Vegetables harboring bacteria resistant to antibiotics are a growing food safety issue. However, data concerning carbapenem-resistant Enterobacteriaceae (CRE) in ready-to-eat fresh vegetables is still rare. In this study, 411 vegetable samples from 36 supermarkets or farmer's markets in 18 cities in China, were analyzed for CRE. Carbapenemase-encoding genes and other resistance genes were analyzed among the CRE isolates. Plasmids carrying carbapenemase genes were studied by conjugation, replicon typing, S1-PFGE southern blot, restriction fragment length polymorphism (RFLP), and sequencing. CRE isolates were also analyzed by pulsed-field gel electrophoresis (PFGE). Ten vegetable samples yielded one or more CRE isolates. The highest detection rate of CRE (14.3%, 4/28) was found in curly endive. Twelve CRE isolates were obtained and all showed multidrug resistance: Escherichia coli, 5; Citrobacter freundii, 5; and Klebsiella pneumoniae, 2. All E. coli and C. freundii carried blaNDM, while K. pneumoniae harbored blaKPC−2. Notably, E. coli with blaNDM and ST23 hypervirulent Klebsiella pneumoniae (hvKP) carrying blaKPC−2 were found in the same cucumber sample and clonal spread of E. coli, C. freundii, and K. pneumoniae isolates were all observed between vegetable types and/or cities. IncX3 plasmids carrying blaNDM from E. coli and C. freundii showed identical or highly similar RFLP patterns, and the sequenced IncX3 plasmid from cucumber was also identical or highly similar (99%) to the IncX3 plasmids from clinical patients reported in other countries, while blaKPC−2 in K. pneumoniae was mediated by similar F35:A-:B1 plasmids. Our results suggest that both clonal expansion and horizontal transmission of IncX3- or F35:A-:B1-type plasmids may mediate the spread of CRE in ready-to-eat vegetables in China. The presence of CRE in ready-to-eat vegetables is alarming and constitutes a food safety issue. To our knowledge, this is the first report of either the C. freundii carrying blaNDM, or K. pneumoniae harboring blaKPC−2 in vegetables. This is also the first report of ST23 carbapenem-resistant hvKP strain in vegetables.

This study investigated the characteristics of Escherichia coli isolates carrying mcr-1-bla NDM from a chicken farm in China. Of the 78 E. coli isolates, 21 clonally unrelated isolates carried mcr-1-bla NDM . Diverse IncI2 plasmids disseminated mcr-1, while the dissemination of bla NDM was mediated by diverse IncB/O plasmids. More striking was the colocalization of resistance genes mcr-1 and bla in an IncHI2/ST3 plasmid, which might pose a great challenge for public health.KEYWORDS high incidence, mcr-1, bla NDM , colocalization, Escherichia coli C arbapenems have been reliable and potent agents against Gram-negative bacteria. The rapid increase of carbapenem-resistant Enterobacteriaceae poses a great threat to public health and has prompted the reconsideration of colistin as a last-resort therapeutic option (1). Recently, a plasmid-borne colistin resistance gene (mcr-1) was identified in Escherichia coli and Klebsiella pneumoniae strains from animals and humans in China (2). This finding foreshadowed the inevitable dissemination of colistin resistance worldwide and was confirmed by the presence of mcr-1 in other countries (3-6).The mcr-1 gene is often associated with the extended-spectrum ␤-lactamase gene and has been found with bla CTX-M in the same plasmid (4,7,8). The mcr-1 gene has also emerged in carbapenem-resistant isolates (4, 9, 10), and the cotransfer of mcr-1 and carbapenem resistance genes is obviously of great clinical concern. Recently, mcr-1 was even found with bla NDM in the same IncX3-X4 hybrid plasmid in E. coli from pets in China (11). However, only a few isolates resistant to both carbapenem and colistin were characterized in those previous studies.Food-producing animals, especially chickens, serve as resistance gene "reservoirs," so it is crucial to identify the origins of multidrug-resistant plasmids in these animals. In this study, we investigated the genetically diverse E. coli isolates carrying mcr-1 and bla NDM from a chicken farm and characterized the plasmids harboring mcr-1 or bla NDM .Seventy-eight E. coli isolates were collected from diseased chickens in four separate barns on a large chicken farm (100,000 animals) in Shandong Province, China in October 2015. Feces from chickens that showed signs of diarrhea were randomly collected and streaked onto MacConkey agar. After incubating at 37°C for 20 h, one colony with typical E. coli morphology was selected from each chicken sample. Although detailed information on antibiotic usage was not available, ceftiofur and colistin were often used for prophylaxis and treatment of bacterial infections on this farm.Susceptibilities to 18 antimicrobials were determined for these 78 isolates by the agar dilution method using the recommended breakpoints (12, 13). The colistin breakpoint (Ն2 g/ml) was used according to recommendations by the European Committee for Antimicrobial Susceptibility Testing.

We report the presence of mcr-1 in Escherichia coli and carbapenemresistant Cronobacter sakazakii from the same diseased chicken. The mcr-1 gene linked with ISApl1 was located on two different IncI2 plasmids, including one multidrug plasmid in E. coli, whereas fosA3-bla NDM-9 was on an IncB/O plasmid in C. sakazakii. The development of the fosA3-bla NDM-9 resistance region was mediated by IS26. The colocation of mcr-1 or bla NDM-9 with other resistance genes will accelerate the dissemination of the two genes.KEYWORDS mcr-1, NDM-9, Cronobacter sakazakii, chicken, insertion sequences S ince the plasmid-mediated colistin resistance gene mcr-1 was first reported in Escherichia coli isolates in China (1), several reports confirmed that mcr-1 has spread in several Enterobacteriaceae species on different continents (2-5). Of great clinical concern are the inevitable concurrence of mcr-1 and carbapenem-resistance genes among Enterobacteriaceae and the massive use of colistin in animals, which could aggravate the selection process, resulting in the emergence of true pan-drug resistance. To date, the emergence of mcr-1 has been reported in carbapenem-resistant E. coli and Klebsiella pneumoniae (3, 6, 7). Here, we report the presence of mcr-1 in E. coli and the carbapenem-resistant Cronobacter sakazakii strain recovered from the same diseased chicken in China. We also report a new gene arrangement of the fosA3-bla NDM-9 resistance region.During a surveillance study in 2015 for E. coli susceptibility to carbapenem on animal farms in Shandong province, China, two carbapenem-resistant Cronobacter sakazakii isolates, WF5-19C and WF5-21C, were collected from two different diseased chickens with diarrhea at a chicken farm. Further information about these animals, the underlying disease, and possible antimicrobial pretreatment in the farm was unfortunately not available. Antimicrobial susceptibilities were assayed according to the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) (8). The breakpoints for each antimicrobial were recommended by the CLSI and veterinary CLSI (8, 9). We found that the two isolates showed resistances to each of the antimicrobial drugs tested, including colistin (MICs, 4 g/ml) (European Committee on Antimicrobial Susceptibility Testing clinical breakpoint for colistin resistance, Ն2 g/ml) (Table 1). Interestingly, E. coli isolate WF5-19 from the same chicken with C. sakazakii WF5-19C was also resistant to colistin and other antimicrobial drugs (except meropenem and imipenem). We genotyped the three carbapenem-and/or colistin-resistant isolates for the presence of carbapenemase genes and mcr-1 using PCR as described previously (1, 10, 11). The mcr-1 gene was detected in all isolates. In addition, sequencing of the bla NDM amplicons confirmed that both C. sakazakii isolates produced the NDM-9

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