Genetic characterization of extraintestinal Escherichia coli isolates from chicken, cow and swine.

Phenotypic determination of antimicrobial resistance in bacteria is very important for diagnosis and treatment, but sometimes this procedure needs further genetic evaluation. Whole-genome sequencing plays a critical role in deciphering and advancing our understanding of bacterial evolution, transmission, and surveillance of antimicrobial resistance. In this study, whole-genome sequencing was performed on nineteen clinically extraintestinal Escherichia coli isolates from chicken, cows and swine and showing different antimicrobial susceptibility. A total of 44 different genes conferring resistance to 11 classes of antimicrobials were detected in 15 of 19 E. coli isolates (78.9%), and 22 types of plasmids were detected in 15/19 (78.9%) isolates. In addition, whole-genome sequencing of these 19 isolates identified 111 potential virulence factors, and 53 of these VFDB-annotated genes were carried by all these 19 isolates. Twelve different virulence genes were identified while the most frequent ones were gad (glutamate decarboxylase), iss (increased serum survival) and lpfA (long polar fimbriae). All isolates harbored at least one of the virulence genes. The findings from comparative genomic analyses of the 19 diverse E. coli isolates in this study provided insights into molecular basis of the rising multi-drug resistance in E. coli.

Newly identified colistin resistance genes, mcr-4 and mcr-5, from upper and lower alimentary tract of pigs and poultry in China.

Antimicrobial resistance against colistin has emerged worldwide threatening the efficacy of one of the last-resort antimicrobials used for the treatment of Enterobacteriaceae. To investigate the presence of the recently identified colistin resistance genes (mcr-4, mcr-5) in China, we established PCRs to detect mcr-4 and mcr-5 on 213 anal and 1,339 nasal swabs from apparently healthy pigs (n = 1,454) in nine provinces, and 1,696 cloacal and 1,647 oropharyngeal samples from poultry (n = 1,836) at live-bird markets in 24 provinces of China. The prevalence of the mcr-4 in swine swabs (41.4%; 642/1,552) was significantly higher than in swabs from poultry (11.5%; 384/3,343). The mcr-4 gene was found in geese (49.5%, 54/109), chickens (17.2%, 257/1,498), pigeons (17.2%, 17/99) and ducks (15.4%, 20/130). In a similar trend, the prevalence of the mcr-5 in swine swabs (33.1%; 514/1552) was significantly higher than in swabs from poultry (5.6%; 187/3,343). The mcr-5 was identified in geese (17.4%, 19/109), chickens (9.9%, 148/1,498), ducks (7.7%, 10/130) and pigeons (3%, 3/99). The mcr-4 prevalence in the nasal swabs from pigs (59.2%, 58/98) was significantly higher than that in anal swabs (29.6%, 29/98) (P<0.001). Similarly, the mcr-5 prevalence in the nasal swabs from pigs (61.2%, 60/98) was significantly higher than in anal swabs (44.9%, 44/98) (P = 0.02), and significantly higher in oropharyngeal swabs (7.2%, 109/1,507) than in the cloacal swabs (3.7%, 56/1,507) (P<0.001). This study further confirms the presence of the mcr-4 and mcr-5 in animals and indicates these genes are prevalent and widespread in food producing animals (pig and poultry) in China. Future studies are needed to characterize the bacteria carrying the mcr-4 and mcr-5 and their locations on plasmids and/or the bacterial chromosomes, and determine co-resistances in the mcr-4 and mcr-5 positive strains.

Molecular detection of colistin resistance genes (mcr-1, mcr-2 and mcr-3) in nasal/oropharyngeal and anal/cloacal swabs from pigs and poultry.

Antimicrobial resistance against colistin has emerged worldwide and is threatening the efficacy of colistin treatment of multi-resistant Gram-negative bacteria. In this study, PCRs were used to detect mcr genes (mcr-1, mcr-2, mcr-3) in 213 anal and 1,339 nasal swabs from pigs (n = 1,454) in nine provinces of China, and 1,696 cloacal and 1,647 oropharyngeal samples from poultry (n = 1,836) at live-bird markets in 24 provinces. The mcr-1 prevalences in pigs (79.2%) and geese (71.7%) were significantly higher than in chickens (31.8%), ducks (34.6%) and pigeons (13.1%). The mcr-2 prevalence in pigs was 56.3%, significantly higher than in chickens (5.5%), ducks (2.3%), geese (5.5%) and pigeons (0%). The mcr-3 prevalences in pigs (18.7%), ducks (13.8%) and geese (11.9%) were significantly higher than in chickens (5.2%) and pigeons (5.1%). In total, 173 pigs and three chickens were positive for all three mcr genes. The prevalences of the mcr were significantly higher in nasal/oropharyngeal swabs than in the anal /cloacal swabs. Phylogenetic studies identified 33 new mcr-2 variants and 12 new mcr-3 variants. This study demonstrates high prevalences of mcr in pigs and poultry in China, and indicates there is need for more thorough surveillance and control programs to prevent further selection of colistin resistance.

Housefly (Musca domestica) and Blow Fly (Protophormia terraenovae) as Vectors of Bacteria Carrying Colistin Resistance Genes.

Flies have the capacity to transfer pathogens between different environments, acting as one of the most important vectors of human diseases worldwide. In this study, we trapped flies on a university campus and tested them for mobile resistance genes against colistin, a last-resort antibiotic in human medicine for treating clinical infections caused by multidrug-resistant Gram-negative bacteria. Quantitative PCR assays we developed showed that 34.1% of Musca domestica (86/252) and 51.1% of Protophormia terraenovae (23/45) isolates were positive for the mcr-1 gene, 1.2% of M. domestica (3/252) and 2.2% of P. terraenovae (2.2%, 1/45) isolates were positive for mcr-2, and 5.2% of M. domestica (13/252) and 44.4% of P. terraenovae (20/45) isolates were positive for mcr-3 Overall, 4.8% (9/189) of bacteria isolated from the flies were positive for the mcr-1 gene (Escherichia coli: 8.3%, 4/48; Enterobacter cloacae: 12.5%, 1/8; Providencia alcalifaciens: 11.8%, 2/17; Providencia stuartii: 4.9%, 2/41), while none were positive for mcr-2 and mcr-3 Four mcr-1-positive isolates (two P. stuartii and two P. alcalifaciens) from blow flies trapped near a dumpster had a MIC for colistin above 4 mg/ml. This study reports mcr-1 carriage in Providencia spp. and detection of mcr-2 and mcr-3 after their initial identification in Belgium and China, respectively. This study suggests that flies might contribute significantly to the dissemination of bacteria, carrying these genes into a large variety of ecological niches. Further studies are warranted to explore the roles that flies might play in the spread of colistin resistance genes.IMPORTANCE Antimicrobial resistance is recognized as one of the most serious global threats to human health. An option for treatment of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) bacteria with multiple drug resistance was the reintroduction of the older antibiotic colistin. However, a mobile colistin resistance gene (mcr-1) has recently been found to occur widely; very recently, two other colistin resistance genes (mcr-2 and mcr-3) have been identified in Belgium and China, respectively. In this study, we report the presence of colistin resistance genes in flies. This study also reports the carriage of colistin resistance genes in the genus Providencia and detection of mcr-2 and mcr-3 after their initial identification. This study will stimulate more in-depth studies to fully elucidate the transmission mechanisms of the colistin resistance genes and their interaction.

Identification and characterization of mcr mediated colistin resistance in extraintestinal Escherichia coli from poultry and livestock in China.

Antimicrobial resistance to colistin has emerged worldwide threatening the efficacy of one of the last-resort antimicrobials used for the treatment of multidrug-resistant Enterobacteriaceae infection in humans. In this study, we investigated the presence of colistin resistance genes (mcr-1, mcr-2, mcr-3) in Escherichia coli strains isolated from poultry and livestock collected between 2004 and 2012 in China. Furthermore, we studied the maintenance and transfer of the mcr-1 gene in E. coli after serial passages. Overall, 2.7% (17/624) of the E. coli isolates were positive for the mcr-1 gene while none were positive for the mcr-2 and mcr-3 genes. The prevalences of mcr-1 were similar in E. coli isolates from chickens (3.2%; 13/404), pigs (0.9%; 1/113) and ducks (6.8%; 3/44) but were absent in isolates from cattle (0/63). The mcr-1 gene was maintained in the E. coli after six passages (equivalent to 60 generations). In vitro transfer of mcr-1 was evident even without colistin selection. Our data indicate the presence of mcr-1 in extraintestinal E. coli from food-producing animals in China, and suggest that high numbers of the mcr-1-positive bacteria in poultry and livestock do not appear to be readily lost after withdrawal of colistin as a food additive.

Antimicrobial resistance in clinical Escherichia coli isolates from poultry and livestock, China.

Poultry and livestock are the most important reservoirs for pathogenic Escherichia coli and use of antimicrobials in animal farming is considered the most important factor promoting the emergence, selection and dissemination of antimicrobial-resistant microorganisms. The aim of our study was to investigate antimicrobial resistance in E. coli isolated from food animals in Jiangsu, China. The disc diffusion method was used to determine susceptibility to 18 antimicrobial agents in 862 clinical isolates collected from chickens, ducks, pigs, and cows between 2004 and 2012. Overall, 94% of the isolates showed resistance to at least one drug with 83% being resistance to at least three different classes of antimicrobials. The isolates from the different species were most commonly resistant to tetracycline, nalidixic acid, sulfamethoxazole, trimethoprim/sulfamethoxazole and ampicillin, and showed increasing resistance to amikacin, aztreonam, ceftazidime, cefotaxime, chloramphenicol, ciprofloxacin. They were least resistant to amoxicillin/clavulanic acid (3.4%) and ertapenem (0.2%). MDR was most common in isolates from ducks (44/44, 100%), followed by chickens (568/644, 88.2%), pigs (93/113, 82.3%) and cows (13/61, 21.3%). Our finding that clinical E. coli isolates from poultry and livestock are commonly resistant to multiple antibiotics should alert public health and veterinary authorities to limit and rationalize antimicrobial use in China.

Contribution of the csgA and bcsA genes to Salmonella enterica serovar Pullorum biofilm formation and virulence.

Salmonella biofilm formation is important to environmental stress resistance and virulence. However, the roles of the csgA and bcsA genes, which affect curli protein and cellulose production, respectively, in Salmonella enterica serovar Pullorum, are unknown. Here we constructed deletions in the csgA and bcsA genes in S. enterica serovar Pullorum strain S6702 and evaluated several aspects of biofilm formation and virulence. ΔcsgA showed decreased production of curli fimbriae, while ΔbcsA had reduced cellulose production. Both mutants had a reduced ability to form biofilms. ΔcsgA was reduced in adhesion and invasion to HeLa cells and exhibited decreased intracellular proliferation in HD11 macrophages. ΔbcsA exhibited increased proliferation in HD11 cells and replicated better in chicken spleens, as compared to the wild-type strain. ΔcsgA virulence was attenuated in assays involving oral challenge of one-day-old chickens.