Multidrug efflux pumps: Expression patterns and contribution to antibioticresistance in Pseudomonas aeruginosa biofilms

Pseudomonas aerginosa biofilms are intrinsically resistant to antimicrobial chemotherapies. At present, very little is known about the physiological c hanges that occur during the transition from the planktonic to biofilm mode of growth. The resistance of P. aeruginosa biofilms to numerous antimicrob ial agents that are substrates subject to active efflux from planktonic cel ls suggests that efflux pumps may substantially contribute to the innate re sistance of biofilms. In this study, we investigated the expression of gene s associated with two multidrug resistance (MDR) efflux pumps, MexAB-OprM a nd MexCD-OprJ, throughout the course of biofilm development. Using fusions to gfp, we were able to analyze spatial and temporal expression of mexA and mexC in the developing biofilm. Remarkably, expression of mexAB-oprM and m exCD-oprJ was not upregulated but rather decreased over time in the develop ing biofilm. Northern blot analysis confirmed that these pumps were not hyp erexpressed in the biofilm. Furthermore, spatial differences in mexAB-oprM and mexCD-oprJ expression were observed, with maximal activity occurring at the biofilm substratum. Using a series of MDR mutants, we assessed the con tribution of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY efflux pumps to P. aeruginosa biofilm resistance. These analyses led to the surprising discovery that the four characterized efflux pumps do not play a role in th e antibiotic-resistant phenotype of P. aeruginosa biofilms.