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ABSTRACTPseudomonas aeruginosabiofilms are intrinsically resistant to antimicrobial chemotherapies. At present, very little is known about the physiological changes that occur during the transition from the planktonic to biofilm mode of growth. The resistance ofP. aeruginosabiofilms to numerous antimicrobial agents that are substrates subject to active efflux from planktonic cells suggests that efflux pumps may substantially contribute to the innate resistance of biofilms. In this study, we investigated the expression of genes associated with two multidrug resistance (MDR) efflux pumps, MexAB-OprM and MexCD-OprJ, throughout the course of biofilm development. Using fusions to gfp, we were able to analyze spatial and temporal expression of mexAandmexCin the developing biofilm. Remarkably, expression ofmexAB-oprMand mexCD-oprJwas not upregulated but rather decreased over time in the developing biofilm. Northern blot analysis confirmed that these pumps were not hyperexpressed in the biofilm. Furthermore, spatial differences in mexAB-oprMandmexCD-oprJexpression were observed, with maximal activity occurring at the biofilm substratum. Using a series of MDR mutants, we assessed the contribution of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY efflux pumps to P. aeruginosabiofilm resistance. These analyses led to the surprising discovery that the four characterized efflux pumps do not play a role in the antibiotic-resistant phenotype of P. aeruginosabiofilms.

Using a gene-replacement strategy and a mutated copy of the Pseudomonas aeruginosa O5 rfc gene, we were able to generate a rfc mutant in P. aeruginosa serotype O2. This mutant, which exhibits the semi-rough (SR) LPS phenotype, was used to isolate the O2 rfc gene. Mobilization of the O2 and O5 rfc genes into SR mutants of the heterologous serotype resulted in ‘cross-polymerization’ of O-repeat units, indicating that the genes are functionally exchangeable. Analysis of the nucleotide sequence of the rfc genes revealed that the two Rfc proteins are identical. The results of this study have enabled us to propose the linkage catalyzed by the O5 O-polymerase enzyme.