Construction of in vitro resistance model of pan-resistant Pseudomonas aeruginosaand investigation of its resistance mechanism

OBJECTIVE To establish in vitro model of pan-resistant Pseudomonas aeruginosa(P.aeruginosa) by in vitro induction of multiple antibiotics and detect its resistance mechanism;to discuss the effect of in vitro induction of combined antibiotics on the drug resistance of P.aeruginosa.METHODS By gradient plate method,4 isolates were inoculated on the agar plates carrying drug of gradient concentrations until the emergence of resistance to this drug.The bacterial susceptibility before and after the induction was determined;the MBL detection was performed using E-test method in the susceptible isolates before induction,the induced pan-resistant isolates,and pan-resistant clinical isolates.The detection of active efflux pumps system of the 4 susceptible isolates before induction,the 4 pan-resistant P.aeruginosa isolates after induction,and 4 clinical isolates was performed by real-time PCR.The four efflux pumps included MexAB-OprM,MexCD-OprJ,MexEF-OprN and MexXY-OprM.RESULTS The susceptibility of 4 susceptible P.aeruginosa isolates to 6 antibiotics after induction decreased,and the value of MIC for 6 antibiotics increased markedly.According to the breaking points of CLSI,all of the 4 strains became pan-resistant.MBL detections were all negative for 4 clinical susceptible P.aeruginosa isolates before and after induction;2 of 4 clinical pan-resistant P.aeruginosa isolates were positive for MBLdetection,and the other 2 strains were negative.Of the 4 susceptibleP.aeruginosa isolates before induction,there were no high expression of 4 efflux pumps;compared with that before the induction,there were high expression of mexA-OprM in 4 induced resistant isolates,high expression of mexCD-OprJ in 1 isolate,high expression of mexEF-OprN in 1 isolate,but high expression of mexXY-OprM were found in none of the isolates.Of all the clinical isolates of pan-resistantP.aeruginosa,there were high expression of 4 efflux pumps in 1 isolate,mexAB-OprM only in 1 isolate without high expression of other 3 efflux pumps,and no high expression of 4 efflux pumps in the other 2 isolates. CONCLUSION Under the pressure of in vitro antibiotics,the drug resistance of pan-resistantP.aeruginosa resulted from the induction of susceptible isolates is stable;pan-resistantP.aeruginosa induced in vitro do not produce metallo-beta-lactamases,therefore the emergence of drug resistance maybe related to active efflux pumps,in particular MexAB-OprM.