C033/2013) was obtained under the National Parks and Wildlife Service and with approval from the University Collage Dublin Animal Research Ethics Committee. Deer faecal samples were collected from Sallygap Co. Wicklow (coordinates 53°150 39.54 North, 6°340 11.22 West). Depending on the host species, that is herring gulls (Larus argentatus), black-headed gulls (Larus ridibundus), lesser black-back gulls (Larus fuscus), hybrid deer species (Cervus elaphus x Cervus nippon) or starlings (Sturnus vulgaris), being sampled, bacteria were isolated from freshly deposited faeces or directly from faecal material in the cloacae of the host, as was the case for starling chicks. Faecal sample collection was carried out using a sterile swab/transport tube system containing nutrient broth. Samples were collected by swirling a sterile cotton swab into freshly deposited faecal material of host species, after which the tip of the swab was placed inside a micropipette tube, which was then placed inside a cooler box and transported to a laboratory where they were stored at 4°C for a maximum of 14 days before E. coli was isolated. Isolation of E. coli and antimicrobial susceptibility testing


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Fig. 1. The location of the sites where samples were collected.

inoculated onto MacConkey agar plates. Inoculated plates were incubated at 37.5°C for 24 h. From each suspected plate, three separate colonies were removed and biochemically tested for their indol and citrase reactions. Colonies that tested positive for indole and negative for citrate were confirmed as E. coli. Susceptibility testing by disc diffusion was performed on the confirmed E. coli isolates, and results were interpreted as recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2013). Susceptibility testing included a panel of seven different antimicrobial agents; six of which were amoxicillin-clavulanic acid (AMC), 20/10 lg; ampicillin (AMP), 10 lg; ciprofloxacin (CIP), 5 lg; penicillin G (P), 5 lg; streptomycin (S), 10 lg; and tetracycline (TET), 30 lg. Penicillin G was used for comparison purposes and to demonstrate the intrinsic resistance of E. coli. Meropenem susceptibility was assessed using the modified Hodge test (MHT) using meropenem 10 lg. The discs were purchased from Oxoid, and a quality control strain, E. coli ATCC 25922, was used to validate the routine susceptibility testing

methods. An isolate with resistance to two or more antimicrobial compounds (excluding penicillian G) was classified as multidrug resistant (IOM, 1998).

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Detection of phylogenetic groups Phylogenetic grouping of E. coli isolates in this study was determined by a previously described triplex PCR-based protocol targeting chuA, yjaA and tspE4.C2 markers (Clermont et al., 2000). Briefly, the method was developed to classify E. coli into four phylogenetic groups designated as A, B1, B2 and D based on the presence or absence of two genes (chuA and yjaA) and an anonymous DNA fragment (tspE4.C2). Total genomic DNA was extracted using a Promega Wizard genomic DNA purification kit (Madison, WI, USA) following the manufacturer’s protocol. Purified DNA served as a template and three specific primer sets were synthesized by MWG-Biotech AG, Ebersberg, Germany. Quality control strains were obtained from a previous study in our laboratory (Wang et al., 2013).

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Detection of antibiotic resistance genes Purified total genomic DNA from confirmed multidrugresistant (MDR) E. coli in this study was obtained to test for the corresponding resistance genes. Detection of antibiotic resistance genes was performed using the primers listed in Table 1. The following resistance determinants were investigated by PCR: blaTEM, blaCTX, blaNDM-1, tet(A), tet (B), tet(C) and strA. All PCRs were performed in a final volume of 25 ll consisting of 2.5 ll 10XPCR buffer (New England BioLabs, Ipswich, MA, USA), 25 pmol of each primer (MWG-Biotech AG, Ebersberg, Germany), deoxynucleoside triphosphates at a concentration of 200 lM (Promega), 1U Taq DNA polymerase (New England BioLabs) and 50 ng of genomic DNA. Plasmid profiling Plasmid DNA profiles of all MDR E. coli isolates were carried out using the S1-nuclease digestion pulsed-field gel

electrophoresis (S1-PFGE) molecular subtyping method (Wang et al., 2013). This protocol includes a lysis step of the bacterial cells embedded in agarose plugs followed by an endonuclease restriction digest using 8 u S1 nuclease for 45 min at 37°C. Finally, after casting agarose gel, plasmid samples were resolved by PFGE in a Chef Mapperâ XA System (Bio-Rad, Hercules, CA, USA) at 14°C with a switch time between 1 and 12 s, at 6 V/cm on a 120° angle in 0.5 X TBE buffer for 18 h. E. coli 39R 861 containing four reference plasmids, of molecular weights 6.9-, 36-, 63- and 147-kb was included to determine the approximate molecular mass of plasmids (Macrina et al., 1978). Results Percentages of antimicrobial resistance (AMR) phenotypes The prevalence of E. coli among host species ranged from 23% to 90%. Overall E. coli was detected in 23% (n = 30) of black-headed gulls (B), 35% (n = 26) of starlings (S), 43% (n = 30) of herring gulls (H), 83% (n = 30) of deer

Table 1. An overview of tested antimicrobials, target genes, PCR primers, product sizes and conditions

Target gene tet(A) tet(B) tet(C) blaCTX blaTEM strA blaNDM-1

Primer directiona

Sequence (50 -30 )

Tm (°C)

Amplicon size (bp)

Reference

F R F R F R F R F R F R F R

GCT ACA TCC TGC TTG CCT TC CAT AGA TCG CCG TGA AGA GG TTG GTT AGG GGC AAG TTT TG GTA ATG GGC CAA TAA CAC CG CTT GAG AGC CTT CAA CCC AG ATG GTC GTC ATC TAC CTG CC CGA TGT GCA GTA CCA GTA A TTA GTG ACC AGA ATC AGC GG TAC GAT ACG GGA GGG CTT AC TTC CTG TTT TTG CTC ACC CA CCT ATC GGT TGA TCA ATG TC GGA GAG TTT TAG GGT CCA CC GGT TTG GCG ATC TGG TTT TC CGG ATT GGC TCA TCA CGA TC

55

210

Ng et al. (2001)

55

659

Ng et al. (2001)

55

418

Ng et al. (2001)

60

585

Batchelor et al. (2005)

53

716

Belaaouaj et al. (1994)

58

250

Faldynova et al. (2003)

55

150

Poirel et al. (2011)

a

F, Forward; R, Reverse.

Table 2. Details of sourced samples, geographic locations, number of samples, number of Escherichia coli isolates, number of E. coli isolates resistant to antimicrobial compounds (excluding penicillin G) and percentage of multidrug-resistant (MDR) isolates (resistance to ≥2 antimicrobial compounds excluding penicillin G)

Wild source of samples

Geographic origin

No. of faecal samples

Black-headed gulls Starlings Herring gulls Lesser black-back gulls Deer Total

Lakelands (Goat Island and Lough Boora) Farmland (Edenderry) Coastal (Howth Harbour) Island (Great Saltee Island) Mountain terrain (Wicklow Mountains) 5

30 26 30 30 30 146


No. of E. coli isolates

No. of E. coli isolates resistant to ≥2 antimicrobial compounds (excluding Penicillin G)

10 17 18 42 28 115

1 5 1 0 1 8

Percentage of MDR E. coli isolates (%) 3.3 19.2 3.3 0 3.3 5.4

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Fig. 2. A map showing strain reference number, characterized on the basis of their plasmid profiles, strain source, phylogenetic group and target genes.

(Cervus) and 90% (n = 30) in lesser black-back gulls (LBB). A total of 115 E. coli isolates were cultured from 81 of 146 samples tested (Table 2). In total, resistance to more than one antimicrobial agent was detected in 10 isolates (5.4%) representing eight out 146 samples, these were S7 (2), S7 (3), S9 (2), S10 (2), S10 (3), H16 (2), B19 (3), LBB23 (3), Cervus 14 and Cervus 16. All isolates were resistant to penicillin G. The dominant resistance type (excluding penicillin G) was tetracycline, which was detected in all 10 isolates (5.4%, n = 146), followed by streptomycin, which was detected in seven isolates (4.7%). Two samples (1.3%, n = 146) were resistant to ampicillin, and these belonged to two different species, herring gull H16 (2) and black-headed gull B19 (3) (Fig. 2). All isolates were susceptible to meropenem, ciprofloxacin and amoxicillin. A conservative approach was utilized, and only isolates classified as resistant were further analysed for AMR-encoding genes. In the replication of sensitivity tests, all 10 isolates were retested and confirmed their original classification of resistance. A total of 5.4% (n = 146) of isolates demonstrated as an MDR phenotype, see Table 2. Occurrence of AMR genes The following genes, tet(A), tet(B), strA and blaTEM, were identified in E. coli isolates that expressed an MDR phenotype (Fig. 2). The blaTEM gene was identified in two samples that were phenotypically resistant to ampicillin, but blaCTX, blaNDM-1 and tet(C) genes were not found in any of the 10 isolates. The genes tet(A) and tet(B), associated with an active efflux system, were identified from 2 and 8 isolates, respectively. Two isolates that were positive for the blaTEM gene were also the only two isolates positive for the tet(A) gene. Seven isolates were resistant to streptomycin and all contained the strA gene (Fig. 2). Cervus 16 and LBB 23(3) were the only two isolates that were found to be positive for the tet(B) gene by PCR, encoding resistance to tetracycline.

tested for the presence of the TspE4.C2 gene. All ten isolates tested positive for the latter gene (data not shown). The Dichotomous decision tree was used to determine the phylogenetic group of all 10 E. coli isolates using the results from the amplification of the chuA and yjaA genes along with the DNA fragment TspE4.C2 (Clermont et al., 2000). All ten isolates were confirmed as belonging to group B1, indicating that they were commensal organisms (Fig. 2). Plasmid profiling Ten isolates were screened for the presence of plasmids using (S1-PFGE). Two plasmids were detected in each isolate, and 8 isolates carried plasmids ranging in size from approximately 40 to 80 kb. S1 nuclease-mediated plasmid analysis revealed that two isolates contained detectable large plasmids ranging in size from 80 to 150 kb. Plasmid profiles of isolates of the same species were homogeneous in nature, as determined by conventional agarose gel electrophoresis with each isolate having a corresponding profile (data not shown). Bacterial isolates from two separate deer samples and a lesser black-back gull had uniformly similar plasmid profiles ranging in size from approximately 40 to 80 kb (Fig. 2). Discussion

All resistant E. coli isolates were tested for the presence of the chuA gene, and those that were negative were further

This study clearly emphasizes that AMR is no longer an issue confined to food-producing animals and humans in Ireland, but is instead a wider environmental issue of public health concern. Although previous studies have demonstrated AMR in wild species (Stedt et al., 2014), the current study examined a broader range of species and probed further into the genomics of the resistant isolates. Plasmidmediated transmission is the most common mechanism of horizontal gene transfer (HGT) and is an almost universal procedure for gene spread among bacteria (Davies and Davies, 2010). In this study, large (>30 kb) plasmids were identified in all samples that exhibited multidrug-resistant phenotypes. Interestingly, uniformly similar plasmid profiles ranging in size from approximately 40 to 80 kb were identified in two divergently related species, hybrid deer

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Phylogenetic group

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and lesser black-back gulls. A number of multidrug-resistant phenotypes have been associated with large transferable plasmids, on which may exist other mobile genetic elements. For example, class 1 integrons frequently found located on plasmids in E. coli, contain several antimicrobial-resistant gene cassettes and so can coselect for resistance to a range of different antimicrobials (Schroeder et al., 2004). The uniformity of plasmid profiles found in two divergently different species in this study demonstrates the versatility and intracellular adaptability of plasmids. Moreover, it demonstrates that no strong distinction exists between plasmids derived from bacteria of species that are unrelated either evolutionarily or ecologically (Kruse and Sorum, 1994). Interpretations drawn from plasmid profiling studies should always be taken with a degree of caution. Without a more detailed subsequent characterization, it may be difficult to determine the true nature of these structures. Nevertheless, this approach while valid needs to be done with a knowledge of its limitations. The rapid movement of antimicrobial resistance genes between taxonomically divergent commensal and pathogenic bacterial strains may pose a threat to food-producing animals sharing similar environments with wildlife. This is concerning, considering that the resistance phenotypes observed most frequently in E. coli recovered from retail meats and in food-producing animals in Ireland have exhibited resistance to ampicillin, streptomycin and tetracycline (Karczmarczyk et al., 2011), the most prevalent resistance phenotypes found in this study. Schroeder et al. (2004) demonstrated that antimicrobial resistance E. coli cultured from food-producing animals entered and survived the human alimentary tract via food consumption. Antimicrobial resistance E. coli was also traced from the gut contents of food-producing animals at slaughter and ultimately shown to colonize the gut of human volunteers handling and eating the meat (Schroeder et al., 2004). The threat then exists that bacteria found in food-producing animals may acquire resistance determinants from wildlife and vice versa and thus enter the human food chain. This ability to share resistance determinants creates a dangerous scenario where commensal E. coli strains may harbour antimicrobial resistance determinants, act as reservoirs of resistance, and at a later stage pass these resistance traits onto pathogenic bacteria (Bonnedahl et al., 2009). The impact on human health resides in the possibility that once the resistance genes have mobilized to human pathogenic or commensal bacteria, the genes will naturally amplify and be transmitted subsequently by horizontal transfer between bacteria of the same and/or different species (Bonnedahl et al., 2009). These new antibiotic resistance bacteria can then be released back into the environment through wastewater or through agricultural application of manure. A typical E. coli spends on average,

half its life cycle in the external environment (Gordon, 2001). Once present in the environment, the resistant bacteria can then be potentially acquired by wild animals and then reintroduced to humans, for example through contamination of agricultural fields and drinking supplies (Bonnedahl et al., 2009). Therefore, the potential of plasmid transfer between strains of the same species or between different bacterial species or genera creates an environmental reservoir of resistance with potentially far reaching impacts for human health (Tenover, 2006). The environmental route by which AMR is transmitted between human beings and animals is often the least explored. Allen et al. (2010) and Dolejska et al. (2007) argue that gulls nesting near waste or agriculture water harbour more AMR bacteria than gulls associated with unpolluted water and that proximity to human activity increases the prevalence of AMR found in wild birds. This coincides with the results of this study, as resistance to more than four antimicrobials was found in a black-headed gull isolate B19 (3), nesting on Goats Island on Lough Derg, a small island surrounded by agricultural land. The land application of animal manure and biological solids is a common practice in this area, and black-headed gulls are frequent visitors to freshly manured fields, often congregating in large flocks. The omnivorous-feeding pattern of blackheaded gulls can predispose them to contamination with antimicrobial resistance E. coli of agricultural origins (Literak et al., 2007; Chee-Sanford et al., 2009). However, the observed resistance to tetracycline noted in a lesser blackback gull E. coli isolate is surprising considering that the gull colony sampled resides in the Saltee islands, an uninhabited landmass off the southeast coast of Ireland. Lesser black-back gulls do winter in Ireland; however, a large proportion of the population that breed here are summer migrants that winter as far south as north-west Africa (Wernham et al., 2002). Due to their migratory nature, the lesser black-back gull may have acquired AMR E. coli during wintering or stops at lower latitudes before migrating to the Saltees, providing a potential explanation for the introduction of AMR onto the island. This study highlights the unique nature of bacterial adaptation and the complexity for dissemination of AMR-encoding genes and the ability of birds to act as long distance vectors of AMR bacteria (Sjolund et al., 2008). In this study, AMR was found in all four-bird species with the highest prevalence in starlings (11.5%, n = 26). The predominant resistant phenotypes in starling isolates in this study were tetracycline and streptomycin. In a similar study, approximately half of all E. coli cultured from starlings showed similar resistant phenotypes (Gaukler et al., 2009). Starlings harbour a range of pathogens, including some that are classified as zoonoses, such as E. coli O157:H7, avian salmonellosis and chlamydiosis (Linz


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et al., 2007). In the United States, starlings have been implicated in spreading infectious diseases that cause illness in both humans and livestock, costing an approximate $800 million in health treatment costs annually (Linz et al., 2007). In Ireland, starling numbers are boosted by those arriving from continental Europe in winter (Crowe et al., 2014), forming large flocks with roosts of up to 50 000– 100 000 (Gaukler et al., 2009). These flocks can travel long distances in a single day at speeds of 60 to 80 km/h and frequently congregate on pastures and agricultural feedlots, especially in winter when food is scarce (Gaukler et al., 2008). The feeding and behavioural ecology of starlings highlights their recognized and important ecological role in the transmission of pathogenic microorganisms to humans and livestock. These could also be considered as sentinel species with a role in the dissemination of resistance (Gaukler et al., 2008). All isolates in this study belonged to phylogenetic group B1, a commensal E. coli group. Gordon and Cowling (2003) reported that in birds, B1 strains predominate and that most of those responsible for intestinal E. coli infections in mammals are derived from strains related to groups A and B1. Furthermore, commensal E. coli strains can cause extraintestinal disease when predisposing factors for infection are present (Karczmarczyk et al., 2011). Two deer samples in this study expressed a MDR phenotype and these were shown to contain both strA and tet(B) resistance genes. However, considering that deer can move up to 80 km and regularly feed on agricultural land bordering forested areas in Ireland, these animals have the potential to act as a reservoir and a source for the dissemination of resistance genes back onto agricultural land (Yokoyama et al., 2000). For each antimicrobial resistance gene, there exist several genetic variants, which potentially can confer resistance. The blaTEM gene, which codes for a ubiquitous enzyme frequently associated with therapeutic failure, was among one of the resistance genes found in the isolates H16(2) and B19(3) (Karczmarczyk et al., 2011). While the blaTEM gene found in this study provides resistance only to b-lactam antibiotics, derivatives from these original genes (e.g. blaTEM-3 to blaTEM-166) confer resistance to extended-spectrum cephalosporin antibiotics, often referred to as ESBL (Alroy and Ellis, 2011). In Ireland, blaCTX-M-mediated ESBL resistance is widespread, as demonstrated by a nationwide survey of Enterobacteriaceae isolates recovered from human clinical cases over an 11-year period (Karczmarczyk et al., 2011). However, all resistant E. coli isolates in this study tested negative for the blaCTX-M gene. Considering the global distribution of the blaCTX-M gene, this observation may correlate with the low rate of AMR (5.4%, n = 146) found in wildlife in Ireland, as reported in this study. However, the blaCTX-M gene has been recovered from gulls in the

Iberian Peninsula; therefore, lesser black-back gulls migrating from this area to Ireland during the summer breeding season have the potential to spread this resistant gene to Irish wildlife (Poeta et al., 2008; Bonnedahl et al., 2009; Simoes et al., 2010). Similarly, the blaNDM-1 gene was not identified in any isolates in this study. The global spread of the New Delhi metallo-beta-lactamase encoding genes and its association with environments such as drinking water and wastewater necessitates that this situation be continuously monitored (Bonnin et al., 2012; Devaraja et al., 2012). In conclusion, this study supported our earlier reported findings that wildlife can serve as reservoirs, vectors and sentinels of AMR in the environment (e.g. Smith et al., 2014; Stedt et al., 2014). Such findings are important, considering that in the last two decades, 75% of emerging human diseases originated in wildlife (Taylor et al., 2001) and that antibiotic treatment is our primary, and in many cases our only method of treatment for infectious diseases (Allen et al., 2010). This study indicates that more detailed studies of environmental reservoirs of AMR are crucial in our fight against infectious diseases, and implementation of appropriate strategies using collective actions to avert the increased morbidity and mortality from AMR.

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Acknowledgements This work derives from M.Sc. studies in Wildlife Conservation and Management at University College Dublin. The authors wish to thank Alex Copland, Declan Manley and Alyn Walsh for their assistance in obtaining samples. The authors also wish to thank the Neale family for their support and encouragement during the study as well as the comments from two anonymous reviewers who improved the manuscript. Assistance from Bernard Kaye was greatly appreciated. Funding for this research project was through University College Dublin Seed Funding 2012- Horizon Scanning and UCD School of Agriculture and Food Science Research and Innovation Committee start up grant 2013. References Allen, H.K., J. Donato, H.H. Wang, K.A. Cloud-Hansen, J. Davies, and J. Handelsman, 2010: Call of the wild: antibiotic resistance genes in natural environments. Nat. Rev. Microbiol. 8, 251–259. Alroy, K., and J.C. Ellis, 2011: Pilot study of antimicrobial-resistant Escherichia coli in herring gulls (Larus argentatus) and wastewater in the Northeastern United States. J. Zoo. Wildl. Med. 42, 160–163. Baquero, F., J.L. Martinez, and R. Canton, 2008: Antibiotics and antibiotic resistance in water environments. Curr. Opin. Biotechnol. 19, 260–265.

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