Prevalent And Resistance Characteristics Of Bla_NDM In Escherichia Coli From Human And Animals

Pathogenic Escherichia coli often causes multiple infections and has become one of the most important pathogens for animal and human infections.Carbapenems are one of the important antibiotics for the treatment of bacterial infections in human medicine.Although these drugs have not been approved in animals in China,carbapenem-resistant genes are still found in E.coli from animals.At present,NDM enzyme is one of the most widely popular among carbapenem hydrolase.The emergence and spread of blaNDM resistance genes of E.coli from different sources have a certain threat to public health safety.In this study,a total of 1013 E.coli from 2007 to 2018 were tested the minimum inhibitory concentration against 15 antibacterial drugs by agar dilution method.The results showed the lowest resistance rate to meropenem was 0.69%,followed by amikacin is 8.88%,less than 10%,The rate of resistance to tetracycline(72.36%)was highest,followed by four antibacterial drugs,ampicillin(71.67%),nalidixic acid(69.79%),co-trimoxazole(65.65%),streptomycin(52.52%).E.coli were from human,pig,chicken,duck,goose and sheep sources.Among them,the resistance rates of E.coli from chicken and pig were generally high,and the resistance of human E.coli was generally low.There was not detected carbapenemase-producing E.coli in human and the resistance rate to amikacin was 3.3%.It found that the drug resistance rates of ampicillin,tetracycline,co-trimoxazole and nalidixic acid had always maintained a high level in the four time periods of 2007-2009,2010-2012,2013-2015 and 2016-2018.632 of 1013 isolates showed resistance to at least 3 antibiotics and up to 62.39%(632/1013),implied that multiple antimicrobial resistance in E.coli isolates was a common phenomenon.The blaNDM gene was detected in meropenem-resistant E.coli isolates by PCR and sequencing.The results showed that all 7 drug-resistant bacteria were blaNDM positive,and a total of 2 NDM variants were obtained,namely blaNDM-1(n=1),blaNDM-5(N=6).Although the number of NDM-positive strains detected from 1013 E.coli was little,the host sources were extensive,covering pig sources(n=2),duck sources(n=1),goose sources(n=2),chicken sources(n=1),sheep sources(n=1).The 7 NDM-positive strains belonged to 5 different STs in total,namely ST6388,ST7386,ST10,and ST5229 and a new ST was also found,indicating that clonal spread was not the major reason for spread of blaNDM in E.coli.The plasmid types of 7 NDM-positive strains were analyzed and 8 plasmid types were detected,including IncHI2,IncX1,Col156,IncY,InxX3,IncFIB,IncN and IncFIC.All of the blaNDM-5 positive strains contained the IncX3 plasmid type.Three isolates namd C1147,C1588 and C1283 were selected to be sequenced.Among them,C1147 was blNDM-1-carried E.coli from goose,C1588 and C1283 both carried blaNDM-5 from duck and goose.The results showed that the blaNDM-1 resistance gene was present on IncHI2,while NDM-5 was present on IncX3 plasmid.To further investigate the biological characteristics of blaNDM-positive plasmids,MIC test,stability test and conjugation frequency were performed.IncX3 had good stability,and the conjugation frequency was 10-6～10-7,while IncHI2 was also stable enough,and the conjugation frequency was about 10-5,all of the recipient strains increased the value of MIC to meropenem 256 times.The plasmids carrying the blaNDM gene in the above three strains based on whole genome data were named pC1588.1,pC1283 and pC1147 respectively,and these different positive plasmids were analyzed.The pC1588.1 plasmid was 21351 bp in size which was too small and retained some of the NDM genetic background:IS5-blaNDM-5-bleMBL-trpF-dsbC-IS26.The pC 1283 plasmid was 46168 bp in size and belonged to the IncX3 type plasmid.The structure was extremely stable,the query coverage and nucleotide identity with most of the IncX3 plasmids carrying NDM were 99%.The blaNDM-5 gene was in the typical structure:IS3000-ISAba125-IS5-blaNDM5-bleMBL-trpF-dsbC-IS26.The pC1147 plasmid was 238291 bp in size and belonged to the IncHI2 type plasmid.The backbone structure was relatively stable and the variable region near the drug resistance gene varied greatly on different plasmids.The genetic background of blaNDM-1 on this plasmid was:IS26-sul1-emrE-catB-blaNDM-1-bleMBL-trpF-folP-Tn2.In conclusion,E.coli from different sources had severe resistance to antibiotics in China,especially from animal sources.7 carbapenem-resistant positive strains were detected in this study.And blaNDM was widely detected in animal host sources,including five animal sources:pig,goose,chicken,duck and sheep sources.Among them,IncX3 plasmids carring blaNDM-5 were widely distributed,and it was the first time to find IncHI2 plasmid carring blaNDM-1 from goose source.The results of biological characteristics of different NDM-positive plasmids revealed that the IncHI2 and IncX3 plasmids were readily to mediate wide dissemination of blaNDM in different sources E.coli.