Research On The Mechanism Of Less Susceptibility To Cefepime Than To Ceftazidime In Enterobacter Cloacae

Objective:To investigate the drug mechanism of Enterobacter cloacae with special resistance phenotype,which has the characteristic of the sensitivity of cefepime less than ceftazidime.Methods:The clinical isolates with the above special phenotypic characteristics were collected and the minimal inhibitory concentration?MIC?of cefepime and ceftazidime was determined by agar dilution method.Agar dilution method was used to determine the minimum inhibitory concentration of the bacteria after the addition of an efflux pump inhibitor.The genomic DNA was extracted by boiling method.Resistant enzyme genes(bla_OXA-1,bla_OXA-10,bla_OXA-31,bla_OXA35,bla_PSE-1)and efflux pump genes?acrA,acrB,tolC?were amplified by polymerase chain reaction?PCR?.Plasmid DNA of positive strains with resistant enzyme gene was extracted,and corresponding genes were amplified by PCR.The strains with positive plasmid DNA were tested for conjugation experiment to observe whether the resistant enzyme genes can be spread horizontally through plasmid.The mRNA of the positive strains with an efflux pump gene was extracted,and transcribed into cDNA by reverse transcription.And the expression of the efflux genes?macrA,acrB,tolC?in Enterobacter cloacae was detected by real-time quantitative polymerase chain reaction?RT-PCR?.The bacterial DNA was used as a template,and the efflux pump regulatory genes?acrR,marA,ramA?were amplified by PCR.Gene sequencing was performed on the positive products of efflux pump genes and efflux pump regulatory genes.A homology analysis was performed by pulsed field gel electrophoresis?PFGE?for the strains with the above special resistant phenotype.Results:There were eight strains with special resistant phenotype?ie,cefepime was less sensitive than ceftazidime?of Enterobacter cloacae,and the MIC of two strains was significantly decreased after adding the pump inhibitor.The results showed that genes bla_OXA-1XA-1 and bla_OXA-31XA-31 were positive simultaneously in three strains,and genes bla_OXA-10XA-10 and bla_OXA-35XA-35 were positive simultaneously in two strains,but gene bla_PSE-1SE-1 was negative in all strains of the eight bacterial strains.Among the five strains with positive resistant enzyme genes in the genome,all of the isolates were positive for plasmid-resistant gene amplification,but only two of them were positive by conjugation experiment.The results of efflux pump gene amplification in genome DNA showed acrA,acrB and tolC of two strains were positive at the same time,while acrA and acrB of the additional two strains were positive at the same time,but the tolC of the other four strains was individually positive.The expression of mRNA in the efflux pump genes showed the expression of acrA decreased in all of the eight strains,while the expression of acrB and tolC wholly increased.The results of efflux pump regulatory genes amplification in genome showed the positive rate?87.5%?of marA gene was the highest,the positive rate?50%?of acrR gene was second,and the ramA gene were all negative in the eight strains.The results of BLAST analysis showed that the homology of acrA gene with sequence number DQ679966.1 in the database of NCBI was 96%,the identity between acrB gene and sequence number DQ679966.1 was 98%,the homology of tolC gene and AM287288.1 was 98%,the homology of acrR gene and EF627524.1 was 98%,and the homology of marA gene and AF411963.1 was100%.Pulsed field gel electrophoresis?PFGE?showed that the homology of the strains of number one and number three was high,which were distributed in the ICU ward.And the homology of strains number two,number four and number seven was high,which were allocated in the respiratory department.Conclusion:There was a special resistance phenotype of the sensitivity of the fourth generation cephalosporins?such as cefepime?lower than the third generation cephalosporins?such as ceftazidime?in Enterobacter cloacae.One of potential mechanisms,which caused the sensitivity of cefepime lower than ceftazidime,was the appearance of resistance gene bla_OXA-1,bla_OXA-10,bla_OXA-31,bla_OXA-35,and there was a risk of horizontal propagation through plasmid conductance,we should take to prevent its outbreak.The efflux pump system AcrAB-TolC was another important mechanism,that leaded to lower susceptibility of cefepime than ceftazidime in Enterobacter cloacae,and it could be affected by regulation gene acrR and marA.