The Study Of Mechanisms In Tigecycline Resistance In Enterobacteriaceae

Enterobacteriaceae including Klebsiella pneumoniae and Escherichia coli have emerged worldwide as an important pathogen of nosocomial infections.Tigecycline is one of the last resort for treating infections caused by carbapenem-resistant Enterobacteriaceae.Tigecycline resistance in Enterobacteriaceae often occurs during clinical use,especially in Klebsiella pneumoniae and Escherichia coli.The mechanism of tigecycline resistance in Enterobacteriaceae has not yet been clearly elucidated.In this study,we investigated the dominant mechanism of tigecycline resistance in Enterobacteriaceae clinical isolates and search for other novel mechanisms of tigecycline resistance.In part Ⅰ,we identified the susceptibility profile of KPC-producing K.pneumoniae to tigecycline and investigated the role of RND efflux pumps in tigecycline resistance.A total of 215 KPC-producing K pneumoniae isolates were collected.The minimum inhibitory concentration(MIC)of tigecycline was determined by standard broth microdilution tests.Isolates showing resistance to tigecycline underwent susceptibility test with efflux pump inhibitors.Expression levels of efflux pump genes(acrB and oqxB)and their regulators(ramA,marA,soxS and rarA)were examined by real-time PCR,and the correlation between tigecycline MICs and gene expression levels were analysed.Our results show that the tigecycline resistance rate in these isolates was 11.2%.Exposure of the tigecycline-resistant isolates to the efflux pump inhibitor NMP resulted in an obvious decrease in MICs and restored susceptibility to tigecycline in 91.7%of the isolates.A statistically significant association between acrB expression and tigecycline MICs was observed,and overexpression of ramA was found in three tigecycline-resistant isolates,further analysis confirmed ramR mutations existed in these isolates.Transformation of one mutant with wild-type ramR restored susceptibility to tigecycline and repressed overexpression of ramA and acrB.These data indicate that efflux pump AcrAB,which can be up-regulated by ramR mutations and subsequent ramA activation,contributed to tigecycline resistance in K.pneumonia clinical isolates.In part Ⅱ,the novel mechanisms for tigecycline resistance in Escherichia coli were investigated using gene deletion and complementation experiment.In this study,we structured an AcrAB efflux pump deletion strain 25922△acrAB from E.coli ATCC 25922.Using serial passage method,tigecycline resistant isolate 25922△acrAB-TGC8 and 25922-TGC8 were obtained from parental strain 25922AacrAB and ATCC 25922 respectively.Mutation in mlaA was found both in 25922△acrAB-TGC8 and 25922-TGC8.Deletion and complementation experiment revealed that mlaA mutation contribute to 8 fold decreased tigecycline susceptibility both in 25922AacrAB and ATCC 25922.Our results revealed that tigecycline resistance can still occur without the association of AcrAB efflux pump in E.coli,and ribosomal S10 protein mutation was also not mandatory for tigecycline resistance.The mlaA mutation may explain why tigecycline resistance generally occurs during tigecycline treatment in Enterobacteriaceae infection to a certain extent.And it seems that multiple mechanisms(Mla system,efflux pump,and ribosomal S10 protein)can accumulate gradually in the development of tigecycline resistance in Enterobacteriaceae.