JAC Advance Access published online on May 2, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn179
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Original research |
Antibiotic resistance integrons and extended-spectrum β-lactamases among Enterobacteriaceae isolates recovered from chickens and swine in Portugal
1 REQUIMTE, Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal 2 CEBIMED, Faculdade de Ciências da Saúde, Universidade Fernando Pessoa, Porto, Portugal 3 Servicio de Microbiología, Hospital Universitario Rámon y Cajal, IMSALUD, Madrid, Spain
* Corresponding author. Tel: +351-22-200-89-46; Fax: +351-91-209-33-77; E-mail: lpeixe{at}ff.up.pt
Received 13 December 2007; returned 23 January 2008; revised 19 March 2008; accepted 31 March 2008
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Abstract |
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Objectives: To investigate the diversity of integrons and extended-spectrum β-lactamases (ESBLs) among Enterobacteriaceae from chickens and swine in Portugal and analyse the clonal relationships between Portuguese ESBL-producing isolates of animal and human origin.
Methods: We analysed samples from faeces of healthy swine (HSF, n = 35), from uncooked chicken carcasses (CM, n = 20) and from faeces of healthy chickens (HCF, n = 20). Samples were plated on MacConkey agar with and without ceftazidime (1 mg/L) or cefotaxime (1 mg/L). ESBLs were characterized by PCR and DNA sequencing. Bacterial identification, antibiotic susceptibility and conjugation assays were performed by standard procedures. Isolate clonal relatedness was established by PFGE and by RAPD for PFGE non-typeable isolates. Escherichia coli phylogenetic groups were identified by a multiplex PCR. Integron analysis was accomplished by PCR-RFLP and sequencing.
Results: ESBL-producing Enterobacteriaceae were identified in 60% of CM, 10% of HCF and 5.7% of HSF samples, respectively, mostly corresponding to E. coli (phylogroups A, D and B1). TEM-52, SHV-2 and CTX-M-1 were detected from chicken and SHV-12 from swine samples. High clonal diversity was observed and most blaESBL genes were transferable (67%). Class 1 and/or class 2 integrons were identified in 80% of CM, 10% of HCF and 63% of HSF samples, with class 1 integrons more common than class 2 integrons (36% versus 12% of the isolates recovered, respectively). Ten class 1 integron types are described, aadA1 and dfrA1-aadA1 being the most frequently found. Two class 1 integron types (aadA13-estX and dfrA14-aadA1-catB2) and one class 2 integron (aadA1) are first reported here.
Conclusions: This study is the first report of ESBLs and integrons from chickens and swine in Portugal and highlights the antibiotic-resistant bacteria and/or resistance genes that might be acquired by humans through the food chain.
Key Words: CTX-M , TEM-52 , SHV , ESBLs , animals
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Introduction |
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Intestinal commensal Enterobacteriaceae of animals reared in high-population flocks are usually under different selective pressures by the use of antibiotics for treating infections, for metaphylaxis and for prophylaxis.1 Several antimicrobial agents used in veterinary and human medicine belong to the same antibiotic families and hence different selective pressures exercised in distinct environments might contribute to the selection and dissemination of similar resistance genes.2,3 Surveillance of antimicrobial resistance in commensal bacteria from food-producing animals is considered as one of the main priorities of the World Health Organization and the European Commission to better control the spread of antimicrobial resistance from food animal products to humans through the food chain.4,5 Recent studies have alerted for the wide presence of extended-spectrum β-lactamases (ESBLs) and integrons in bacteria recovered from a diversity of animals and food products in different countries;6–16 however, data on the occurrence of integrons and/or ESBLs among commensal Enterobacteriaceae from food-producing animals are very scarce in Portugal.17 In this study, we analysed the occurrence and diversity of integrons and ESBLs among Enterobacteriaceae from faeces of healthy food-producing animals and raw chicken meat samples. Moreover, as ESBLs are widely disseminated in Portuguese hospitals,18,19 we also investigated the clonal relationships between ESBL-producing Enterobacteriaceae from animal and human origin.
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Materials and methods |
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Bacterial isolates
Samples from swine (n = 35) and chickens (n = 40) were analysed. Swine samples included faeces from healthy swine (HSF) freshly slaughtered at a slaughterhouse of the central region of Portugal (n = 21, 1998) or reared at two non-intensive-production farms in the north (n = 14, 2004). Chicken samples included uncooked chicken carcasses (CM) from two butcher shops corresponding to chickens from intensive-production farms of five different brands widely commercialized throughout Portugal (n = 20, 2003 and 2005) and faeces from healthy chickens (HCF) reared at two non-intensive-production farms (n = 20, 2005). The faecal samples collected in 2004 and 2005 were obtained from healthy animals of farms without records of antibiotic use in the 3 months preceding the sample recovery. Samples were processed immediately after collection. Rectal swabs were immersed in the transport medium and faeces were suspended in 1 mL of saline. Chicken carcasses (25 g) were pre-enriched in 500 mL of buffered peptone water for 18 h at 37°C. An aliquot of 0.2 mL from these suspensions was inoculated on MacConkey agar with and without ceftazidime (1 mg/L) or cefotaxime (1 mg/L). Presumptive Enterobacteriaceae were selected and identified by using the automated WIDER system (Fco. Soria Melguizo, Madrid, Spain) or API ID 32GN galleries (bioMérieux, Marcy l'Étoile, France). Representative isolates of ESBL-producing Enterobacteriaceae recovered from Portuguese hospitals were also included for establishing clonal relationships.
ESBL detection and antimicrobial susceptibility
Each different morphotype growing on MacConkey agar supplemented with ceftazidime or cefotaxime was screened for ESBL production by the standard double disc synergy test18 and underwent susceptibility testing to additional non-β-lactam antibiotics using the standard CLSI disc diffusion method.20 Non-β-lactam antibiotics tested were the following: gentamicin, tobramycin, amikacin, streptomycin, spectinomycin, netilmicin, neomycin, apramycin, kanamycin, sulphonamides, trimethoprim, tetracycline, chloramphenicol, ciprofloxacin and nalidixic acid. Morphotypes recovered from MacConkey agar with and without ceftazidime or cefotaxime, but identified as non-ESBL producers, underwent susceptibility testing to streptomycin, gentamicin, trimethoprim and sulphonamides using the standard CLSI disc diffusion method.20 All resistant and intermediate-susceptible isolates were considered as non-susceptible.
ESBL characterization was performed by PCR using specific primers for blaTEM, blaSHV and blaCTX-M and further sequencing of both strands of each amplified bla gene using ABI Prism 377 automated sequencer (Applied Biosystems, Perkin-Elmer, Foster City, CA, USA), as previously described.21 The presence of ISEcp1 in the genetic environment of blaCTX-M was searched for by PCR.22
Clonal relationships among ESBL-producing isolates
These were established by PFGE, according to the Tenover et al. criteria,23 using XbaI as restriction enzyme (Amersham, Life Sciences, Uppsala, Sweden) and the following electrophoresis conditions: 10–40 s for 24 h, 14°C, 6 V/cm2.18 As some isolates were not typeable by PFGE, randomly amplified polymorphic DNA analysis (RAPD) using primer M13 (5'-GAG GGT GGC GGT TCT-3') and Ready-To-Go RAPD analysis beads (Amersham Biosciences, Amersham, Portugal) were also performed, as described.24 Isolates with RAPD patterns showing two or more band differences were considered as unrelated.25 The PFGE and RAPD patterns were visually analysed and were also compared with each clonal type/subtype of each ESBL-producing species identified in Portuguese hospitals.18 The phylogenetic groups of ESBL-producing Escherichia coli isolates were determined by a multiplex PCR assay described by Clermont et al.26
Transfer of β-lactam resistance was performed by the filter mating method using E. coli BM21R (nalidixic acid- and rifampicin-resistant, lactose fermentation positive and plasmid-free) as recipient.21 Mating experiments were performed overnight at 37°C. Transconjugants were selected on MacConkey agar plates containing 100 mg/L rifampicin and 2 mg/L cefotaxime or ceftazidime.21
Analysis and characterization of integrons
Isolates resistant to streptomycin, gentamicin, trimethoprim and/or sulphonamides were screened for the presence of integrons, as these phenotypes are commonly associated with these genetic structures.21 The PCR was used in the detection of class 1 and class 2 integrons using genomic DNA from wild-type and the corresponding transconjugant strains, and primers and conditions previously described for amplification of intI1, intI2, 5'CS-3'CS class 1 integron variable region and attI2-orfX variable region of class 2 integrons.21 Typing of class 1 and class 2 integrons was performed by restriction fragment length polymorphism (RFLP) using AluI and TaqI restriction endonucleases, respectively.21 An amplified DNA fragment corresponding to the variable regions of each distinct class 1 or class 2 RFLP-type integron was sequenced using an ABI Prism 377 automated sequencer (Applied Biosystems, Perkin-Elmer, Foster City, CA, USA). Nucleotide sequences were compared with sequences in the GenBank and EMBL databases using the BLASTN local alignment search tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Integrons were designated by roman numbers and a subindex indicates the class to which each integron belongs, as previously described.21
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Epidemiological background
Isolates with decreased susceptibility to expanded-spectrum cephalosporins were recovered from all chicken meat samples and from two HCF and three HSF samples. A total of 149 isolates representing different colony morphotypes and antibiotic susceptibility patterns were obtained from HSF (n = 48), HCF (n = 33) and CM (n = 68). Resistance to at least one antibiotic was observed in 113 isolates, being commonly resistant to streptomycin (65%), sulphonamides (51%) or trimethoprim (40%). Non-susceptibility rates for sulphonamides and trimethoprim were similar among isolates from chickens and swine (50% versus 54% for sulphonamides and 43% versus 35% for trimethoprim). Non-susceptibility rates for non-β-lactam antibiotics were more frequently found among ESBL producers than among non-ESBL-producing isolates: streptomycin (83% versus 63%), sulphonamides (67% versus 49%) and trimethoprim (61% versus 37%).
We identified 18 ESBL-producing Enterobacteriaceae corresponding to TEM-52 (11 E. coli and 1 Klebsiella pneumoniae from HCF or CM), SHV-2 (3 K. pneumoniae from CM), SHV-12 (2 Citrobacter freundii from HSF) and CTX-M-1 (1 E. coli from CM; blaCTX-M-1 located downstream of ISEcp1). These isolates were frequently resistant to aminoglycosides, sulphonamides, trimethoprim, tetracyclines, chloramphenicol or quinolones (Table 1). Genes encoding ESBLs were transferred from 67% of the isolates, often associated with genes encoding resistance to non-β-lactam antibiotics (Table 1). A great heterogeneity of PFGE patterns was observed, although some clones were persistently recovered, as TEM-52-producing E. coli PFGE type C and RAPD type D, isolated in 2003 and 2005, or SHV-2-producing K. pneumoniae PFGE type H isolated from different brands in 2003 (Table 1). When comparing these isolates with those previously published with ESBL from hospitalized patients in Portugal,18 we did not find any relationship between PFGE or RAPD profiles. ESBL-producing E. coli isolates belonged to phylogenetic groups A (n = 7/12), D (n = 4/12) or B1 (n = 1/12).
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Analysis of integrons
Isolates carrying class 1 and/or class 2 integrons were recovered from 80% of chicken meat, 10% of chicken faecal samples and 63% of swine faecal samples. Class 1 and/or class 2 integrons were detected in 40% (n = 60/149) of the isolates studied, being more common among isolates from swine than those from chickens: 48% versus 31% for class 1 integrons and 21% versus 8% for class 2 integrons (Table 2). Among ESBL-producing isolates, class 1 integrons were identified in isolates harbouring blaCTX-M-1, blaSHV-2 or blaTEM-52. Co-transfer of integrons and bla genes was only observed for a CTX-M-1-producing E. coli and for an SHV-2-producing K. pneumoniae. Class 2 integrons were only detected among TEM-52-producing E. coli and were not transferred. Simultaneous presence of class 1 and class 2 integrons was observed in 8% of the isolates studied, all lacking blaESBL (n = 12/149). The presence of class 1 and/or class 2 integrons among non-ESBL-producing isolates was not a rare event (39%, 51/131), particularly in isolates recovered from swine and chicken meat samples. Ten different class 1 integron types were found, mostly corresponding to non-ESBL-producing isolates (Table 3). Gene cassettes coding for aminoglycoside (aadA1, aadA1a, aadA2, aadA5, aadA13) and/or trimethoprim (dfrA1, dfrA12, dfrA14, dfrA17) resistance were the most commonly identified. Integron types I1 (aadA1) and II1 (dfrA1-aadA1) were the most prevalent. Types I1' (aadA1a), III1 (dfrA12-orfF-aadA2), V1 (aadA2) and XX1 (blaP1) were confined to isolates from CM, whereas types XXII1 (aadA13-estX) and XXIII1 (dfrA14-aadA1-catB2) were only distributed among HSF. Three class 2 integron types were observed: type II2, identical to that described for Tn7, and type III2 (estX-sat2-aadA1-orfX) were the most widely distributed. Type IV2 (aadA1) is first reported in this study. Gene cassettes were not identified for a number of non-ESBL-producing isolates harbouring intI1 (15/149, 10%) or intI2 (1/149, 0.7%).
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Discussion |
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ESBLs frequently identified in humans associated with either hospitals or community settings in European countries are now being increasingly detected among animals.8–10,16,27 The frequent recovery of Enterobacteriaceae producing ESBLs and/or carrying class 1 and/or class 2 integrons from faecal samples from food-producing swine and chickens, and from both different commercial brands and chickens reared at different farms from Portugal, confirms the role of animals as possible reservoirs for dissemination of resistance genes in the community. The presence of ESBLs among chicken samples from both different commercial brands and chickens reared at different farms might be caused by contamination of the few genetic lines of primary breeding flocks that are sold by the same producer, as also supported by previous studies on poultry enterococci populations.28 The ESBLs were more frequently observed among E. coli mainly belonging to the phylogenetic group A (58.3%), which is associated with animal or human commensal E. coli strains, in agreement with other studies.26 However, we also detected ESBL-producing E. coli of phylogenetic group D, which is associated with extra-intestinal human infection, mirroring an animal reservoir for these invasive strains.
All ESBL genes identified in this study have already been identified in Portuguese hospitals, and the most common ESBLs among animals, TEM-52 and SHV-12, are also the most frequent types among humans.18 The blaTEM-52 has also been detected among food-producing and wild animals, pets and healthy humans from Belgium, France, Greece, Portugal or Spain8–10,29–31 and associated with epidemic conjugative plasmids among animal samples in areas with high prevalence of this ESBL type.29 Our results increase the number of hosts of blaTEM-52 in Portugal9,10,18 and might reflect the successful spread of an epidemic plasmid of the IncI group.32 This possibility is also supported by the absence of relationship between ESBL-producing isolates from animals and from patients at Portuguese hospitals located in the same regions studied.18 The other ESBL genes found, blaCTX-M-1, blaSHV-2 or blaSHV-12, are widely disseminated among Enterobacteriaceae from different continents by both clonal and plasmid spread and have recently been detected in Portugal.8–10,18,27,29,33 The clonal diversity of isolates carrying these genes suggests that horizontal gene transmission may be responsible for the recent and fast spread of these variants in our country.
Most ESBL-producing isolates exhibited resistance to antibiotics used in intensive animal production, mainly streptomycin, sulphonamides and trimethoprim. In agreement with other studies, integrons were commonly identified and corresponded to a few integron types that have been mostly found among Enterobacteriaceae from food animals, hospitalized patients and/or healthy humans.6,7,11,12,34–37 Some of them are globally disseminated as type I1 (aadA1), type II1 (dfrA1-aadA1), type III1 (dfrA12-orfF-aadA2), type VI1 (dfrA17-aadA5) or class 2 integron types II2 and III2, whereas others are first reported in this study as type XXII1 (aadA13-estX), type XXIII1 (dfrA14-aadA1-catB2) or type IV2 (aadA1). Type XXI1 (estX-aadA1) has been recently described to be associated with human community isolates.38,39 Differences in integron dissemination might be explained by the association of specific integron types with particular globally disseminated plasmids and/or transposons potentially exchanged among humans and animals.37 It is to be noted that the simultaneous presence of class 1 and class 2 integrons in 8% of the isolates and the finding of a new class 2 integron (type IV2, aadA1), carrying one of the most common gene cassettes identified in class 1 integrons, highlight a possible variety of recombinatorial events among these genetic platforms.
In summary, we describe the presence and diversity of integrons and ESBLs in Enterobacteriaceae from chickens and swine in Portugal. The findings are worrisome as transmission to humans via the food chain of bacteria resistant to practically all antimicrobial classes cannot be dismissed.13,40,41 Some of the ESBLs and integrons we found have already been detected among Enterobacteriaceae from different ecological niches in Portugal and other countries,6,18,34,37 suggesting that the genetic mobile structures harbouring them are widespread. More strict veterinary antibiotic policies are needed in order to prevent emergence and dissemination of these strains among animals and humans, limiting future problems of therapy failure.
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R. C.: grants from Wyeth and consultancies from Novartis. Other authors: none to declare.
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Funding |
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E. M. was supported by a fellowship from Fundação para a Ciência e a Tecnologia de Portugal (SFRH/BD/11304/2002). This work was partially supported by REQUIMTE and by research grants from FSE/FEDER (POCI/AMB/61814/2004).
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Acknowledgements |
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We are very grateful to Dr Jorge Silva (Departamento de Medicina Veterinária, Câmara Municipal de Fafe, Portugal) and to Cristina Réu (Faculdade de Farmácia da Universidade do Porto, Portugal) for support in the sample recruitment.
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References |
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