Science of the Total Environment 410-411 (2011) 266–268

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Short Communication

Detection of vancomycin-resistant enterococci from faecal samples of Iberian wolfand Iberian lynx, including Enterococcus faecium strains of CC17 and the newsingleton ST573

Alexandre Gonçalves a,b,c,d, Gilberto Igrejas a,b, Hajer Radhouani a,b,c,d, María López e, Ana Guerra f,Francisco Petrucci-Fonseca f, Eva Alcaide g, Irene Zorrilla g, Rodrigo Serra h,Carmen Torres e, Patrícia Poeta c,d,⁎a Institute for Biotechnology and Bioengineering/Center of Genomics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugalb Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro; Vila Real, Portugalc Center for Animal Science and Veterinary, Vila Real, Portugald Veterinary Science Department, University of Trás-os-Montes and Alto Douro, Vila Real, Portugale Biochemistry and Molecular Biology Area, University of La Rioja, Logroño, Spainf Grupo Lobo, Lisbon Faculty of Sciences, University of Lisbon, Lisbon, Portugalg Center for Analysis and Diagnosis of Wildlife (CAD), Ministry of Environment (EGMASA), Junta de Andalucía, Spainh National Center for Captive Breeding of the Iberian Lynx, Silves, Portugal

⁎ Corresponding author at: Department of Veterinaros-Montes and Alto Douro, Vila Real, Portugal. Tel.: +259350629.

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

0048-9697/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.scitotenv.2011.09.074

a b s t r a c t

a r t i c l e i n f o

Article history:Received 29 July 2011Received in revised form 26 September 2011Accepted 27 September 2011Available online 20 October 2011

Keywords:VREIberian wolfIberian lynxAntibiotic resistance

The aim of this study was to perform the molecular characterization of vancomycin resistant enterococci (VRE)within the faecal flora of Iberian wolf and Iberian lynx. The association with other resistance genes and the de-tection of virulence genes were also analysed. From 2008 to 2010, 365 faecal samples from Iberian wolf and Ibe-rian lynx were collected and tested for VRE recovery. Mechanisms of resistance to vancomycin and otherantibiotics, as well as genes encoding virulence factors were detected through PCR. Multilocus Sequence Typing(MLST) was performed for Enterococcus faecium strains. VRE were recovered in 8 of the 365 analysed samples.The vanA gene was identified in two E. faecium isolates recovered from Iberian wolf faecal samples and theremaining six showed intrinsic resistance (3 vanC1–E. gallinarum and 3 vanC2–E. casseliflavus, from Iberianwolf and Iberian lynx faecal samples, respectively). One vanA-containing isolate showed tetracycline and eryth-romycin resistance [with erm(B) and tet(L) genes] and the other one also exhibited ampicillin and kanamycin re-sistance [with erm(B), tet(M) and aph(3′)-III genes]. One of the vanA-isolates revealed a new sequence typenamed ST573 and the other one belonged to the CC17 clonal complex (ST18). The hyl gene was detected inone E. casseliflavus and three E. gallinarum but not among vanA-positive isolates, and the occurrence of cylAand cylL genes was confirmed in two E. casseliflavus isolates. A low prevalence of VRE has been detected in faecalsamples of Iberian wolf and Iberian lynx and strains with an acquired mechanism of resistance to vancomycinhave not been detected among Iberian lynx.

y Sciences, University of Trás-351 259350466; fax: +351

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© 2011 Elsevier B.V. All rights reserved.

1. Introduction

The emergence of vancomycin-resistant enterococci (VRE) in Europewas associated with the widespread use of the antimicrobial avoparcinas feed additive in food-producing animals until it was banned in 1997in the European Union (Radhouani et al., 2010). On the other hand, awide dissemination of vancomycin-resistant Enterococcus faecium isolatesof the CC17 clonal complex seems to be responsible for the worldwide

emergence of nosocomial vancomycin-resistant E. faecium isolates. Clonalspread of this high-risk complex has been reported from hospitals inEurope, Asia, America, Africa and Australia (Lopez et al., 2009). Still,studies concerning the incidence of VRE in wild animals are limited(Figueiredo et al., 2009; Poeta et al., 2007; Radhouani et al., 2010),and the flow of resistant microorganisms and resistant genes from live-stock and humans to wildlife remains poorly understood despite thefact that wild animals may act as reservoirs of resistant genetic ele-ments that could be spread across the environment (Radhouani et al.,2010). Therefore, the purpose of this study was to determine the prev-alence of VRE in faecal samples of Iberianwolf and Iberian lynx. Themo-lecular characterization of vancomycin resistance mechanisms, theassociation with other resistance genes, as well as the detection of viru-lence genes will also be addressed in this study.

Table 1Characteristics of the 8 VRE isolates recovered from Iberian wolf and Iberian lynx identified in this study.

Isolate Origin ofthe isolate

Enterococcusspecies

Vancomycin-resistantgenedetected

MICa (mg/L) Resistant phenotypeb Genes detected by PCR MLSTc ST (CC) Virulencegenesdetected

VAN TEI

W50 Iberian wolf E. faecium vanA N128 N16 TET, TEI, VAN, AMP, ERY, KAN tet(M), erm(B); aph(3′)-IIIa 18 (17) –

W58 Iberian wolf E. faecium vanA N128 N16 TET, TEI, VAN, ERY tet(L); erm(B) 573 (Singleton) –

W63 Iberian wolf E. gallinarum vanC1 8 1 TET tet(M); Tn916 – hylW71 Iberian wolf E. gallinarum vanC1 8 1 TET tet(M); Tn916 – hylW166 Iberian wolf E. gallinarum vanC1 8 1 TET tet(M); – hylL127 Iberian lynx E. casseliflavus vanC2 8 1 TET, Q–D, ERY, STR tet(M), erm(B) – hyl, cylA, cylLL148 Iberian lynx E. casseliflavus vanC2 8 1 Q–D – – cylLL172 Iberian lynx E. casseliflavus vanC2 8 1 TET, ERY, STR tet(M), erm(B) – –

a MIC: Minimal inhibitory concentrations.b Antimicrobial abbreviations: TET: tetracycline; Q–D: quinupristin–dalfopristin; ERY: erythromycin; STR: streptomycin; TEI: teicoplanin; VAN: vancomycin; AMP: ampicillin;

KAN: kanamycin.c Multilocus sequence typing; ST: sequence type; CC: clonal complex.

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2. Material and methods

2.1. Faecal samples and bacterial isolates

237 fresh faecal sampleswere obtained fromwild Iberianwolf (Canislupus signatus) during 2008 and 2009. The faecal sampleswere collectedon the north side of Douro River in five locations (Falperra, Alvão,Minhéu, Padrela and Candedo), in the Northeast of Portugal. Samplegathering was carried out during surveillance studies performed bytheWolf Group. This group is a Portuguese non-governmental, indepen-dent and nonprofit association that works for the conservation of theIberian wolf and its ecosystem in Portugal.

From 2008 to 2010, 98 and 30 fresh faecal samples were obtainedfrom captive and wild Iberian lynx (Lynx pardinus), respectively. Eachfaecal sample collected belonged to a different specimen. Sample col-lection from captive animals originated from the breeding facilities inthe Centre of Analysis and Diagnosis of the Wildlife (CAD), in DoñanaNational Park, South Spain, and took place during clinical or manage-ment procedures. Thirty faecal samples from wild animals wereobtained in Doñana National Park and in Sierra Morena, duringsmall procedures, e.g. placement of radio tracking systems.

Samples were seeded in Slanetz–Bartley agar plates supplementedwith vancomycin (4 μg/mL). Colonies with typical enterococcal mor-phology were identified to the genus and species level by Gram-staining, catalase test, bile-aesculin reaction, and by biochemicaltests using the API ID20 Strep system (BioMérieux, La Balme, LesGrottes, France). Species identification was confirmed by polymerasechain reaction (PCR) using primers and conditions for the differententerococcal species (Poeta et al., 2005).

2.2. Antimicrobial susceptibility testing

Susceptibility for 11 antimicrobial agents (vancomycin; teicoplanin;ampicillin; streptomycin; gentamicin; kanamycin; chloramphenicol; tet-racycline; erythromycin; quinupristin–dalfopristin; and ciprofloxacin)was performed by the disk diffusion method according to the criteria ofthe CLSI (CLSI, 2010). High-level resistance was evaluated for aminogly-cosides. Minimal inhibitory concentrations (MICs) of vancomycin andteicoplanin were also determined by the agar dilution method (CLSI,2010). E. faecalis ATCC 29212 and Staphylococcus aureus ATCC 25923strains were used for quality control.

2.3. Characterization of antibiotic resistance mechanisms

Vancomycin resistance mechanisms were analysed by PCR usingspecific primers for amplification of the vanA, vanB, vanC-1, vanC-2/3,and vanD genes (Torres et al., 2003). The vanA cluster of genes was fur-ther characterized in all vanA-positive isolates (Woodford et al., 1998).

Resistance genes for other antibiotics, including erm(A), erm(B), tet(M), tet(K), tet(L), aph(3′)-IIIa and the presence of Tn916- and Tn5397-specific sequences were analysed by PCR (Aarestrup et al., 2000; Agersoet al., 2006; De Leener et al., 2004; Sutcliffe et al., 1996; Torres et al.,2003). The presence of genes encoding different virulence factors (gelE,agg, ace, cpd, fsr, esp, hyl and cylLLLSABM) was also studied by PCR(Creti et al., 2004; Eaton and Gasson, 2001; Klare et al., 2005; Pillai etal., 2002). Positive and negative controls from the collection of strainsof the University of Trás-os-Montes and Alto Douro (Portugal) were in-cluded in all PCR assays.

2.4. Multilocus sequence typing (MLST)

Vancomycin-resistant E. faecium isolates were characterized byMLST. TheMLST characterizationwas performed as previously described(Homan et al., 2002).

2.5. Assay of gelatinase and beta-haemolytic activities

Evaluation of gelatinase and haemolysin production was per-formed as previously reported (Lopez et al., 2009).

3. Results and discussion

This is the first study reporting the presence of VRE strains in Iberianwolf and Iberian lynx. VRE were recovered in 3 of the 128 faecal samplesfrom Iberian lynx and in 5 of the 237 faecal samples of Iberian wolf ana-lysed (2.3% and 2.1%, respectively) (Table 1). All the Iberian lynx VRE iso-lates were recovered from faecal samples of captive animals. The vanAgene was identified in two E. faecium isolates from Iberian wolf (W50and W58) and the remaining six isolates showed intrinsic vancomycinresistance (3 vanC1–E. gallinarum and 3 vanC2–E. casseliflavus, recoveredfrom Iberianwolf and Iberian lynx faecal samples, respectively) (Table 1).Various studies reported the colonization by VRE in different wild ani-mals (Figueiredo et al., 2009; Poeta et al., 2007; Radhouani et al., 2010).In this study, vancomycin resistant strains with an acquired mechanismof resistance were only detected among faecal samples of Iberian wolf.These strains showed multiresistance phenotypes that, simultaneouslywith the VanA mechanism, represent a larger public health problem.Travelling large distances exposes the Iberianwolf to food remains or fae-calmaterial from farm animals or even fromhumans thatmight carry re-sistant strains (Figueiredo et al., 2009). These resistant strains or genescould be transmitted to thewild animals (Poeta et al., 2007). Also, Iberianwolf may possibly be contaminated through the food chain, as the pres-ence of VRE strains has been previously demonstrated in their primarypreys, wild boars and wild rabbits, (Figueiredo et al., 2009; Poeta et al.,2007).

268 A. Gonçalves et al. / Science of the Total Environment 410-411 (2011) 266–268

The complete Tn1546 structurewas identified in one vanA-containingisolate (W58). Other authors also found this type A structure in vanA-positive enterococci from animals and healthy humans suggesting a pos-sible exchange of VREbetween these sources (Biavasco et al., 2007; Lopezet al., 2009). In the other vanA-containing isolate (W50), the Tn1546structurewas also detected but the orf1 sequence located in its upstreamregion was lacking. The orf1 gene, together with the orf2 gene, is associ-ated with transposition functions playing a crucial role in the dissemina-tion of vancomycin resistance. Thus, the lack of orf1 gene may possiblyinfluence the ability of Tn1546 to disseminate (Woodford et al., 1998).No insertion sequences were detected within this structure in any ofthese vanA-positive strains.

One of the two vanA-containing E. faecium isolates (W50)detected in our study was assigned to the ST18 sequence type (in-cluded in CC17 clonal complex), and the other isolate (W58) showeda new allelic combination (atpA 9; ddl 40; gdh 12; purK 3; gyd 1; pstS33; and adk 1) being assigned to a new sequence type, that was in-cluded in the MLST database (www.mlst.net) and registered asST573, corresponding to a singleton (Table 1). The results obtainedin this study associated with those performed in food samples, in fae-cal samples from food-producing animals, and in wild animals mayindicate a higher dissemination rate of the clonal lineage CC17 thanpreviously assumed (Lopez et al., 2009). Also, the W58 E. faecium iso-late was included in a new sequence type named ST573. Consequent-ly, different lineages of vancomycin-resistant E. faecium isolatessimilar to that found in humans seem to be disseminated in wild an-imals and their evolution in the different ecosystems is a subject ofrelevance that should be tracked in the future.

Generally, E. faecium isolates are devoid of virulence determinantsand the same situation is observed in E. durans and E. hirae (Eaton andGasson, 2001). As expected, none of the vanA–E. faecium isolates re-covered in this study presented genes encoding virulence factors.On the other hand, the hyl gene, encoding a putative glycoside hydro-lase, was detected in four isolates with intrinsic mechanisms of van-comycin resistance. The absence of beta-haemolytic activity in ourtwo E. casseliflavus isolates with cylA and/or cylL genes could be dueto the absence of the whole cyl operon; required for haemolytic activ-ity (Poeta et al., 2007). Also, as expected, none of our isolates showedgelatinase activity since this activity is most frequent in E. faecalis andnot in other enterococcal species (Lopez et al., 2009).

A low prevalence of VRE has been detected in faecal samples ofIberian wolf and Iberian lynx. Fortunately, strains with an acquiredmechanism of resistance to vancomycin have not been detectedamong Iberian lynx. Different food sources and larger territoryrange of Iberian wolf could be the reason why vanA-strains wereonly detected in the faecal flora of this species. Still, further studiesmust be performed in order to better understand the disseminationof vanA-strains and their epidemiology in wild ecosystems.

Acknowledgements

Alexandre Gonçalves has a PhD fellowship granted by FCT—Fundaçãopara a Ciência e a Tecnologia (SFRH/BD/47833/2008). Hajer Radhouani

has a PhD fellowship granted by FCT-Fundação para a Ciência e a Tecno-logia (SFRH/BD/60846/2009). María López has a fellowship from theGobierno de La Rioja of Spain.

References

Aarestrup FM, Agerso Y, Gerner-Smidt P, Madsen M, Jensen LB. Comparison of antimicro-bial resistance phenotypes and resistance genes in Enterococcus faecalis andEnterococcus faecium from humans in the community, broilers, and pigs in Denmark.Diagn Microbiol Infect Dis 2000;37:127–37.

Agerso Y, Pedersen AG, Aarestrup FM. Identification of Tn5397-like and Tn916-liketransposons and diversity of the tetracycline resistance gene tet(M) in enterococcifrom humans, pigs and poultry. J Antimicrob Chemother 2006;57:832–9.

Biavasco F, Foglia G, Paoletti C, Zandri G, Magi G, Guaglianone E, et al. VanA-type en-terococci from humans, animals, and food: species distribution, population struc-ture, Tn1546 typing and location, and virulence determinants. Appl EnvironMicrobiol 2007;73:3307–19.

CLSI. Performance standards for antimicrobial susceptibility testing. Informational sup-plement. CLSI document M100-S20Twentieth edition. . Wayne, PA, USA: Clinicaland Laboratory Standards Institute; 2010.

Creti R, Imperi M, Bertuccini L, Fabretti F, Orefici G, Di Rosa R, et al. Survey for virulencedeterminants among Enterococcus faecalis isolated from different sources. J MedMicrobiol 2004;53:13–20.

De Leener E, Martel A, Decostere A, Haesebrouck F. Distribution of the erm (B) gene,tetracycline resistance genes, and Tn1545-like transposons in macrolide- andlincosamide-resistant enterococci from pigs and humans. Microb Drug Resist2004;10:341–5.

Eaton TJ, Gasson MJ. Molecular screening of Enterococcus virulence determinants andpotential for genetic exchange between food and medical isolates. Appl EnvironMicrobiol 2001;67:1628–35.

Figueiredo N, Radhouani H, Goncalves A, Rodrigues J, Carvalho C, Igrejas G, et al. Genet-ic characterization of vancomycin-resistant enterococci isolates fromwild rabbits. JBasic Microbiol 2009;49:491–4.

Homan WL, Tribe D, Poznanski S, Li M, Hogg G, Spalburg E, et al. Multilocus sequencetyping scheme for Enterococcus faecium. J Clin Microbiol 2002;40:1963–71.

Klare I, Konstabel C, Mueller-Bertling S, Werner G, Strommenger B, Kettlitz C, et al.Spread of ampicillin/vancomycin-resistant Enterococcus faecium of the epidemic-virulent clonal complex-17 carrying the genes esp and hyl in German hospitals.Eur J Clin Microbiol Infect Dis 2005;24:815–25.

Lopez M, Saenz Y, Rojo-Bezares B, Martinez S, del Campo R, Ruiz-Larrea F, et al. Detec-tion of vanA and vanB2-containing enterococci from food samples in Spain, includ-ing Enterococcus faecium strains of CC17 and the new singleton ST425. Int J FoodMicrobiol 2009;133:172–8.

Pillai SK, Sakoulas G, Gold HS, Wennersten C, Eliopoulos GM, Moellering Jr RC, et al.Prevalence of the fsr locus in Enterococcus faecalis infections. J Clin Microbiol2002;40:2651–2.

Poeta P, Costa D, Saenz Y, Klibi N, Ruiz-Larrea F, Rodrigues J, et al. Characterization ofantibiotic resistance genes and virulence factors in faecal enterococci of wild ani-mals in Portugal. J Vet Med B Infect Dis Vet Public Health 2005;52:396–402.

Poeta P, Costa D, Igrejas G, Rojo-Bezares B, Saenz Y, Zarazaga M, et al. Characterizationof vanA-containing Enterococcus faecium isolates carrying Tn5397-like and Tn916/Tn1545-like transposons in wild boars (Sus scrofa). Microb Drug Resist 2007;13:151–6.

Radhouani H, Poeta P, Pinto L, Miranda J, Coelho C, Carvalho C, et al. Proteomic charac-terization of vanA-containing Enterococcus recovered from Seagulls at the Berlen-gas Natural Reserve, W Portugal. Proteome Sci 2010;8:48.

Sutcliffe J, Grebe T, Tait-Kamradt A, Wondrack L. Detection of erythromycin-resistantdeterminants by PCR. Antimicrob Agents Chemother 1996;40:2562–6.

Torres C, Tenorio C, Portillo A, Garcia M, Martinez C, Del Campo R, et al. Intestinal col-onization by vanA- or vanB2-containing enterococcal isolates of healthy animals inSpain. Microb Drug Resist 2003;9(Suppl. 1):S47–52.

Woodford N, Adebiyi AM, Palepou MF, Cookson BD. Diversity of VanA glycopeptide re-sistance elements in enterococci from humans and nonhuman sources. AntimicrobAgents Chemother 1998;42:502–8.