Characterization And Transfer Mechanism Of ESBL And Mcr-1 Carrying E.coli Isolates Recovered From Food Animals In Jiangsu,China

Antimicrobial resistance(AMR)has been considered as one of the major global threats to human and animal health,as warned by World Health Organization(WHO)and World Organization for Animal Health(OIE)on several occasions.One such threat is the emergence and spread of multidrug-resistant(MDR)pathogens like extended-spectrum beta-lactamase(ESBL)-producing and colistin-resistant Escherichia coli in both the field of animal settings and human medicine.This dissertation studied the prevalence and characteristics of ESBLproducing and colistin-resistant E.coli in food animals(pigs,cattle,and poultry)in Jiangsu province,China,to monitor the current status of antibiotic resistance related with beta-lactam antibiotics and colistin in E.coli in this region.We also investigated the clonal dissemination and horizontal transfer mechanisms of ESBL and mcr-1 genes in E.coli to evaluated the transmission risks.Study ?:High incidence of multidrug-resistant Escherichia coli coharboring mcr-1 and blaCTX-M-15 recovered from pigsThe coexistence of ESBL with mcr-1 gene in E.coli has become a serious threat globally.The aim of this study was to investigate the increasing resistance to colistin(COL)and in particular,its coexistence with ESBL-producing E.coli recovered from pig farms in Jiangsu Province.Susceptibility testing,PCR and sequencing technology were used to detect COLresistant genes,mcr-1 to-5,as well as ESBL genes,blaCTX-M,blaSHV,and blaTEM.Conjugation experiment,plasmid replicon typing,S1-PFGE and DNA southern hybridization were performed to study the transferability of these genes.Overall,275 E.coli isolates were recovered from a total of 432 cloacal and nasal swabs.More than 90%of the isolates were MDR,of which 70.18%were resistant to COL.Of these 275 isolates,mcr-1 was identified as the most predominant gene carried by 71.63%(197/275)of isolates,39.59%(78/197)of the isolates harbored both mcr-1 and at least one of the ESBL genes(blaCTX-M,blaSHV,and blaTEM).ESBL genotyping showed that blaCTX-M was the most predominant ESBL gene(68.49%,followed by blaSHV(16.4%)and blaTEM(15%).Sequencing revealed that blaCTX-M15(69%)was the predominant allele.IncHI2,IncFIB,IncFIC,IncN,and IncX4 were found to be the most common Inc-types and were associated with the transfer of the mcr-1 and ESBL genes.The coexistence of the mcr-1-and blaCTX-M-15-carrying isolates displaying high MDR,recovered from E.coli of pig origin,is a major concern for both humans and veterinary medicine.Study ?:Characterization and virulence factors distribution of blaCTX-M and mcr1 carrying Escherichia coli isolates from bovine mastitisThis study aimed to investigate the resistance mechanisms and genetic relationships of ESBL-producing and colistin-resistant E.coli in milk with bovine mastitis.Five hundred and thirty-five mastitic milk samples were collected.Antimicrobial susceptibility testing and PCRs were used for the detection of ESBL genes(blaCTX-M,blaSHV,and blaTEM),mcr-(1-9),phylogenetic grouping and for virulence genes(ompC,fimH,ECs3703,ompF,irp2,fyuA,iucD,colV,eaeA,and ler).MLST,MLS A,and PFGE were used for clonal relatedness.Among ESBL positive E.coli isolates,more than 85%isolates were multidrug resistance ones.Genotypic characterization indicated a dominance of CTX-M-1 group and the prevalent alleles were in sequence of blaCTX-M-28(38.37%),blaCTX-M-14(17.44%),blaCTX-M-66(13.95%),blaCTX-M-55(10.46%),and blaCTX-M-69(6.97%),blaCTX-M-15(4.65%),blaCTX-M-177(4.65%),and blaCTX-M-148(3.48%).Phylogenetic analysis showed that phylogroup A was the most predominant group with 54.73%,followed by group B1 and B2(24.21%,10.52%)respectively.All tested isolates belonged to group A,B1,B2,C,D,and E presented the virulence factors.The most prevalent gene was ompC detected in 97.89%(n=93)of the isolates,then followed byfimH in 61.05%(n=58),ECs3703 57.89%(n=55),ompF 50.52%(n=48),irp2 31.57%(n=30),fyuA 21.05%(n=20),iucD 15.78%(n=15),and 2.10%(n=2)of the isolates were positive for colV.MLST and PFGE of the tested E.coli isolates were determined for clonal relatedness and yielded clear fingerprint patterns.There were 16 different clusters sharing?80%similarity in the PFGE pattern.The majority of the E.coli isolates carrying ESBL and mcr-1 isolates were clonally unrelated.ST 58 and ST410 were the most predominant comprising 2 each(2/20=20%).The most prevalent plasmid types found were IncHI2(n=11),FIB(09),FIC(07),and X4(05).Conclusively,this study provides important insight into the genetic features and virulence attributes of ESBLs-producing and colistin-resistant E.coli of bovine mastitis milk origin.Study ?:Characterization and resistant determinants related MGEs of ESBLproducing and mcr-1 positive Escherichia coli isolates recovered from the chicken originThe spread of AMR in E.coli is a complex process that is linked with various mobile genetic elements(MGEs)like plasmids,transposons,and integrons.The aims of this study were to determine the antibiotic resistance,ESBL,and mcr genes and their association with MGEs in diarrheagenic E.coli isolates of chicken origin.Two hundred and ten(n=210)E.coli isolates were recovered from different commercial chicken farms.Resistance profiles,detection of various resistant genes,and the presence of integrons,and IS elements were determined.Multiplex PCRs were used to detect phylogenetic grouping and various diarrheagenic virulence genes(DAEC,EIEC,EAEC,EPEC,EHEC,and ETEC)among the isolates.Mating assay and replicon typing were performed to know the transferability of resistant genes and MGEs.The results showed that 83.33%of the strains presented MDR and the prevalence of blaCTX-M,blaSHV,blaTEM,and mcr-1 was found 30%,10.95%,8.09%,and 36.66%,respectively.Among all the mcr-1 positive isolates,70.12%were found co-harboring ESBL genes.The most common CTX-M alleles were blaCTX-M-55(46%),then followed by blaCTX-M-14(31%),and blaCTX-M-15(13%).Detective rates of ISEcp1,ISCR1,ISApl1,and int1 were 27.77%,53.70%,51.85%,and 70.37%respectively.Most CTX-M,SHV and TEM positive strains were found associated with ISEcp1,ISCR1,and Integron 1.While the majority(51.80%)of mcr-1-harboring isolates had the ISAPl1-mcr-1 segment.Phylogenetic group A was the most prevalent phenotype of the mcr-1-positive strains(77.77%),while the common diarrheagenic E.coli(DEC)identified in these strains were EHEC 59.25%,followed by EAEC 12.96%and EPEC 9.25%.Conjugation assay also confirmed that multiple genetic elements were borne on narrow host-range plasmids such as FIB,IncHI2.This study determined the prevalence of selected genetic elements concerned in the evolution of resistance determinants in E.coli from a chicken origin in Jiangsu province,China.In conclusion,the current study reports on the high incidence of multidrug-resistant E.coli harboring mcr-1 and blaCTX-M as a predominant gene,particularly blaCTX-M-15 in pigs,blaCTX-M-28 in cattle and blaCTX-M-55 was prevalent in chicken isolates.This study determined the prevalence of selected genetic elements concerned in the evolution of resistance determinants in E.coli and the propagation of such MDR strains carrying multiple resistance elements among the bacterial population is a threat of worry.This study investigated the drug resistance rate and molecular epidemiology in a large scale of MDR ESBL-producing and mcr-1 positive E.coli isolates from food animals,and found a high incidence of mcr-1 harboring blaCTX-M genes.The current study also reported the phylogenetic analysis,genetic environment and horizontal transfer mechanisms of ESBL and mcr-1 carrying E.coli isolates.