JAC Advance Access originally published online on March 18, 2009
Journal of Antimicrobial Chemotherapy 2009 63(6):1286-1288; doi:10.1093/jac/dkp101
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Research letters |
Comparative analysis of extended-spectrum-β-lactamase-carrying plasmids from different members of Enterobacteriaceae isolated from poultry, pigs and humans: evidence for a shared β-lactam resistance gene pool?
1 Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium 2 Department of Bacteriology and Immunology, CODA-CERVA-VAR, Groeselenberg 99, 1180 Brussels, Belgium 3 Department of Reproduction, Obstetrics and Herd Health, Veterinary Epidemiology Unit, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium 4 Institute for Agricultural and Fisheries Research, Technology and Food Unit, Brusselsesteenweg 370, B-9090 Melle, Belgium 5 INRA, UR1282, Infectiologie Animale et Santé Publique, IASP, F-37380 Nouzilly, France 6 Department of Clinical Microbiology, Faculty of Medecin, Ghent University, De Pintelaan 185, 9000 Ghent, Belgium 7 Scientific Institute of Public Health, J. Wytsmanstraat 14, 1050 Brussels, Belgium
* Corresponding author. Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium. Tel: +32-9264-7435; Fax: +32-9264-7494; E-mail: Annemieke.Smet{at}Ugent.be
Keywords: RFLP , ESBLs , conjugation
β-Lactam antibiotics are extensively used in human and veterinary medicine. The detection rate of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolated from humans has increased rapidly worldwide.1 In addition, ESBLs have been increasingly described in bacterial populations circulating in animals.2,3 Recently, a high diversity of ESBLs in Escherichia coli was reported in Belgian poultry farms. In that instance, CTX-M enzymes were the predominant ESBL family.4 CTX-M-2-producing Salmonella enterica serovar Virchow strains and TEM-52-producing S. enterica serovar Infantis strains have also been isolated from Belgian poultry.2,3 This raises a potential public health concern. Moreover, the presence of ESBLs in the microbiota of food-producing animals may pose a human health hazard since these bacteria may represent a reservoir of resistance genes for pathogens causing disease in humans and animals.4 Therefore, to demonstrate whether a common ESBL gene pool exists among isolates in different hosts, we characterized the plasmids and determined the location and transfer possibilities of the ESBLs blaTEM-52, blaCTX-M-2 and blaCTX-M-15 that were present in different members of Enterobacteriaceae isolated from humans, broilers and pigs.
Fourteen blaTEM-52-, blaCTX-M-2- or blaCTX-M-15-carrying clonally unrelated strains were used in this study (Table 1). These strains were isolated in Belgium from humans, pigs and broilers. The human E. coli strains were isolated from patients hospitalized at the Ghent University Hospital. All isolates from poultry were obtained from the faeces of healthy broilers. The E. coli and Klebsiella pneumoniae isolates were obtained as described previously.4 The S. enterica isolates from poultry were collected in the framework of mandatory Salmonella monitoring programmes in Belgium. The two porcine E. coli isolates originated from pigs with diarrhoea. The ESBL gene of each isolate was characterized as described previously by isoelectric focusing, PCR and sequencing.4 Plasmid transfer experiments were carried out as described previously.2 The antimicrobial susceptibility of the parental strains and their E. coli transconjugants was determined by the Kirby–Bauer disc diffusion test (Neo-Sensitabs, Rosco Diagnostica, Taastrup, Denmark) (Table 1).4 For the parental strains and their E. coli transconjugants, plasmid profiles were determined and the size of each ESBL-carrying plasmid was estimated.2 The incompatibility (Inc) group of each ESBL-carrying plasmid was defined by the PCR-based replicon typing method.5 Restriction fragment length polymorphism (RFLP) fingerprint analysis and Southern blot hybridization were performed as described previously.2
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In order to better understand the spread and persistence of mobile β-lactam resistance plasmids among different members of Enterobacteriaceae isolated from different reservoirs, a closer look at the pool of conjugative plasmids was appropriate and timely.
All isolates tested here contained high-molecular-weight ESBL-carrying plasmids (
150 kb) and, for all these isolates, E. coli transconjugants were obtained. The blaCTX-M-2-, blaTEM-52- and blaCTX-M-15-carrying plasmids belonged to IncHI2, IncI1 and IncI1, respectively (Table 1), as has already been demonstrated in previous reports.2,3,6 RFLP analysis of plasmid DNA from the transconjugants revealed, in most cases, closely related fingerprints for plasmids carrying the same ESBL gene. All blaTEM-52-carrying plasmids showed the same fingerprint pattern analysis, suggesting that this is a rather stable plasmid circulating in different members of the Enterobacteriaceae, present in different animal reservoirs and in humans. Southern blot hybridization with a blaTEM-52 probe revealed two PstI fragments of 2.75 and 2.9 kb, as has already been shown in a previous report.3 The spread of a blaCTX-M-2-carrying multiresistant plasmid among E. coli and S. enterica isolates from pigs and broilers was demonstrated. Only the plasmid from the human E. coli isolate differed in RFLP fingerprint pattern from the other blaCTX-M-2-carrying plasmids. Southern blot hybridization with a blaCTX-M-2 probe revealed a >10 kb EcoRI fragment in the plasmids from the porcine and broiler E. coli isolates and from the Salmonella Virchow CODA-1 isolate. Two EcoRI fragments of 6 and 10 kb were found in the plasmid from the human E. coli isolate and in the plasmid from the Salmonella Virchow 142-1 isolate.2 For the blaCTX-M-15-carrying plasmids, results of RFLP analysis were identical except for the plasmid from the human E. coli strain. For the animal strains, Southern blot hybridization with a blaCTX-M-15 probe revealed two EcoRI fragments of 6.5 and 7 kb and one 5 kb PstI fragment. The plasmid from the human E. coli strain showed 5 and >10 kb EcoRI fragments and a 5.5 kb PstI fragment. The Southern blot results suggest the existence of two copies of the tested ESBLs on their 150 kb plasmids.
The differences seen in the RFLP analyses for the blaCTX-M-2- and blaCTX-M-15-carrying plasmids may possibly reflect the rapid evolution of these plasmids as they were exposed to different environmental stresses. The human, porcine and poultry environments may be experienced by bacteria in different ways.
In summary, ESBL resistance plasmids appear to move readily between different microorganisms and different ecosystems. TEM-52-carrying cephalosporin-resistant organisms may have been transmitted from food animals to humans, or vice versa.3
For the CTX-M-2- or CTX-M-15-carrying cephalosporin-resistant organisms, exchange between food animals and humans, however, remains unclear, mainly due to the unknown plasticity and evolutionary speed of the plasmids carrying them.
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This work was supported by a grant from the Federal Public Service of Health, Food Chain Safety and Environment (grant number RT 06/3 ABRISK).
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None to declare.
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Acknowledgements |
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We are grateful to A. Carattoli for providing the plasmid incompatibility group controls. We thank Hanne Vereecke and Danielle Vandergheynst for their skilled technical assistance.
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References |
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1 Pitout JDD, Laupland KB. Extended-spectrum β-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet (2008) 8:159–66.[Web of Science]
2
Bertrand S, Weill FX, Cloeckaert A, et al. Clonal emergence of extended-spectrum β-lactamase (CTX-M-2)-producing Salmonella enterica serovar Virchow isolates with reduced susceptibilities to ciprofloxacin among poultry and humans in Belgium and France (2000 to 2003). J Clin Microbiol (2006) 44:2897–903.
3
Cloeckaert A, Praud K, Doublet B, et al. Dissemination of an extended-spectrum-β-lactamase blaTEM-52 gene-carrying IncI1 plasmid in various Salmonella enterica serovars isolated from poultry and humans in Belgium and France between 2001 and 2005. Antimicrob Agents Chemother (2007) 51:1872–5.
4
Smet A, Martel A, Persoons D, et al. Diversity of extended-spectrum β-lactamases and class C β-lactamases among cloacal Escherichia coli in Belgian broiler farms. Antimicrob Agents Chemother (2008) 52:1238–43.
5 Carattoli A, Bertini A, Villa L, et al. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods (2005) 63:219–28.[CrossRef][Web of Science][Medline]
6
Hopkins KL, Liebana E, Villa L, et al. Replicon typing of plasmids carrying CTX-M or CMY β-lactamases circulating among Salmonella and Escherichia coli isolates. Antimicrob Agents Chemother (2006) 50:3203–6.
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