Mechanism On Antibiotic-resistance Transmission Associated With CTX-M-55 Extended-Spectrum β-Lactamase Mediated By Plasmid In Enterobacteriaceae

Emergence of bacterial antibiotic resistance is a growing problem,which might associate with the worldwide use of antimicrobial agents in livestock production,veterinary medicine and human clinical treatment.Extended spectrumβ-lactamases（ESBLs）are enzymes that could confer resistance to mostβ-lactam antibiotics,especially to the third-generation cephalosporins.In this study,ESBLs producing Salmonella screening,antimicrobial susceptibility and subtype of Salmonella enteritidis isolates recovered from different sources（i.e.Clinical Salmonella infected patients in different hospitals in different treatment times,retail foods and environmental samples in the communities where patients live）were investigated.Meanwhile,the genotype of CTX-M-55-producing isolates were further studied via pulsed field gel electrophoresis（PFGE）,and the transmission of bla _CTX-M-55gene was detected to preliminary reveal the mechanism of plasmid-mediated antibiotic-resistance transmission among bla_CTX-M-55-positive Enterobacteriaceae strains.The mainly acquired results are as follows:（1）Two hundred forty-two（78.83%）Salmonella enteritidis isolates were screened and identified as ESBLs producing strains via double-disk synergy test.Based on the results of disk diffusion method,all（100%）isolates were detected resisted to cefotaxime.Other antibiotics that the strains resisted to and the corresponding rates of antibiotic resistant isolates were as followings:ampicillin（91.74%）,cefoperazone（91.32%）,nalidixic acid（91.32%）,ceftazidime（82.64%）,streptomycin（51.24%）,sulphafurazole（45.45%）,tetracycline（44.21%）,chloramphenicol（30.58%）,sulfamethoxazole/trimethoprim（19.83%）,gentamycin（12.81%）,trimethoprim（12.81%）,amoxicillin/clavulanate（4.55%）,ciprofloxacin（1.24%）and ofloxacin（1.24%）.（2）ESBLs encoding genes including bla_TEM,bla_CMY,bla_CTX-M,bla_OXA,bla_PSE,bla_PER,bla _IMP,bla_VIM,bla_ACC and bla_GES would be carried by 242 ESBLs-producing isolates were screened via polymerase chain reaction（PCR）combined DNA sequencing.Among 242ESBLs producing Salmonella,159（65.70%）were detected carrying bla_CTX-M-55,which was the most common ESBLs encoding gene detected among the isolates.Sixty-four（25.62%）,4（1.65%）and 4（1.65%）ESBLs producing isolates were detected harboring bla_TEM-1,bla _CTX-M-64 and bla_CTX-M-123,respectively.（3）Sixty-four PFGE profiles were observed among 159 CTX-M-55-producing isolates after PFGE analysis.Seventy-four isolates showed genetic similarity of 98.89%,1 isolate recovered from retail chicken showed 100%genetic similarity to some clinical isolates.Meanwhile,some clinical isolates recovered in different years,clinical patients and hospitals also showed 100%genetic similarity.High genetic similarity of these ESBLs producing Salmonella indicated that retail chicken might be the one of the most common sources of foodborne salmonellosis and these S.enteritidis isolated from different hospitals at different times may evolved from some clones with higher genetic homology.（4）Eight representative Enterobacteriaceae strains,which carried bla_CTX-M-55 on different plasmids,were selected as donor.One Salmonella,1 Escherichia coli and EC600were selected as recipient in conjugation experiments.All donor strains could successfully transfer their bla_CTX-M-55 and antibiotic resistance phenotype to the recipient cells,no difference was found among the conjugation frequencies when different donor and recipient were combined for conjugation.Results of S1-PFGE and southern blot hybridization showed that each of the transconjugants carried one to three plasmids with sizes ranging from～70 kb to～335 kb,bla_CTX-M-55 was predominantly located on an～80 kb plasmid.