Science of the Total Environment 408 (2010) 4871–4876

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Molecular characterization of antimicrobial resistance in enterococci and Escherichiacoli isolates from European wild rabbit (Oryctolagus cuniculus)

Nuno Silva a, Gilberto Igrejas c,d, Nicholas Figueiredo a,b,c,d, Alexandre Gonçalves a,b,c,d, Hajer Radhouani a,b,c,d,Jorge Rodrigues a,b, Patrícia Poeta a,b,⁎a Centre of Studies of Animal and Veterinary Sciences, Vila Real, Portugalb Veterinary Science Department, University of Trás-os-Montes and Alto Douro, Vila Real, Portugalc Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugald Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal

⁎ Corresponding author. Department of Veterinary SMontes and Alto Douro, Vila Real, Portugal. Tel.: +259350629.

E-mail address: ppoeta@utad.pt (P. Poeta).

0048-9697/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.scitotenv.2010.06.046

a b s t r a c t

a r t i c l e i n f o

Article history:Received 30 April 2010Received in revised form 16 June 2010Accepted 18 June 2010Available online 10 July 2010

Keywords:Antimicrobial resistanceEscherichia coliEnterococciEuropean rabbits

A total of 44 Escherichia coli and 64 enterococci recovered from 77 intestinal samples of wild Europeanrabbits in Portugal were analyzed for resistance to antimicrobial agents. Resistance in E. coli isolates wasobserved for ampicillin, tetracycline, sulfamethoxazole/trimethoprim, streptomycin, gentamicin, tobramy-cin, nalidixic acid, ciprofloxacin and chloramphenicol. None of the E. coli isolates produced extended-spectrum beta-lactamases (ESBLs). The blaTEM, aadA, aac(3)-II, tet(A) and/or tet(B), and the catA genes weredemonstrated in all ampicillin, streptomycin, gentamicin, tetracycline, and chloramphenicol-resistantisolates respectively, and the sul1 and/or sul2 and/or sul3 genes in 4 of 5 sulfamethoxazole/trimethoprimresistant isolates. Of the enterococcal isolates, Enterococcus faecalis was the most prevalent detected species(39 isolates), followed by E. faecium (21 isolates) and E. hirae (4 isolates). More than one-fourth (29.7%) ofthe isolates were resistant to tetracycline; 20.3% were resistant to erythromycin, 14.1% were resistant tociprofloxacin and 10.9% were resistant to high-level-kanamycin. Lower level of resistance (b10%) wasdetected for ampicillin, quinupristin/dalfopristin and high-level-gentamicin, -streptomycin. No vancomycin-resistance was detected in the enterococci isolates. Resistance genes detected included aac(6′)-aph(2″), ant(6)-Ia, tet(M) and/or tet(L) in all gentamicin, streptomycin and tetracycline-resistant isolates respectively.The aph(3′)-IIIa gene was detected in 6 of 7 kanamycin-resistant isolates, the erm(B) gene in 11 of 13erythromycin-resistant isolates and the vat(D) gene in the quinupristin/dalfopristin-resistant E. faeciumisolate. This survey showed that faecal bacteria such as E. coli and enterococci of wild rabbits could be areservoir of antimicrobial resistance genes.

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1. Introduction

The selective pressure caused by intensive use of antimicrobialdrugs in human and veterinary medicine, livestock, aquaculture,agriculture and food technology, associated with several mechan-isms for bacteria genetic transfer, could have contributed to theemergence and spread of resistance in different bacteria groups(Barbosa and Levy, 2000; Coque et al., 2008; Werner et al., 2008).Antimicrobial agents exert a selection pressure not only onpathogenic, but also on commensal bacteria of the intestinal tractof humans and animals. Resistant commensal bacteria constitute areservoir of resistance genes for facultative and obligate pathogenic

bacteria (Guardabassi et al., 2004; van den Bogaard and Stobberingh,2000).

Escherichia coli and Enterococcus spp. colonize the gastrointestinaltract of many animals and are also commonly found in soil, plants andwater. These microorganisms are considered important as “indicatorbacteria” that could be used to track the evolution of antibioticresistance in different ecosystems (van den Bogaard and Stobberingh,2000). Furthermore, they have also emerged as important causes ofnosocomial and community-acquired infections (Murray, 1998; Pater-son and Bonomo, 2005). Although these bacteria generally do not causedisease, they might act as reservoir of antimicrobial resistance genesthat could be transmitted to other pathogenic bacteria and for thisreason might represent a worldwide problem with large repercussionsin public health. Furthermore, studies in antimicrobial resistance ofintestinal enterococci and E. coli isolates in wild animals are scarce andgenerally restricted to the analysis of vancomycin-resistant enterococciand extended-spectrum β-lactamases (ESBL) containing E. coli (Literaket al., 2009; Mallon et al., 2002; Poeta et al., 2005a; Poeta et al., 2009).

4872 N. Silva et al. / Science of the Total Environment 408 (2010) 4871–4876

The European wild rabbit (Oryctolagus cuniculus), an endemicspecies from the Iberian Peninsula, is one of the key species in theIberian ecosystems, since it is an important prey for predators,including the Iberian lynx (Lynx pardinus) and the imperial eagle(Aquila adalberti) (Delibes-Mateos et al., 2008). In addition, wild rabbithas a great economic value, since it is an important game species insport hunting (Garcia-Bocanegra et al., 2010), and in contrary of othergame species, the wild rabbit is part of the human food chain.

The aim of the present study was to analyze the prevalence ofantimicrobial resistance and the mechanisms implicated in faecalE. coli isolates and enterococci of wild European rabbits in Portugal.

2. Material and methods

2.1. Faecal samples and bacterial isolates

Antimicrobial resistance in E. coli isolates and enterococci wasstudied in 77 faecal samples recovered from wild European rabbits(one sample per animal). Samples were collected from October 2007to February 2008 in the North of Portugal during rabbit huntingseason organized by different corporations of hunters. This hunting issupervised by the Agriculture, Rural development and FisheryMinistry of Portugal under the Decree-Law no. 202/2004. Briefly,the faecal samples were recovered from the intestinal tract of thehunted rabbits, with the aid of sterile gloves, and transported to thelab in Carry–Blair medium where they were processed.

For E. coli isolation, faecal samples were seeded in Levine agarplates and incubated 24 h at 37 °C. One colony per sample with typicalE. coli morphology was selected and identified by classical biochem-ical methods (Gram-staining, catalase, oxidase, indol, Methyl-Red-Voges-Proskauer, citrate and urease), and by the API 20E system(BioMérieux, La Balme Les Grottes, France).

For enterococcal isolation, faecal samples were diluted andsampled in Slanetz–Bartley agar plates and were incubated for 48 hat 37 °C. Colonies with typical enterococcal morphology (one persample) were identified by cultural characteristics, Gram-staining,catalase test, bile-aesculin reaction and by biochemical tests using theAPI ID20 Strep system (BioMérieux, La Balme Les Grottes, France).Furthermore, enterococci were identified to the species level by PCRusing primers and conditions for the different enterococcal species(Torres et al., 2003).

2.2. Antimicrobial susceptibility test

Antibiotic susceptibilitywas tested by the agardiskdiffusionmethodas recommended by the Clinical and Laboratory Standards Institute(CLSI) (CLSI, 2008). The susceptibility of the E. coli isolateswas tested for16 antibiotics: [ampicillin (10 μg), amoxicillin+clavulanic acid (30 μg),cefoxitin (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), aztreonam(30 μg), imipenem (10 μg), gentamicin (10 μg), amikacin (30 μg),tobramycin (10 μg), streptomycin (10 μg), nalidixic acid (30 μg),ciprofloxacin (5 μg), sulfamethoxazole-trimethoprim (25 µg), tetracy-cline (30 μg), andchloramphenicol (30 μg)].E. coliATCC25922wasusedas a quality control strain. Additionally, screening test for detection ofESBLs was carried out by the double disk diffusion test (Bradford, 2001;CLSI, 2008).

On the other hand, the susceptibility of the enterococcal isolateswas tested for 11 antibiotics: [vancomycin (30 μg), teicoplanin(30 μg), Ampicillin (10 μg), streptomycin (300 μg), gentamicin(120 μg), kanamycin (120 μg), chloramphenicol (30 μg), tetracycline(30 μg), erythromycin (15 μg), quinupristin-dalfopristin (15 μg), andciprofloxacin (5 μg)] by the disk diffusion method (CLSI, 2008). Onlythe category of high-level resistance (HLR) was considered forstreptomycin, gentamicin and kanamycin. E. faecalis strain ATCC29212 and Staphylococcus aureus strain ATCC 25923 were used asquality controls.

2.3. Characterization of resistance genes

E. coli isolates with resistance to one or more antibiotics wereselected for the characterization of antibiotic-resistance genes. Thepresence of tet(A), and tet(B) genes were studied by PCR for thetetracycline-resistant isolates. The following genes were also studiedby PCR: blaTEM (in ampicillin-resistant isolates), aac(3)-II (ingentamicin-resistant isolates), aadA (in streptomycin-resistant iso-lates), catA (in chloramphenicol-resistant isolates) and sul1, sul2 andsul3 (in SXT-resistant isolates) (Saenz et al., 2004).

The quinolone resistance-determining region (QRDR) of the gyrAgene, as well as the analogous region of the parC gene, was amplifiedby PCR in all quinolone-resistant E. coli isolates (Saenz et al., 2003).Amplified fragments were purified (Qiagen), and both strands wereautomatically sequenced by the Applied Biosystem 3730 sequencer(Genome Express, France), using the same set of primers as for thePCR reactions. Sequences obtained were compared with thosepreviously reported for gyrA (GenBank accession number X06373)and parC genes (M58408 with the modification included in L22025).

On the other hand, resistant enterococci isolates were tested by PCRfordetectionof the followingresistancegenes: erm(B) (inerythromycin-resistant isolates), tet(M) and tet(L) (in tetracycline-resistant isolates),aph(3′)-IIIa (in kanamycin-resistant isolates), aac(6′)-aph(2″) (ingentamicin-resistant isolates), ant(6)-Ia (in streptomycin-resistantisolates), vat(D) and vat(E) (in quinupristin/dalfopristin-resistantisolates), using primers and conditions previously reported (Aarestrupet al., 2000; Leener et al., 2005; Torres et al., 2003). Furthermore, specificPCR assays for detection of tdnX and int genes were also used in tet(M)-positive isolates, to demonstrate the presence of the Tn5397-like andTn916/Tn1545-like transposons, respectively (Agerso et al., 2006).

Positive and negative controls were used in all PCRs, from thestrain collection of the University of Trás-os-Montes and Alto Douro(Portugal). DNA sequencingwas used to verify the identity of the geneproducts of at least one isolate randomly selected for each gene.

3. Results

3.1. Bacteria isolation

E. coli isolates were detected in 44 of the 77 wild European rabbitfaecal samples (57.1%), while enterococci were obtained from 64(83.1%) of the samples analyzed. E. faecalis was the most prevalentdetected species (39 isolates), followed by E. faecium (21 isolates) andE. hirae (4 isolates). The distribution of E. coli and enterococci isolatesfrom European wild rabbits are shown in Fig. 1. The majority of theisolates were recovered in the Vila Real and Montalegre regions.

3.2. Resistance among E. coli

The antimicrobial resistance detected in the E. coli isolates isshown in Table 1. Antibiotic resistance was detected in six of the totalE. coli isolates. Ampicillin, sulfamethoxazole/trimethoprim and tetra-cycline resistance was observed in five E. coli isolates, streptomycinresistance in three isolates, while gentamicin, tobramycin, nalidixicacid, ciprofloxacin and chloramphenicol resistance was found in oneE. coli isolate. No resistances were found to other antimicrobial agents.

The resistance phenotypes detected among the 64 E. coli isolatesare shown in Table 2. It is interesting to underline that four of the sixE. coli-resistant isolates were resistant to 3 or more antibiotic classestested.

Table 3 shows the presence of antibiotic resistance genes in E. coliisolates in relation to their specific phenotype of resistance. TheblaTEM gene was identified by PCR in all ampicillin-resistant isolates.The aac(3)-II gene was detected in the E. coli isolate with resistance togentamicin, and the aadA gene was found in the three streptomycin-resistant isolates. In addition, the tet(A) or tet(B) gene, associatedwith

Fig. 1.Distribution of 44 E. coli and 64 enterococci isolates from Europeanwild rabbits in the north of Portugal, and location of antimicrobial resistant isolates. Black open cycles, E. coliisolates without antibiotic resistance; Black filled cycles, E. coli isolates resistant to at least one antibiotic; Black open boxes, Enterococcus spp. isolates without antibiotic resistance;Black filled boxes, Enterococcus spp. isolates resistant to at least one antibiotic.

4873N. Silva et al. / Science of the Total Environment 408 (2010) 4871–4876

an active efflux system, was identified in all five tetracycline-resistantisolates, and both genes were detected in two of them.

The sul1 and sul2 or sul3 genes were detected in four of the E. coliisolates with sulfamethoxazole/trimethoprim resistance. It is ofinterest to remark that three isolates harbored simultaneously thesul1 and sul2 genes. In addition, the cmlA gene was identified in thechloramphenicol-resistant isolate.

One E. coli isolate showed resistance to ciprofloxacin and nalidixicacid, and the mutations in gyrA and parC genes were analysed to

Table 1Distribution of antibiotic resistance in E. coli and Enterococcus spp. isolated from faecalsamples of wild rabbits in Portugal.

Antimicrobial agent Number ofE. coli isolates(n=44)

Percentages (%) and number of enterococciisolates distributed by species

E. faecalis(n=39)

E. faecium(n=21)

E. hirae(n=4)

Total(n=64)

Ampicillin 5 0 9.5 (2) 0 3.1 (2)Amoxicillin/clavulanic acid

0 NT NT NT NT

Cefoxitin 0 NT NT NT NTCefotaxime 0 NT NT NT NTCeftazidime 0 NT NT NT NTAztreonam 0 NT NT NT NTImipenem 0 NT NT NT NTGentamicin 1 10.3 (4) 0 0 6.3 (4)Amikacin 0 NT NT NT NTTobramycin 1 NT NT NT NTStreptomycin 3 2.6 (1) 9.5 (2) 0 4.7 (3)Nalidixic acid 1 NT NT NT NTCiprofloxacin 1 7.7 (3) 28.6 (6) 0 14.1 (9)Sulfamethoxazole/trimethoprim

5 NT NT NT NT

Tetracycline 5 23.1 (9) 28.6 (6) 100.0 (4) 29.7 (19)Chloramphenicol 1 0 0 0 0Vancomycin NT 0 0 0 0Teicoplanin NT 0 0 0 0Kanamycin NT 10.2 (4) 14.2 (3) 0 10.9 (7)Erythromycin NT 23.1 (9) 19.0 (4) 0 20.3 (13)Quinupristin/dalfopristina

NT – 4.8 (1) 0 1.6 (1)

Susceptibles toall antibiotics

38 66.7 (26) 38.1 (8) 0.0 (0) 53.1 (34)

NT, not tested.a Susceptibility for this drug combination was not tested in E. faecalis isolates.

determine the mechanism of quinolone resistance. Two amino acidchanges in GyrA protein (Ser83Leu+Asp87Asn) and one change inParC protein (Ser80Ile) were identified in this isolate.

3.3. Resistance among Enterococcus spp.

Table 1 shows the distribution of enterococcal species isolates andthe percentages of antibiotic resistance. Antibiotic resistance wasdetected in about half of the enterococci isolates (46.9%).

Table 2Resistance phenotypes of E. coli and enterococci isolates obtained from faecal samplesof wild rabbits in Portugal.

Bacteria Resistance phenotypea Number of isolates

E. coli AMP-CHL 1TET-SXT 1AMP-TET-SXT 1AMP-STR-TET-SXT 2AMP-GEN-TOB-STR-TET-CIP-NAL-SXT 1Susceptible to all tested antibiotics 38

Enterococcal speciesE. faecalis TET-QD 1

ERY-QD 1CIP-QD 3TET-ERY-QD 4TET-ERY-GEN-KAN-QD 3TET-ERY-GEN-KAN-STR-QD 1Susceptible to all tested antibiotics 26

E. faecium TET 1ERY 1AMP 1CIP 3KAN 1QD 1TET-CIP 2TET-ERY-KAN-STR 2TET-CIP-AMP-ERY 1Susceptible to all tested antibiotics 8

E. hirae TET 4Susceptible to all tested antibiotics 0

a QD, quinupristin/dalfopristin; TET, tetracycline; CIP, ciprofloxacin; ERY, erythro-mycin; STR, streptomycin; GEN, gentamicin; AMP, ampicillin; KAN, kanamycin; SXT,Sulfamethoxazole/trimethoprim, TOB, tobramycin; NAL, nalidixic acid; CHL,chloramphenicol.

Table 3Resistance genes detected in antibiotic resistant E. coli and enterococci isolates obtainedfrom faecal samples of wild rabbits in Portugal.

Bacteria Phenotype ofresistance

Numberofisolates

Genes detected by PCR

Resistance genesand geneticelements analyzed

Number ofisolates

E. coli Ampicillin 5 blaTEM 5Gentamicin 1 aac(3)-II 1Tobramycin 1 – –

Streptomycin 3 aadA 3Sulfamethoxazole/trimethoprim

5 sul1+ sul2 3sul3 1

Tetracycline 5 tet(A) 5tet(A)+ tet(B 2

Chloramphenicol 1 cmlA 1

Enterococcal species

E. faecalis Gentamicin 4 aac(6′)-aph(2″) 4Streptomycin 1 ant(6)-Ia 1Ciprofloxacin 3 – –

Tetracycline 9 tet(M) 8tet(L), 9tet(M)+ tet(L) 8tet(M)+ tet(L)+Tn916

6

Kanamycin 4 aph(3′)-IIIa 3Erythromycin 9 erm(B) 7

E. faecium Ampicillin 2 – –

Streptomycin 2 ant(6)-Ia 2Ciprofloxacin 6 – –

Tetracycline 6 tet(M)+ tet(L) 6tet(M)+ tet(L)+Tn916

1

Kanamycin 3 aph(3′)-IIIa 3Erythromycin 4 erm(B) 4Quinupristin/Dalfopristin

1 vat(D) 1

E. hirae Tetracycline 4 tet(M)+tet(L)+Tn916

4

4874 N. Silva et al. / Science of the Total Environment 408 (2010) 4871–4876

Higher level of resistance was observed for tetracycline (29.7%),erythromycin (20.3%) and ciprofloxacin (14.1%). Lower level ofresistance was found to other antimicrobial agents (b10%). Nochloramphenicol, vancomycin or teicoplanin-resistance was demon-strated in our enterococcal isolates. Low percentages of HLR forgentamicin or streptomycin (HLR-G or HLR-S, respectively) weredetected in our enterococci (6.3 and 4.7%, respectively), and relativehigher percentage was found for kanamycin (HLR-K) (10.9%). It isinteresting to refer that none of our enterococci showed glycopeptidesresistance.

Table 2 shows the antimicrobial resistance phenotypes detected inthe series of 64 enterococci isolates. More than half of the E. faecalisisolates (66.7%) were susceptible to all tested antibiotics, but onlyeight E. faecium isolates (38.1%) were susceptible to all testedantimicrobial agents. Moreover, all the four E. hirae were resistantto tetracycline. Furthermore, seven of the enterococci isolates weremultiresistant to the tested antibiotics.

The presence of antibiotic resistance genes was studied by PCRin all resistant enterococci (Table 3). All the erythromycin-resistantenterococcal isolates harbored the erm(B) gene, with the exceptionof two E. faecalis in which no macrolide resistance genes weredetected.

The combination tet(M)+ tet(L) genes were found in all ourtetracycline-resistant isolates, with the exception of one E. faecalisisolate which did not contain the tet(M) gene. Moreover, 75% of ourtet(M)-positive enterococci carried specific genes of Tn916 transposon.

The vat(D), associated with streptogramin A resistance, wasdetected in the quinupristin/dalfopristin-resistant E. faecium. Fur-

thermore, the enterococcal isolates showing HLR-G and HLR-Scontained the aac(6′)-aph(2″) and ant(6)-Ia genes, respectively. Inaddition, the aph(3′)-IIIa gene was found in 3 of 4 HLR-K E. faecalisisolates.

4. Discussion

4.1. Bacteria isolation

The low percentage of E. coli isolates recovered from the 77 faecalsamples of European wild rabbits, may be due to the fact that E. coli isa Gram-negative bacterium and thus, in general, more sensitive to theenvironment conditions than Gram-positive bacteria (Wan et al.,2009). On the other hand, the detection of E. faecalis and E. faecium asthe predominant enterococcal species in the faecal samples of wildrabbits shows strong similarities with data previously reported forfaecal enterococci of farmer's rabbits (Linaje et al., 2004). Further-more, E. faecalis (52.1%) and E. faecium (32.1%) have been reported asthe predominant species in the faecal samples in other wild animalsobtained in different Natural Parks of the north and center of Portugal(Poeta et al., 2005b).

4.2. Resistance among E. coli

The low frequency of resistance found in our E. coli isolatescontrast with higher levels of antibiotic resistance described previ-ously in E. coli isolates from wild animals (Costa et al., 2008), where71.4% of the isolates demonstrated resistance to one or moreantibiotics, and in food-producing animals, where higher levels ofnalidixic acid resistance (88%), and tetracycline resistance (95.6%)were found in E. coli isolated from broilers and pigs, respectively(Saenz et al., 2001; Teshager et al., 2000). This could be explained bythe fact wild rabbits are herbivores, adapting its diet according toplant items availability (Alves et al., 2006). Furthermore, contrary tothe food-producing animals, where there is an extensive use ofantibiotics (Mathew et al., 2007), the wild rabbits were not subjectedto the selective pressure associated with the use of antimicrobialdrugs. In addition, none of the E. coli isolates were shown to produceESBLs using the double disk diffusion test.

In our study the blaTEM gene was detected in all the ampicillin-resistant E. coli. The TEM β-lactamase is the most often mechanism ofampicillin resistance in E. coli, and itwaspreviously shown in ampicillin-resistant E. coli isolates from different origins (Brinas et al., 2002).

Four classes of AAC(3) acetyltransferases have been reportedassociated with gentamicin resistance in E. coli (Costa et al., 2008). Inour gentamicin-resistant isolate, the gene aac(3)-II was detected. Thismechanism of resistance has been also indentified in a gentamicin-resistant E. coli recovered from animals (Boerlin et al., 2005; Saenz etal., 2004). In general, with the exception of streptomycin, E. colishowed lower percentages of resistant to aminoglycosides. As referredby others (Barreto et al., 2009; Jouini et al., 2009), the aadA gene wasdetected in all the E. coli isolates with streptomycin-resistance.

Thedetectionof tet(A) or tet(B) genes in all our tetracycline-resistantisolates indicates that themainmechanism of tetracycline resistance inwild rabbits E. coli isolates is by active efflux. To date, fourteen differenttet genes encoding for efflux proteins have been studied in Gram-negative bacteria from different animals and humans origins, beingtet(A) and tet(B) reported tobemore associated in tetracycline resistance(Bryan et al., 2004). In addition, the sul1 and/or sul2 and/or sul3 geneswere detected in all of the 21 sulfamethoxazole/trimethoprim-resistantE. coli isolates obtained from wild animals from different Natural Parksof the north and center of Portugal (Costa et al., 2008).

The resistance to nalidixic acid and ciprofloxacin has been relatedwith the presence of amino acid changes in GyrA and ParC proteins inboth human and animal E. coli isolates (Saenz et al., 2003; Sayah et al.,2005).

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4.3. Resistance among Enterococcus spp.

In general, and with few exceptions, E. faecalis and E. faeciumspecies showed similar percentages of antimicrobial resistance,mainly associated with streptomycin, tetracycline, kanamycin anderythromycin, while E. hirae shows resistance only to tetracycline.Ciprofloxacin resistance was more associated with the E. faecium andsimilar results were described in other study of our group inenterococcal isolates recovered from 67 faecal samples of wildboars, collected from December 2005 to February 2006 in North ofPortugal during the wild boar hunting season (Poeta et al., 2007).

None of our enterococci showed glycopeptides resistance. How-ever, vancomycin-resistant enterococcus (VRE) were detected ineleven enterococci isolates recovered from faecal samples of wildrabbits in a previous study carried out by our group usingvancomycin-supplemented agar plates (4 mg/L) for enterococciisolation (Figueiredo et al., 2009). This fact could indicate that VREmight be present within the faecal enterococcal population of wildanimals but in a lower proportion in respect to the vancomycin-susceptible ones, and could not be detectedwhen non-supplemented-plates were used for bacterial isolation.

Eleven of the total 13 erythromycin- resistant, showed the presenceof the erm(B) gene. Our results are similar to those previously detectedin enterococci isolates from animal and human (Jensen et al., 1999).Nevertheless, other mechanisms of resistance can be present in thoseisolates in which no tested macrolide resistant genes were found.

The tet(M) and tet(L) genes are frequently reported to be asso-ciated with tetracycline resistance in enterococci isolates (Aarestrupet al., 2000; del Campo et al., 2003), and in our study at least one ofthese genes was found in all our tetracycline-resistant isolates.Moreover, the tet(M) gene is usually associated with conjugativetransposons related to the Tn916 family (Rice, 1998), and in this study75% of our tet(M)-positive enterococci carried specific genes of Tn916transposon, although the association of tet(M) gene with thistransposon was not analysed.

Previous reports have described the higher frequency of vat(E)that the vat(D) in quinupristin/dalfopristin resistance in E. faeciumisolates (Jensen et al., 2002; Werner et al., 2000). However, in ourstudy only the vat(D) gene was detected in the quinupristin/dalfopristin-resistant E. faecium isolate. In addition, the aac(6′)-aph(2″), ant(6)-Ia and aph(3′)-IIIa genes detected in our high-levelaminoglycoside-resistant enterococci isolates, were also found in aprevious report among HLR-G, HLR-S and HLR-K enterococci isolatesfrom healthy humans, pets and poultry in Portugal (Poeta et al., 2006).

5. Conclusion

Few studies are available in the literature about the susceptibilityto antibiotics in E. coli and Enterococcus spp. of healthy wild animalsand in particular in wild rabbits. In the present study, we show that E.coli and enterococci of the intestinal tract of wild European rabbits canconstitute a reservoir of antimicrobial resistant genes, and that playsan important role in the spread of antimicrobial resistance into theenvironment. However, the resistance is in general lower than thepreviously reported for food-producing animals and could reflect alow antimicrobial pressure in this type of animals. Nevertheless, thewild rabbits are an important food source for many animals, andunlike other game species, they are used in human diet, making this apotential transmitter of genes or resistant bacteria to other animals oreven to humans. Therefore, the possible transmission of theseresistant bacteria to humans as well as the fact that the antibioticresistances found in this study belonging to the same classes ofantimicrobial agents used in human medicine might represent apublic health problem, leading to the ineffectiveness of theseantibiotics in the therapy. Infections caused by resistant microbesfail to respond to treatment, resulting in prolonged illness and greater

risk of death. Treatment failures also lead to longer periods ofinfectivity, which increase the numbers of infected people moving inthe community and thus expose the general population to the risk ofcontracting a resistant strain of infection.

On the other hand, the genes detected in E. coli and enterococci ofwild rabbits are similar to those found in the same bacteria of humanorigin demonstrating the possible circulation of bacteria andresistance genes between the animal and human ecosystems.Monitoring the level of antimicrobial resistance in commensalbacteria allows a comparison between the prevalence and evolutionof resistance patterns, over time. Future studies should be carried outto analyze the evolution of colonization in different ecosystems,including wild animals of different species and countries.

Acknowledgment

We thank the different hunter associations of north of Portugalfor their contribution to the collection of samples.

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