Mechanism Of Plasmids IncI1 And IncN Mediated Salmonella Antimicrobial Resistance And Transmission

Salmonella is one of major foodborne pathogens that can produce human disease worldwide.Multidrug-resistant（MDR）Salmonella have become a national public health problem,their antibiotic resistance was verified closely linked to extensive usage of antimicrobial agents in food-animal and livestock production as well as in human clinical treatment.The acquisition of drug resistance gene is an important way for Salmonella to develop antibiotic resistance phenotype,which is mainly expressed in two aspects,the bacterial DNA mutation and plasmid conjugation.As a self-replicating mobile genetic element,plasmid was widely presented in bacteria and can be horizontally transferred between different microorganisms to spread drug resistance.In this study,Salmonella and Escherichia coli that collected in difference sources and districts were used to determine the prevalence of plasmids IncI1,IncN,and ten antimicrobial-resistance genes,to reveal mechanisms of the two plasmid-mediated drug resistance and transmission.The results of this study were as followings:（1）Screening of plasmids IncI1 and IncNTwo incompatible plasmids IncI1 and IncN in foodborne Salmonella were screened by PCR replicon typing method.Five IncN positive strains（0.47%）and 52 IncI1 positive strains（4.86%）were identified among the 1069 Salmonella strains.In addition,2 IncN positive strains and 13 IncI1 positive strains were denoted by related researchers.（2）Antibiotic susceptibility and related genes carried by IncN and IncI1 positive SalmonellaSeven IncN positive and 27 IncI1 positive strains that selected from Salmonella were tested for their antimicrobial susceptibility to 16 common antibiotics including fluoroquinolones,aminoglycosides and cephalosporins.Resistance were most frequently observed to ceftiofur（100%）,nalidixic acid（88.2%）,ampicillin（88.2%）,tetracycline（88.2%）,cefoperazone（82.4%）,and sulfamethoxazole/trimethoprim（82.4%）,followed by ceftriaxone（79.4%）,streptomycin（64.7%）,chloramphenicol（64.7%）,and a lesser extent to amoxicillin/clavulanic acid（14.7%）,cefoxitin（11.8%）,and amikacin（2.9%）.Results showed that the strains carried IncI1 plasmid have a broader antibiotic-resistance profiles and higher resistance ability to cephalosporin than IncN plasmid positive strains,whereas IncN plasmid positive strains were more tolerant to aminoglycoside antibiotics.qnrA,qnrB,qnrS,qepA,acc（6’）-Ib,bla_TEM,bla _CTX-M,bla _ACC,bla _OXA,and bla _PSE,which were related to antibiotic resistance,were not frequently detected from 34 Salmonella,most strains carried one to three kinds of resistance genes.The qep A and bla _ACC gene were not detected in all tested strains.（3）Conjugation experiments and change of MIC to transconjugantsTwo IncN positive strains and four IncI1 positive strains were used as donors,one Escherichia coli and one Salmonella were selected as recipient strains in conjugation experiments.The 6 donor strains transferred their plasmids to Salmonella at conjugation frequency ranging from 3.2×10^-5to 2.0×10^-3 transconjugant per recipient cell,while donors strains transferred their plasmids to E.coli at conjugation frequency ranging from 8.7×10^-7 to9.6×10^-5.Antibiotic resistance genes including qnrB,acc（6’）-Ib-cr,bla_TEM and bla _CTX-Mwere transferred from donor strains to the recipient Salmonella and E.coli strains.Recipient strains asquired antibiotic resistance phenotypes to kanamycin,ceftiofur,ceftriaxone,ampicillin,streptomycin,and gentamicin after conjugation.（4）Transfered plasmids identified via S1-PFGEThe results of PFGE showed that most of the donor and recipient strains were genetically diverse except N3,I14 and I22,which exhibited high genomic similarities.S1-PFGE was performed on donor strains,recipient strains,and transconjugants,respectively.Plasmids with sizes of 54.6～76.8 kb could be transferred from donor strains（N7,I1,I14,I22,and I24）to the recipients,as well as genes of qnrB,acc（6’）-Ib-cr,bla_CTX-M,and bla _TEM.The results of antimicrobila resistance of donor strains and recipient strains in this study demonstrated that the transfer of acc（6’）-Ib-cr gene mediated reisitance to streptomycin and gentamicin to transconjugants corresponded to N7.