Molecular Mechanisms Of Salmonella Enteritidis That Show Resistance To Cefepime

Salmonella is one of the most common foodborne pathogens with more than 2600serotypes,among which,Salmonella Enteritidis ranked top 3 serotypes that can lead to foodborne disease both in human and animals.But compared to other predominant serotype like Salmonella Typhimurium and Salmonella Derby,Salmonella Enteritidis show susceptibility to most new type antibiotics and clinical first-line medicines.Cefepime is the first fourth-generation cephalosporin applied in our country's clinical practice,which has high activity,low toxicity and any other advantages.However,Salmonella Enteritidis has starting to show resistance to it based on our epidemiology data.So,we carried out the following research to figure out the reasons for its prevalence and resistance mechanism.In this study,2914 Salmonella Enteritidis were isolated from diarrhoeal patients and animal-source food by Shanghai CDC from 2005 to 2014,all these strains were tested for antimicrobial susceptibility with 17 antibiotics,the result showed that altogether 38 strains were resistant to cefepime.36 were isolated from human-source and another 2 were isolated from chicken wings and duck breast respectively.The percentage of multi-resistance was as high as 100.00%and the types of resistance spectrum was up to 19.All these strains were totally resistant to Ceftazidime,Cefotaxime,Cefepime and Ampicillin and they also showed high resistance to Nalidixic acid?97.37%?,Sulfafurazole?76.32%?,Streptomycin?63.16%?and Trimethoprim-sulfamethoxazole?52.63%?.The presence of ESBLs gene were analysed by PCR and sequencing,the result showed that the number of ESBLs resistance genes varies from 1 to 5 in different strains,including bla_CTX-M?100.00%,38/38??bla_SHV?65.79%,25/38??bla_TEM?52.63%,20/38??bla_ACC?18.42%,7/38?and bla_PSE?5.26%,2/38?,among them,bla_CTX-MTX-M is shared by all strains and the subtype bla_CTX-M-55TX-M-55 is in the majority.It is indicated that different types of ESBLs genes have been widely spread in the cefepime-resistance Salmonella Enteritidis and bla_CTX-M-55TX-M-55 is the predominant one,the different combinations of ESBLs are the main factors that endow them with drug resistance.Then we cultured the drug-resistant bacterias and the receptor bacterias together,ultimately,35 conjugants were obtained,of which the transfer efficiency was 92.11%.Among these strains,SH12G402 could realize the common transfer of bla_CTX-M-3TX-M-3 and bla_TEM-1EM-1 gene,and SH12G1079 could realize the common transfer of bla_CTX-M-55TX-M-55 and bla_SHV-1gene and the other 33 strains could only transfer one bla_CTX-MTX-M gene in to the receptors.As a result,the horizontal transfer of resistance genes increased the MICs of conjugants to cephalosporins from 4²048,which were between the donor bacterias and receptor bacterias.It was indicated that the ESBLs resistance gene,especially bla_CTX-M-55TX-M-55 could also spread among different strains through horizontal transfer,thus increasing the chance of it to show resistance to cefepime.In order to understand the characteristics of bla_CTX-MTX-M gene,we carried out several experiments including plasmid subtyping,S1-PFGE,southern blot and gene environment detection.The result showd that there were totally 7 different kinds of plasmid replicons existed in the conjugants,including IncFrepB?n=20??IncI1?n=11??IncFII1?n=11??IncFIIB?n=11??IncN?n=2??IncL/M?n=1?and IncY?n=1?and there were 1 or 2 plasmids transferred into the conjugants together,the sizes of which were between 76.8 kb and 138.9kb.The bla_CTX-MTX-M genes were located on one of the plasmids and coud be transferred into the receptor bacterias with the help of insertion sequence ISEcp1 or IS903,which was highly detected in the upstream and downstream of bla_CTX-MTX-M gene and played an important role in the effective expression and diffusion of bla_CTX-MTX-M genes.PFGE typing method was used to study the homology of the 38 cefepime-resistant Salmonella Enteritidis.We totally got 18PFGE types.89.47%strains homology were up to 85%,indicating that there was a certain clonal transmission relationship between these cefepime-resistance Salmonella Enteritidis.And the strains showed 100%homology also had the some bla_CTXTX gene,indicating that the process of vertical transmission has promoted the proliferation of drug-resistant genes.In conclusion,bla_CTX-M-55TX-M-55 was popular in cefepime-resistant Salmonella Enteritidis,it has an important role in the emergence of drug resistance.bla _CTX-M-55TX-M-55 can propagated vertically and can also be transmitted horizontally by various plasmids like IncFrepB?IncI1?IncFII1 and IncFIIB,ISEcp1 plays as a mobile element in this process.The strong diffusion ability of bla_CTX-M-55TX-M-55 poses a potential threat to the effectiveness of fourth-generation cephalosporins in clinical treatment.