Study On Molecular Characteristic Of The AmpC-producing Escherichia Coli And Function Of PBP4in The Resistance Mechanisms

Objective To study the molecular characteristic of the Escherichia coli (E.coli)producing AmpC β-lactamase in Shanghai sixth People’s Hospital；To explore the effect ofpenicillin-binding protein4（PBP4)in the resistant mechanisms of E.coli produced AmpCβ-lactamase.Methods1.Three-dimensional test was initially used for the detection of AmpCbeta-lactamase and further polymerase chain reaction (PCR) were used for the detectionthe positive isolates.2.Phylo-group was examined by multiplex PCR.3. The isolates wasgenotyped by Multilocus sequence typing(MLST).4. PCR was used for amplification geneencoded non-essential PBPs.5.Realtime PCR was used for the detection of the transcriptionlevel of PBP4gene dacB and AmpC β-lactamase gene ampC.6. Susceptibility testing wasdetected by broth microdilution method.7.Gene dacB was converted into E.coli ATCC25922by DNA recombination, the expression level of dacB mRNA and ampC mRNAwere detected by realtime PCR method and the MIC changes to antibiotics were observed.Results1. Cefoxitin screening test was performed in the1783isolates of Escherichiacoli and299isolates were positive,34isolates had AmpC β-lactamase.2.Phylogeneticanalysis showed that17.64%were group A,14.71%of B1,14.71%of B2,52.94%ofD.3.MLST analysis of34isolates identified14STs,including1unknow STs.14STsrevealed6ST clonal complexes and ST38Cplx was was the most of these,it accounted for17.65%of the total.4.The gene dacB、dacA、dacC、ampH、ampC and pbpG were positiveby the PCR.5.26of34isolates had overexpression of dacB mRNA（76.47%）and8of34isolates had no overexpression of dacB mRNA（23.53%）,the transcription of ampC in thehigh level expression group is higher than the low level expression group，P=0.011.6. There was lower susceptibility among the isolates that were high expression level of dacBmRNA.7.The gene ampC transcription level and the resistance of recombinant haveincreased,especially first and second generation cephalosporins.Conclusions Phylogenetic group D was predominant in Escherichia coli producingAmpC β-lactamase;this isolates belonged to14different ST and the most was ST38,fiveDHA producers belonged to ST131/B2clone;we speculate that overexpression of PBP4may be helpful to introduce high level resistance by triggering the overexpression ofAmpC β-lactamase among clinical E.coli.