The Prevalence And Characterization Of Bla_NDM-harboring E.coli Isolates Among Poultry

In the treatment of infection caused by multidrug-resistant bacteria in human clinic,carbapenems are very important because it's one of the few drugs which are efficacious.The bla _NDMgene,which can confer resistance to carbapenems,has disseminated all over the world and has also been detected in animals.The spread of bla _NDM in food animals,which are the meat source for human,brings huge risks to food safety and public health.This study investigated the prevalence and characteristics of bla _NDM-harboring E.coli among a chicken farm in Shandong province,a veterinary station,a professional chicken cooperative,and two chicken farms in Guangdong province.We isolated bla _NDM-harboring isolates from 35 organ samples collected from chicken farm in Shandong province,182,87,63,103 organ samples collected from veterinary station A,professional chicken cooperative B,chicken farm C and chicken farm D separately during 2016 and 2017.The results showed that the bla _NDM positive rates of the samples from five places are 20.0%(7/35),14.3%(26/182),1.1%(1/87),3.2%(2/63)and 0%(0/103),respectively.PCR assays and Sanger sequencing were also done to determin the subtype of bla _NDMand detect resistance genes in these bla _NDM-harboring E.coli strains,antibiotic susceptibility tests were also conducted on these strains.The results showed that positive rates of bla _NDM-1?bla_NDM-5and bla _NDM-₉were 3.3%,72.7%and 24.2%,respectively.Positive rates of other kinds of beta-lactamases genes,mcr-1,fos A3,and PMQR genes were93.9%,30.3%,51.5%,69.7%respectively with bla_TEM(72.7%)and bla_CTX-M(63.6%)being the most common two kinds of beta-lactamases genes.Resistance rates of these strains was 48.5%to aztreonam and were over 93.0%to tetracycline,doxycycline,chloramphenicol,florfenicol,ciprofloxacin,trimethoprim-sulfamethoxazole,and trimethoprim.However,resistance rates to gentamicin,tigecycline,colistin and amikacin,which were 36.4%,18.2%,33.3%and 6.1%,respectively,were relatively low.Pulsed filed gel electrophoresis(PFGE),conjugation and transformation assays,Southern blot and plasmid replicon typing assays were also performed to study the dissemination of bla _NDM and characterize bla _NDM-harboring plasmids.The results showed that bla _NDM mainly transferred horizontally,and as well vertically in a small range.The bla _NDM in all E.coli strains were located on plasmids,the sizes of which range from 45 kb to 400 kb.Seven replicon types including Inc X3,Inc B/O/K/Z,Inc Y,Inc HI2,Inc I1,Inc I1-HI2,and Inc X1-FIB(K)were detected.87.9%of these bla_NDM-harboring plasmids are transferrable at a frequency range from 7.59×10^-6 to 1.93×10^-2 with Inc X3bla _NDM-5-positive plasmid being the most common(60.6%,20/33).To study the impact of the bla _NDM-harboring plasmids on the host bacteria,we select 8different plasmids and transfered them respectively into host E.coli.Plasmid stability assay,growth kinetics and competition assays were conducted on these conjugants.The results showed that all of the bla _NDM-harboring plasmids were stably preserved in the host.Inc X3and Inc Y plasmids had no significant influence on the host while Inc I1 plasmid and two different Inc B/O/K/Z plasmids can even enhance the fitness of their host,however Inc HI2and Inc I1-HI2 plasmids reduced the fitness of their host.To further study of the molecular mechanism of bla _NDM gene transfer,some of the bla _NDM-harboring plasmids were sequenced and analyzed,the results showed that IS26-Intl1-dfr A2-hp-aad A2-qac E?1-sul1-hp-ISCR1-dct-tat-trp F-ble _MBL-bla _NDM-ISAba125-IS26and?umu D-?Tn2-IS3000-?ISAba125-IS5-?ISAba125-bla _NDM-ble _MBL-trp F-tat-?dct-IS26-?umu D were the main structures,the first structure was discovered in Inc B/O/K/Z plasmids,and the second structure was mainly discovered in Inc X3 plasmids.In addition,we discovered a new bla _NDM transfer structure IS3000-?ISAba125-IS5-?ISAba125-bla _NDM-ble _MBL-trp F-tat-?dct-IS26-?umu D-hp-?ISL3-IS3000.The mobile genetic elements such as IS26 and IS3000 accelerated the spread of bla _NDM.This study showed that the positive rate of bla _NDM in different pouitry farms maybe utterly different and the bla _NDM maybe highly prevalent in some poultry farms.The dissemination of bla _NDM were mainly involved in horizontal transfer conferred by transferrable plasmids,as well as vertical transmission in a small range.bla _NDM-harboring E.coli always carry many resistance genes and were resistant to many kinds of antibiotics and thus showed a severe resistance phenomenon.Fitness of many bla _NDM-harboring plasmids enhanced the further dissemination of this gene.The emergence and prevalence of bla _NDM-harboring E.coli in poultry reveal the risk of further dissemination of bla _NDM in food animals,and thus further investigation of carbapenem-resistant bacterias among food animals are in urgent need.