Overproduction of efflux pumps caused reduced susceptibility to carbapenem under consecutive imipenem-selected stress in .

PURPOSE

is an important pathogen in the nosocomial infections worldwide. Combining with carbapenemases, efflux pumps and outer membrane proteins (OMPs) have been thought to affect the development of carbapenem resistance in . This study aimed to investigate the contributions of different efflux pumps and OMPs in developing carbapenem resistance in a clinical isolate of and reveal the possible mechanism of overproduction of main efflux pumps.

PATIENTS AND METHODS

In this study, an imipenem-susceptible clinical isolate was identified as and named SZE. Several common carbapenemases were detected by polymerase chain reaction (PCR). Imipenem-selected mutants were selected from SZE by serial subcultivations on Mueller-Hinton agar, and the minimum inhibitory concentration (MIC) was detected. Gene expressions of four families of efflux pumps, five OMPs, and OXA-51 were determined by reverse transcription quantitative PCR, and comparisons were made between SZE strain and the imipenem-selected mutants. The system in SZE and its mutant was sequenced and aligned.

RESULTS

Under consecutive imipenem-selected stress, the MIC to imipenem increased gradually from 0.125 μg/mL to 8 μg/mL. The effect of resistance inducement was almost neutralized when treated with an efflux pump inhibitor. The expression of efflux pumps, , , and , was increased by 6.9-, 4.0-, and 2.1-fold in mutants, respectively, compared to SZE. A single mutation (G to A) at position 58 was detected in the regulatory system and possibly upregulated the expression, and then affected the carbapenem resistance in strains.

CONCLUSION

In conclusion, under consecutive imipenem-selected stress in vitro, strain evolved the ability to reduce susceptibility to a variety of antimicrobials by overproduction of efflux pumps. Especially, the resistance-nodulation-cell division super family and a nucleotide mutant in regulating system caused the overexpression of .