BackgroundThe association of PMQR and ESBLs in negative-bacteria isolates has been of great concern. The present study was performed to investigate the prevalence of co-transferability of oqxAB and bla CTX-M genes among the 696 Escherichia coli (E. coli) isolates from food-producing animals in South China, and to characterize these plasmids.MethodsThe ESBL-encoding genes (bla CTX-M, bla TEM and bla SHV), and PMQR (qnrA, qnrB, qnrS, qnrC, qnrD, aac(6’)-Ib-cr, qepA, and oqxAB) of these 696 isolates were determined by PCR and sequenced directionally. Conjugation, S1 nuclease pulsed-field gel electrophoresis (PFGE) and Southern blotting experiments were performed to investigate the co-transferability and location of oqxAB and bla CTX-M. The EcoRI digestion profiles of the plasmids with oqxAB-bla CTX-M were also analyzed. The clonal relatedness was investigated by PFGE.ResultsOf the 696 isolates, 429 harbored at least one PMQR gene, with oqxAB (328) being the most common type; 191 carried bla CTX-M, with bla CTX-M-14 the most common. We observed a significant higher prevalence of bla CTX-M among the oqxAB-positive isolates (38.7%) than that (17.4%) in the oqxAB-negative isolates. Co-transferability of oqxAB and bla CTX-M was found in 18 of the 127 isolates carrying oqxAB-bla CTX-M. These two genes were located on the same plasmid in all the 18 isolates, with floR being on these plasmids in 13 isolates. The co-dissemination of these genes was mainly mediated by F33:A-: B- and HI2 plasmids with highly similar EcoRI digestion profiles. Diverse PFGE patterns indicated the high prevalence of oqxAB was not caused by clonal dissemination.Conclusion bla CTX-M was highly prevalent among the oqxAB-positive isolates. The co-dissemination of oqxAB-bla CTX-M genes in E. coli isolates from food-producing animals is mediated mainly by similar F33:A-: B- and HI2 plasmids. This is the first report of the co-existence of oqxAB, bla CTX-M, and floR on the same plasmids in E. coli.

Tigecycline serves as one of the antibiotics of last resort to treat multidrug-resistant (including carbapenem-resistant) pathogens. However, the recently emerged plasmid-mediated tigecycline resistance mechanism, Tet(X), challenges the clinical efficacy of this class of antibiotics. In this study, we detected 180 tet(X)-harboring Acinetobacter isolates (8.9%, n = 180) from 2,018 samples collected from avian farms and adjacent environments in China. Eighteen tet(X)-harboring isolates (10.0%) were found to cocarry the carbapenemase gene blaNDM-1, mostly from waterfowl samples (94.4%, 17/18). Interestingly, among six Acinetobacter strains, tet(X) and blaNDM-1 were found to colocalize on the same plasmids. Moreover, whole-genome sequencing (WGS) revealed a novel orthologue of tet(X) in the six isolates coharboring tet(X) and blaNDM-1. Inverse PCR suggested that the two tet(X) genes form a single transposable unit and may be cotransferred. Sequence comparison between six tet(X)- and blaNDM-1-coharboring plasmids showed that they shared a highly homologous plasmid backbone even though they were isolated from different Acinetobacter species (three from Acinetobacter indicus, two from Acinetobacter schindleri, and one from Acinetobacter lwoffii) from various sources and from different geological regions, suggesting the horizontal genetic transfer of a common tet(X)- and blaNDM-1-coharboring plasmid among Acinetobacter species in China. Emergence and spread of such plasmids and strains are of great clinical concern, and measures must be implemented to avoid their dissemination.

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.

The results suggest that both clonal expansion and horizontal transmission of IncHI2-type plasmids containing oqxAB and aac(6')-Ib-cr may be involved in the spread of oqxAB in Salmonella Typhimurium isolates in food-producing animals in China. There is a great need to monitor the potential dissemination of this multiresistance gene.

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

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.

In this study, the prevalence of plasmid-mediated quinolone resistance (PMQR) was investigated in 495 Escherichia coli isolates from diseased food-producing animals in Guangdong province, China. The quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were analysed for mutations in 55 isolates harbouring only oqxAB and all isolates harbouring other PMQR genes. Overall, 282 (57.0 %) E. coli isolates had at least one PMQR gene. oqxAB was detected in 215 isolates and predominated the PMQR genes, followed by qnrS (63 isolates), aac(69)-Ib-cr (56 isolates), qnrB (39 isolates) and qepA (18 isolates). qnrA, qnrC and qnrD were not found in any of the isolates. The rates of resistance to ciprofloxacin, enrofloxacin, levofloxacin and nalidixic acid were 75.2, 81.0, 70.5 and 97.4 %, respectively, among the 495 isolates. Eight types of mutation in gyrA were detected in 154 PMQR-positive isolates, and 147 isolates were found to have mutations in parC. PFGE analysis indicated that the PMQR-positive E. coli isolates were genetically diverse. This study demonstrated that the number of mutations in QRDRs of gyrA and/or parC was significantly associated with the MICs of quinolones (P,0.01). The rates of resistance to ciprofloxacin, enrofloxacin and nalidixic acid in PMQR-positive isolates were significantly higher than those in PMQR-negative isolates (P,0.05). In addition, the prevalence of oqxAB had significant Spearman correlation coefficients in relation to the MICs of all four tested quinolones (P,0.01). INTRODUCTIONEscherichia coli is a common pathogen and certain strains can cause intestinal or extra-intestinal infections in humans and animals. Fluoroquinolones have been used effectively against most bacterial infections because of their broadspectrum antimicrobial activity (Hopkins et al., 2005). However, increasing use of these agents has led to rising rates of resistance to fluoroquinolones in E. coli worldwide (Threlfall et al., 1997). In enterobacteria, the major mechanisms of resistance to quinolones involve mutations of chromosomal genes encoding DNA gyrase and/or topoisomerase IV, mutations of genes regulating the expression of efflux pumps (Hooper, 1999) and a decrease in the permeability of the bacterial cell wall (Nikaido, 2003), all of which are chromosomally mediated. In addition, a number of plasmid-mediated quinolone resistance (PMQR) mechanisms have been described: the pentapeptide repeat family Qnr proteins (QnrA, QnrB, QnrS, QnrC and QnrD;Martínez-Martínez et al., 1998; Hata et al., 2005;Jacoby et al., 2006;Cavaco et al., 2009;Wang et al., 2009), AAC(69)-Ib-cr, an aminoglycoside acetyltransferase that confers reduced susceptibility to ciprofloxacin by modifying ciprofloxacin (Robicsek et al., 2006b), and QepA, an efflux pump belonging to the major facilitator subfamily (Yamane et al., 2007). Recently, OqxAB, a multidrug efflux pump that confers resistance to multiple agents, has also been reported to reduce susceptibility to ciprofloxacin and nalidixic acid (Hansen et al., 2007). The...

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