Interaction of biofilm and efflux pump in clinical isolates of carbapenem resistant P. aeruginosa.

OBJECTIVE

Carbapenem-resistant P. aeruginosa (CRPA) is particularly worrisome because of its resistance against multiple antimicrobial agents which reduces treatment options. The efflux pump decreases antibiotic abundance, and biofilm impairs the penetration of antibiotics. The aim of the present study was to evaluate the role and relationship of efflux pump and biofilm formation in CRPA isolates obtained from different clinical samples.

PATIENTS AND METHODS

A total of 110 different clinical samples were collected from three tertiary medical hospitals. The samples were subjected to isolation and identification by standard operating procedures. Species level were identified using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system. Antibiotic susceptibility testing was done by broth microdilution method. Crystal violet (CV) staining for observing the biofilm forming ability and amplification of efflux pump mexA gene were also performed on clinical CRPA isolates. Three efflux pump MexAB-OprM regulatory genes were analyzed using sequencing methods. The expression of mexA gene both in biofilm and planktonic bacteria was observed by Quantitative real-time PCR (qRT-PCR).

RESULTS

The results showed that 110 samples were CRPA and among them 83 (75.5%) were MDR isolates. The CV staining showed 105 (95.5%) isolates as biofilm producers while 78 (74.3%) MDR isolates showed biofilm formation. mexA hyperexpression was detected in 27 (24.5%) CRPA isolates while 26 (96.3%) in biofilm forming isolates and 96.3% (26/27) in MDR P. aeruginosa. Multiple mutations in nalC, nalD, and mexR genes were detected. The distinct difference confirmed that the expression of mexA gene in P. aeruginosa biofilm producer was significantly higher than that of planktonic bacteria in vitro, and the efflux pump inhibitor PAβN significantly inhibited biofilms in CRPA isolated from clinical samples.

CONCLUSIONS

The biofilm and efflux pumps might be two intertwined processes involved in CRPA isolates. Their synergistic effect magnified the drug resistance characteristics of P. aeruginosa.