JAC Advance Access originally published online on August 24, 2006
Journal of Antimicrobial Chemotherapy 2006 58(4):844-847; doi:10.1093/jac/dkl337
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Detection and characterization of extended-spectrum ß-lactamases in Salmonella enterica strains of healthy food animals in Spain
1 Area de Bioquímica y Biología Molecular, Universidad de La Rioja 26006 Logroño, Spain 2 Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid Madrid, Spain
*Corresponding author. Tel: +34-941299750; Fax: +34-941299721; E-mail: carmen.torres{at}daa.unirioja.es
Received 19 February 2006; returned 10 June 2006; revised 14 June 2006; accepted 26 July 2006
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionTransparency declarationsReferences |
|---|
Objectives: To carry out the characterization of the genes encoding extended-spectrum ß-lactamases (ESBLs) and their genetic environments in four expanded-spectrum cephalosporin-resistant Salmonella enterica isolates (serovars: two Virchow, one Enteritidis, one Rissen) recovered during the monitoring programmes performed in Spain by the VAV Network from faecal samples of pigs, poultry and laying hens at the slaughterhouse level.
Methods: The presence and characterization of ESBL genes as well as their genetic environments in the four S. enterica isolates were investigated by PCR and sequencing. The presence of other resistance genes was also analysed by PCR and sequencing.
Results: Three avian S. enterica isolates (two Virchow and one Enteritidis) harboured the blaCTX-M-9 gene combined with blaTEM-1b. The blaCTX-M-9 gene was included in these three isolates in a class 1 integron with the following 5'
3' structure: integron 1 variable region (dfrA16-aadA2 gene cassettes)-qacE
1-sul1-orf513-blaCTX-M-9-orf3-like-orf1005. The sul2 gene was also detected in these three blaCTX-M-9-containing isolates and tet(A) in one of them. The two serovar Virchow isolates showed an indistinguishable PFGE pattern, although they were recovered from different animal species (broiler and laying hen). A porcine ESBL-positive isolate (serovar Rissen) harboured the blaSHV-12 gene combined with blaTEM-1b. This blaSHV-12-containing isolate also harboured the tet(A), aadA and sul1 genes.
Conclusions: The emergence of ESBL-producing S. enterica isolates among food animals is described for the first time in Spain, with those of the CTX-M group being the predominant ESBLs detected.
Keywords: S. enterica , ESBLs , CTX-M-9 , integrons , SHV-12
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsReferences |
|---|
Salmonella enterica is a zoonotic bacteria transmitted through the food chain. Salmonella isolates harbouring extended-spectrum ß-lactamases (ESBLs) have emerged worldwide during the last decade, with the CTX-M group being particularly important.1 In addition, the occurrence of different genes encoding CTX-M enzymes within integron structures that facilitate its dissemination in different environments has been reported.1,2 A large number of studies have investigated the presence of ESBLs in Salmonella strains from human patients,3 but only a few studies have been carried out in strains of food-producing animals or food products,4–6 and none of them in Spain. The characterization of genes encoding ESBLs and their genetic environments has been carried out in the present study in four S. enterica isolates recovered from faecal samples of food-producing animals in Spain.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsReferences |
|---|
The Spanish Veterinary-Antimicrobial-Resistance-Surveillance Network (VAV) is monitoring antimicrobial resistance of S. enterica obtained from faecal samples of healthy food animals from 1999 at the slaughterhouse level in Spain (pigs, 1999–2004: n = 436; laying hens, 2003: n = 44; and broilers, 2003–2004: n = 76). Four of these 556 S. enterica isolates showed a resistant phenotype for expanded-spectrum cephalosporins (cefotaxime and/or ceftazidime) and also a positive ESBL synergy test (synergy between clavulanic acid and cefotaxime or ceftazidime), and were included in the study for ESBL genetic characterization. These four isolates were recovered as follows: one (serovar Rissen) porcine isolate from 2003 (n = 129); one (serovar Virchow) laying hen isolate from 2003 (n = 44); and two broiler isolates (serovars Virchow and Enteritidis) from 2004 (n = 36).
Susceptibility testing to 26 antimicrobials (ampicillin, amoxicillin, amoxicillin/clavulanic acid, ticarcillin, cefalotin, cefoxitin, cefotaxime, cefotaxime/clavulanic acid, ceftazidime, imipenem, aztreonam, gentamicin, apramycin, tobramycin, amikacin, streptomycin, neomycin, nalidixic acid, ciprofloxacin, chloramphenicol, florfenicol, tetracycline, fosfomycin, sulphonamides, trimethoprim and trimethoprim/sulfamethoxazole) was determined in the four ESBL-positive isolates by agar dilution and/or disc diffusion methods (NCCLS).
The presence of genes encoding TEM (forward, 5'-TTCTTGAAGACGAAAGGGC-3'; reverse, 5'-ACGCTCAGTGGAACGAAAAC-3'), SHV (forward, 5'-CACTCAAGGATGTATTGTG-3'; reverse, 5'-TTAGCGTTGCCAGTGCTCG-3'), OXA-1 (forward, 5'-ACACAATACATATCAACTTCGC-3'; reverse, 5'-AGTGTGTTTAGAATGGTGATC-3'), OXA-2 (forward, 5'-TTCAAGCCAAAGGCACGATAG-3'; reverse, 5'-TCCGAGTTGACTGCCGGGTTG-3'), OXA-10 (forward, 5'-CGTGCTTTGTAAAAGTAGCAG-3'; reverse, 5'-CATGATTTTGGTGGGAATGG-3'), CTX-M-9 group (forward, 5'-GTGACAAAGAGAGTGCAACGG-3'; reverse, 5'-ATGATTCTCGCCGCTGAAGCC-3'), CTX-M-3 group (forward, 5'-GTTACAATGTGTGAGAAGCAG-3'; reverse, 5'-CCGTTTCCGCTATTACAAAC-3'), CTX-M-10 (forward, 5'-CCGCGCTACACTTTGTGGC-3'; reverse, 5'-TTACAAACCGTTGGTGACG-3'), CMY-type (forward, 5'-GATTCCTTGGACTCTTCAG-3'; reverse, 5'-TAAAACCAGGTTCCCAGATAGC-3') and PSE (forward, 5'-TGCTTCGCAACTATGACTAC-3'; reverse, 5'-AGCCTGTGTTTGAGCTAGAT-3') ß-lactamases was analysed by PCR and sequencing.7,8 The sequences were compared with those included in the GenBank database in order to ascribe the specific type of ß-lactamase gene.
The presence of class 1 and class 2 integrons as well as the characterization of their gene cassettes were studied by PCR and sequencing of the variable regions in the four ESBL-positive S. enterica isolates.8–10 In addition, other genes associated with tetracycline [tet(A) and tet(B)] or sulfamethoxazole resistance (sul1, sul2 or sul3) were also analysed by PCR and sequencing.8
The clonal relationship among the isolates was studied by PFGE using XbaI enzyme for chromosomal DNA restriction.
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsReferences |
|---|
The characteristics of the four ESBL-positive S. enterica isolates are shown in Table 1. The three avian S. enterica isolates harboured the blaCTX-M-9 gene combined with blaTEM-1b and showed higher MIC values of cefotaxime (
256 mg/L) than of ceftazidime (1–2 mg/L). They also presented resistance to ampicillin, ticarcillin, amoxicillin, cefalotin, trimethoprim, trimethoprim/sulfamethoxazole, streptomycin, sulphonamides and nalidixic acid, and one of them also to tetracycline.
|
The variable regions of class 1 integrons were amplified by PCR in the three S. enterica isolates harbouring a blaCTX-M-9 gene and the sequences of the obtained amplicons showed the combination of the dfrA16 plus aadA2 gene cassettes in the three isolates (Table 1). In order to know the possible inclusion of the blaCTX-M-9 gene into this integron structure, a wide variety of primers based on the In60 structure (GenBank accession number AF174129 [GenBank] ) were used,8–10 to amplify the upstream and the downstream regions of the blaCTX-M-9 gene. All the obtained amplicons were sequenced. Figure 1 shows the confirmed structure found in these three strains after the comparison of the sequences obtained with those included in the GenBank database. The variable region of class 1 integrons (containing the dfrA16 and aadA2 gene cassettes) followed by the 3' conserved region (including the qacE
1 and sul1 genes) and orf513 were detected upstream of the blaCTX-M-9 gene in these three S. enterica isolates (Figure 1). The orf3-like sequence followed by the orf1005 sequence were detected downstream of the blaCTX-M-9 gene. Curiously, the intI1 gene was not found by PCR in any of these three CTX-M-9-harbouring isolates; in addition, negative PCR results were also obtained when degenerate primers for integrases of classes 1, 2 and 3 were used (forward, 5'-TGCGGGTYAARGATBTKGATTT-3'; reverse, 5'-CARCACATGCGTRTARAT-3').
|
The sequence of both the variable region of class 1 integrons and the open reading frame orf513 in our three blaCTX-M-9-harbouring S. enterica isolates was 100% identical to the corresponding sequence of the In36 integron (GenBank accession no. AY259085 [GenBank] ) and 99% identical to the corresponding sequence of In60 (GenBank accession no. AF174129 [GenBank] ). Similar sequences have been previously found in S. enterica serovar Virchow strains from poultry isolates.6 Nevertheless, the sequence of the downstream region of blaCTX-M-9 in our strains was 100% identical to In60, first reported by Sabaté et al.2 in an Escherichia coli strain harbouring the blaCTX-M-9 gene. In36 and In60 belong to the In6–In7 integron family and both contain a second copy of the 3'- conserved region. Both integrons have the same structure in their 5' region that includes the genes intI1-dfrA16-aadA2-qacE
1-sul1-orf513, but they differ in their 3' region.2 Both Virchow isolates showed an indistinguishable PFGE pattern, although they were recovered from different animal species (broiler and laying hen), indicating the presence of the same clone from different sources.
The porcine ESBL-positive isolate (serovar Rissen) harboured the blaSHV-12 gene combined with blaTEM-1b (Table 1). This isolate presented a high MIC value of both cefotaxime and ceftazidime (256 mg/L), also showing resistance to ampicillin, ticarcillin, amoxicillin, aztreonam, cefalotin, streptomycin, sulphonamides and tetracycline. The sul1, tet(A) and aadA genes were detected in this isolate, although it did not harbour class 1 or class 2 integrons.
The first ß-lactamase of the CTX-M group was detected in a cefotaxime-resistant E. coli strain in Japan in 1986 and was designated as FEC-1, followed by the detection of CTX-M-1 in a clinical E. coli strain in Germany in 1989.1 In 1992, the first Salmonella strain harbouring a CTX-M ß-lactamase (CTX-M-2) was reported in South America, and later this type of resistance was detected in other countries of different continents.1 The first report of the ß-lactamase CTX-M-9 was in 1996 in a clinical E. coli strain in Spain, being first found in S. enterica in 2000.3 To our knowledge, there are only three previous reports (carried out in the Netherlands, Greece and France) in which genes encoding CTX-M ß-lactamases were detected in Salmonella strains of animal origin,4–6 the genes detected being blaCTX-M-2, blaCTX-M-9 and blaCTX-M-32. This is the first time that S. enterica of animal origin, harbouring genes encoding ESBL, have been found in Spain.
In the present study, we report the emergence of ESBL-producing S. enterica isolates, mainly of the CTX-M group, in food-producing animals at the slaughterhouse in Spain. It is important to underline that the blaCTX-M-9 gene in these isolates is incorporated in the structure of an integron of class 1, associated with genes that confer resistance to antimicrobials that could be extensively used among animals and humans (such as trimethoprim, sulfamethoxazole or streptomycin) and that could be an important factor for selection of multiresistant S. enterica strains harbouring blaCTX-M genes. More studies should be carried out in the future in order to track the evolution of this type of resistance among S. enterica living in different ecosystems (humans, animals and food).
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsReferences |
|---|
Nothing to declare.
|
Acknowledgements |
|---|
This work was supported in part by the Project AGL 2002-02637. The Spanish Ministerio de Agricultura, Pesca y Alimentación is financing VAV Network.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsReferences |
|---|
1 Bonnet R. (2004) Growing group of extended-spectrum ß-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother 48:1–14.
2
Sabaté M, Navarro F, Miró E, et al. (2002) Novel complex sul1-type integron in Escherichia coli carrying blaCTX-M-9. Antimicrob Agents Chemother 46:2656–61.
3
Simarro E, Navarro F, Ruiz J, et al. (2000) Salmonella enterica serovar Virchow with CTX-M-like ß-lactamase in Spain. J Clin Microbiol 38:4676–8.
4
Hasman H, Mevius D, Veldman K, et al. (2005) ß-Lactamases among extended-spectrum ß-lactamase (ESBL)-resistant Salmonella from poultry, poultry products and human patients in The Netherlands. J Antimicrob Chemother 56:115–21.
5
Politi L, Tassios PT, Lambiri M, et al. (2005) Repeated occurrence of diverse extended-spectrum ß-lactamases in minor serotypes of food-borne Salmonella enterica subsp. enterica. J Clin Microbiol 43:3453–6.
6
Weill FX, Lailler R, Praud K, et al. (2004) Emergence of extended-spectrum-ß-lactamase (CTX-M-9)-producing multiresistant strains of Salmonella enterica serotype Virchow in poultry and humans in France. J Clin Microbiol 42:5767–73.
7
Briñas L, Moreno MA, Zarazaga M, et al. (2003) Detection of CMY-2, CTX-M-14, and SHV-12 ß-lactamases in Escherichia coli fecal-sample isolates from healthy chickens. Antimicrob Agents Chemother 47:2056–8.
8
Sáenz Y, Briñas L, Domínguez E, et al. (2004) Mechanisms of resistance in multiple-antibiotic resistant Escherichia coli strains of human, animal, and food origins. Antimicrob Agents Chemother 48:3996–01.
9 Lartigue MF, Poirel L, Nordmann P. (2004) Diversity of genetic environment of blaCTX-M genes. FEMS Microbiol Lett 234:201–7.[CrossRef][Web of Science][Medline]
10 Saladin M, Cao VT, Lambert T, et al. (2002) Diversity of CTX-M ß-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. FEMS Microbiol Lett 209:161–8.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  L. Yuan, J.-H. Liu, G.-Z. Hu, Y.-S. Pan, Z.-M. Liu, J. Mo, and Y.-J. Wei Molecular characterization of extended-spectrum {beta}-lactamase-producing Escherichia coli isolates from chickens in Henan Province, China J. Med. Microbiol., November 1, 2009; 58(11): 1449 - 1453. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Vinue, M. Lantero, Y. Saenz, S. Somalo, I. de Diego, F. Perez, F. Ruiz-Larrea, M. Zarazaga, and C. Torres Characterization of extended-spectrum {beta}-lactamases and integrons in Escherichia coli isolates in a Spanish hospital J. Med. Microbiol., July 1, 2008; 57(7): 916 - 920. [Full Text] [PDF]
|
|
|
|
 |
|
 A. Smet, A. Martel, D. Persoons, J. Dewulf, M. Heyndrickx, B. Catry, L. Herman, F. Haesebrouck, and P. Butaye Diversity of Extended-Spectrum {beta}-Lactamases and Class C {beta}-Lactamases among Cloacal Escherichia coli Isolates in Belgian Broiler Farms Antimicrob. Agents Chemother., April 1, 2008; 52(4): 1238 - 1243. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. E. Warren, V. M. Ensor, P. O'Neill, V. Butler, J. Taylor, K. Nye, M. Harvey, D. M. Livermore, N. Woodford, and P. M. Hawkey Imported chicken meat as a potential source of quinolone-resistant Escherichia coli producing extended-spectrum {beta}-lactamases in the UK J. Antimicrob. Chemother., March 1, 2008; 61(3): 504 - 508. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||