Article

Molecular and Phenotypic Characteristics of CMY-2 β-Lactamase-Producing Salmonella enterica Serovar Typhimurium Isolated from Cattle in Japan

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Abstract

Isolates of extended-spectrum cephalosporin (ESC)-resistant Salmonella enterica serovar Typhimurium obtained from two different farms in Fukushima Prefecture, Japan, in 2007 were characterized in order to determine the genetic basis of resistance. ESC resistance in the two isolates was mediated by an AmpC β-lactamase encoded by the bla(CMY-2) gene, which is located in a large self-transmissible plasmid in each isolate. The sizes of the bla(CMY-2)-carrying plasmids were different. The replicon types of the plasmids were I1-Iγ and A/C. The results of macrorestriction analysis and phage typing suggest a close relationship between both isolates. This is the first report of ESC-resistant S. Typhimurium isolated from cattle in Japan.

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... Current mechanisms include the plasmid mediated class C beta-lactamases encoded by bla(CMY-2) genes, which are important for the spread of cephalosporin resistance and were detected in Salmonella spp. isolated from retail meat or animals in several countries such as the US, Japan and China (Dahshan et al., 2010;Glenn et al., 2011;Heider et al., 2009;M'ikanatha N et al., 2010;Sugawara et al., 2011;Yang et al., 2010). Plasmid-mediated quinolone resistant qnr-positive Salmonella isolates have been found in different countries (Veldman et al., 2008). ...
... Third generation cephalosporins are the drug of choice for invasive Salmonella infections treatment, especially in children and growing adolescents where treatment with fluoroquinolones is not recommended (Scientific Advisory Group on Antimicrobials of the Committee for Medicinal Products for Veterinary Use, 2009). Resistance to extended-spectrum cephalosporins has been associated with bacterial production of extended-spectrum β-lactamases (ESBLs, particularly CTX-M, TEM, and SHV), and plasmid-mediated AmpC β-lactamases (particularly CMY-2) (Fabre et al., 2009;Pardos de la Gandara et al., 2011;Rotimi et al., 2008;Smet et al., 2008;Sugawara et al., 2011). The genes encoding these enzymes are located on transferable genetic elements such as plasmids, transposons and integrons; therefore spread of resistance can occur both through dissemination of clones, and through horizontal transmission (Scientific Advisory Group on Antimicrobials of the Committee for Medicinal Products for Veterinary Use, 2009). ...
... Co-resistance to other structurally unrelated antimicrobials such as aminoglycosides, tetracyclines, and sulphonamides has been reported (Pardos de la Gandara et al., 2011; Scientific Advisory Group on Antimicrobials of the Committee for Medicinal Products for Veterinary Use, 2009). In addition, multiresistant CTX-M-producing strains from humans have been shown to carry transferable quinolone resistance genes (Canton and Coque, 2006;Fabre et al., 2009) which is a cause of concern as resistance to these important antibiotics could be spread easily among organisms (Aouf et al., 2011;Archambault et al., 2006;Doublet et al., 2009;Sjolund-Karlsson et al., 2010;Sugawara et al., 2011). In Singapore, ceftriaxone-resistant Salmonella spp. ...
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Antimicrobial resistance is a global problem. It is most prevalent in developing countries where infectious diseases remain common, the use of antibiotics in humans and animals is widespread, and the replacement of older antibiotics with new generation antibiotics is not easy due to the high cost. Information on antibiotic resistance phenotypes and genotypes of Salmonella spp. in food animals and humans in different countries and geographic regions is necessary to combat the spread of resistance. This will improve the understanding of antibiotic resistance epidemiology, tracing of new emerging pathogens, assisting in disease treatment, and enhancing prudent use of antibiotics. However, the extent of antibiotic resistance in food-borne pathogens and humans in many developing countries remains unknown. The goal of this review is to discuss the current state of antibiotic resistance of non-typhoid Salmonella spp. in food-producing animals, retail meat and humans from South East Asia. It is focused on resistance characteristics of traditional and "critically important" antibiotics in this region, and the emergence of multidrug resistant strains and genetic elements that contribute to the development of multidrug resistance, including integrons and the Salmonella Genomic Island (SGI).
... Recently, Salmonella has developed resistance to cephalosporin through the transmission of PABL [12], of which CMY-2 is the most common. CMY-2 was first reported in the USA and is the most widely distributed PABL, with cases also reported in France, Germany, Greece and the United Kingdom; indeed, it was recently isolated from a cow in Japan and from pigs in China [1,3,[12][13][14]. In most cases, the CMY-2 gene is present in large plasmids, of which several genetic types have been reported. ...
... A study of 283 Salmonella sp isolated from Korean chickens between 2002 and 2010 showed that 17 of the ceftiofur-resistant isolates were positive for genes encoding CTX-M-14 and CTX-M-15 [9]. Another study found that two S. Typhimurium strains isolated from cattle in Japan harbored both TEM-1 and CMY-2 [14]. Plasmid-mediated AmpC-β-lactamases are frequently identified in human Salmonella isolates in South Korea [17]; however, until now, CMY-2 has not been isolated from cattle or pigs. ...
... These plasmids can be classified according to size, composition, and incompatibility (Inc) type, and by plasmid multilocus sequence typing [12,14,18]. More recently, the Inc type has been used to classify plasmids. ...
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Abstracts Background Salmonella resistant to third-generation cephalosporin has been isolated from an increasing number of animals worldwide. The purpose of this study was to examine ESBL (extended-spectrum β-lactamases)-producing and PABL (plasmid-mediated AmpC β-lactamases)-producing Salmonella isolates from pigs in South Korea. Results Salmonella Typhimurium KVCC-BA1300259 was resistant to ampicillin, amoxicillin/clavulanic acid, cephalothin, chloramphenicol, florfenicol, cefoxithin, gentamicin, nalidixic acid, trimethoprim/sulfamethoxazole, tetracycline, and ceftiofur. The results of a double-disk synergy test and PCR confirmed that the isolate produced CMY-2 (PABL). Analysis of plasmid incompatibility (Inc) groups revealed the presence of IncA/C and IncFIB, indicating antimicrobial resistance. This study is the first to identify S. Typhimurium isolates harboring CMY-2 in pigs in South Korea. Conclusions The presence of CMY-2 in pigs poses a significant threat of possible horizontal spread between animals and humans.
... One of the studies involving Salmonella enterica serovar Typhimurium (S. Typhimurium) identified blaCMY-2 associated with self-transmissible IncI1-Iγ and A/C plasmids (Sugawara et al., 2011). Another study revealed a novel chromosomally integrated multi-drug resistance genomic island harboring blaCMY-2 among clonally related S. Typhimurium isolates (Shahada et al., 2011). ...
... Salmonella carrying the blaCMY-2 gene were recently recovered from bovine and porcine salmonellosis cases (Dahshan et al., 2010; Sugawara et al., 2011). Besides, S. Infantis isolates harboring the blaTEM-52 gene were reported from broilers for the first time in the year 2004 (Shahada et al., 2010a). ...
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Epidemiologic surveillance study was conducted in southern Japan to determine the antimicrobial resistance phenotypes and characterize the β-lactamase genes and the plasmids harboring these genes in Salmonella enterica serovar Infantis (S. Infantis) isolates from broilers. Between January, 2007 and December, 2008, a total of 1,472 fecal samples were collected and examined at the Laboratory of Veterinary Public Health, Kagoshima University, Japan. In 93 (6.3%) isolates recovered, 33 (35.5%) isolates showed resistance to cefotaxime, an extended-spectrum cephalosporin (ESC), conferred by TEM-20, TEM-52 and CTX-M-25 extended-spectrum β-lactamases (ESBLs). In addition to ESC-resistance, eight (8.6%) isolates exhibited resistance to cefoxitin mediated by CMY-2 AmpC β-lactamase. Plasmid analysis and polymerase chain reaction replicon typing revealed the bla TEM-20 and bla CMY-2 genes were associated with IncP plasmids, bla TEM-52 was linked with a non-typable plasmid and bla CTX-M-25 was carried by an IncA/C plasmid. Non-β-lactam resistance to streptomycin, sulfamethoxazole, and oxytetracycline encoded by the aadA1, sul1, and tet(A) genes, respectively, was found in 86 (92.5%) isolates. Resistance to kanamycin and ofloxacin was exhibited in 12 (12.9%) and 11 (11.8%) isolates, respectively, the former was mediated by aphA1-Iab. These data indicate that S. Infantis isolates producing ESBLs and AmpC β-lactamase have spread among broiler farms in Japan. These data demonstrated that the incidence of ESC-resistant S. Infantis carrying bla TEM-52 remarkably increased and S. Infantis strains harboring bla CMY-2, bla TEM-20, or bla CTX-M-25 genes emerged from broilers in Japan for the first time in 2007 and 2008.
... In the present study, IncFIB plasmids with multidrug resistance genes carried bla CTX-M-15 , bla TEM-1 and qnrS1, whereas IncA/C plasmids carried bla TEM-1 and bla CMY-4 . In Japan, bla CMY-2 -haboring IncA/C plasmids carrying multidrug resistance genes have been identified in BSC-resistant E. coli and S. typhimurium from broiler chickens and cattle, respectively (23,34). In contrast, IncF, including IncFIB, is a replicon type detected in bla CTX-M-15 -haboring plasmids (33). ...
Article
Although antimicrobial products are essential for the treatment of bacterial disease, antimicrobial treatment selects for antimicrobial-resistant (AMR) bacteria. The aim of this study was to determine the effects of first-generation cephalosporin administration on the development of resistant Escherichia coli in dog feces. The proportions of cephalexin (LEX)-resistant E. coli in fecal samples of three healthy dogs treated intravenously with cefazolin before castration and then orally with LEX for 3 days post-operation (PO) were examined using DHL agar with or without LEX (50 μg/mL). LEX-resistant E. coli was rapidly found within 3 days PO, accounted for 100% of all identified E. coli 3-5 days PO in all dogs, and was predominantly found until 12 days PO. LEX-resistant E. coli isolates on DHL agar containing LEX were subjected to antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) genotyping, β-lactamase typing, and plasmid profiling. All isolates tested exhibited cefotaxime (CTX) resistance (CTX minimal inhibitory concentration > 4 μg/mL). We classified seven PFGE profiles into five groups and three β-lactamase combinations (blaCMY-4-blaTEM-1, blaTEM-1-blaCTX-M-15, and blaTEM-1-blaCTX-M-15-blaCMY-4). All of the isolates exhibited identical PFGE profiles in each dog 4-5 days PO and subsequently showed divergent PFGE profiles. Our results indicate the existence of two selection periods for AMR bacteria resulting from the use of antimicrobials. Thus, continuous hygiene practice is necessary to prevent AMR bacteria transfer via dog feces after antimicrobial administration.
... These two Inc group plasmids are also associated with the spread of bla CMY and bla CTX-M in food-producing animals. In Japan, Inc A/C, Inc I1, and Inc P plasmids carrying bla CMY-2 have been identified in Salmonella isolates from cattle, and broilers (14,15). Though limited in number, those Inc types of plasmids were detected among NTS isolates harboring bla CMY-2 from food handlers in addition to diarrheic patients, implying concerns about future increase of third-generation cephalosporin-resistance among invasive NTS which bring an important issue affecting clinical and public health. ...
Article
Detection and characterization of β-lactamase genes in a total of ten non-typhoidal Salmonella (NTS) clinical isolates exhibiting resistance to the third-generation cephalosporins collected during 2012 -2014 in Japan were conducted. Among five strains with cefotaxime MIC (minimum inhibitory concentration) ≥64 μg/ml and positive results of clavulanic acid inhibition effect, blaCTX-M-2 was detected in three strains (serotypes Stanley and Muenchen), and each of blaTEM-52 (serotype Manhattan) and blaSHV-12 (serotype Infantis) was found in one strain. The blaCMY-2 was detected in all of the remaining five strains (serotypes Infantis, Rissen, Newport, and Saintpaul) with cefotaxime MICs of 4 to 32 μg/ml and positive results of cloxacillin- and 3-aminophenylboronic acid-based inhibition tests. ISEcp1 was located upstream of the blaCMY-2 in four strains and of the blaCTX-M-2 in one strain. Incompatibility (Inc) A/C, Inc P, and Inc I1 plasmids were present in strains harboring blaCMY-2 which were detected predominantly in this study. Acquisition of resistance to the third-generation cephalosporins by invasive NTS may well limit therapeutic options for severe systemic infections, and would cause serious public health problem. Though such resistant clinical isolates are still rare in Salmonella species in Japan, our findings revealed the presence of cephem-resistant NTS among food handlers, thus underline the necessity of more systematic nationwide investigation.
... bla CTX-M2 was found in E. coli from cattle in Japan from 2000 to 2001 [30]. bla CMY-2 was found in I1-Ic and A/C plasmids in Salmonella enterica serovar Typhimurium isolated from cattle in Japan in 2007 [31]. bla CTX-M-14 was found in S. enterica serovar Enteritidis from chicken meat imported from China and sold by a retailer in Japan in 2004 [32]. ...
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The use of extended-spectrum cephalosporins in food animals has been suggested to increase the risk of spread of Enterobacteriaceae carrying extended-spectrum β-lactamases to humans. However, evidence that selection of extended-spectrum cephalosporin-resistant bacteria owing to the actual veterinary use of these drugs according to criteria established in cattle has not been demonstrated. In this study, we investigated the natural occurrence of cephalosporin-resistant Escherichia coli in dairy cattle following clinical application of ceftiofur. E. coli isolates were obtained from rectal samples of treated and untreated cattle (n = 20/group) cultured on deoxycholate-hydrogen sulfide-lactose agar in the presence or absence of ceftiofur. Eleven cefazoline-resistant isolates were obtained from two of the ceftiofur-treated cattle; no cefazoline-resistant isolates were found in untreated cattle. The cefazoline-resistant isolates had mutations in the chromosomal ampC promoter region and remained susceptible to ceftiofur. Eighteen extended-spectrum cephalosporin-resistant isolates from two ceftiofur-treated cows were obtained on ceftiofur-supplemented agar; no extended-spectrum cephalosporin-resistant isolates were obtained from untreated cattle. These extended-spectrum cephalosporin-resistant isolates possessed plasmid-mediated β-lactamase genes, including blaCTX-M-2 (9 isolates), blaCTX-M-14 (8 isolates), or blaCMY-2 (1 isolate); isolates possessing blaCTX-M-2 and blaCTX-M-14 were clonally related. These genes were located on self-transmissible plasmids. Our results suggest that appropriate veterinary use of ceftiofur did not trigger growth extended-spectrum cephalosporin-resistant E. coli in the bovine rectal flora; however, ceftiofur selection in vitro suggested that additional ceftiofur exposure enhanced selection for specific extended-spectrum cephalosporin-resistant β-lactamase-expressing E. coli clones.
... (13), and poultry meat is most frequently contaminated with CMY-2-producing Salmonella (14). Indeed, CMY-2-producing S. Infantis and S. Typhimurium isolates have been found in retail chicken and cattle in Japan (15,16). Because of the clinical importance of third-and fourth-generation cephalosporin in human and veterinary medicine, it is of further concern that insufficient testing might lead to antimicrobial-resistant Salmonella isolates being missed. ...
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Whole genome sequencing of non-H(2)S-producing Salmonella enterica serovar Typhimurium and Infantis isolates from poultry meat revealed a nonsense mutation in the phsA thiosulfate reductase gene and carriage of a CMY-2 ß-lactamase. The non-production of H(2)S might lead to the incorrect identification of S. enterica isolates carrying antimicrobial resistant genes.
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