Molecular basis of azithromycin-resistant Pseudomonas aeruginosa biofilms

Abstract

Pseudomonas aeruginosa biofilms are extremely recalcitrant to antibiotic treatment. Treatment of cystic fibrosis patients with azithromycin (AZM) has shown promise. We used DNA microarrays to identify differentially expressed transcripts in developing P. aeruginosa biofilms exposed to 2 mug/ml AZM. We report that transcripts for multiple restriction-nodulation-cell division (RND) efflux pumps, known to be involved in planktonic antibiotic resistance, and transcripts involved in type III secretion were upregulated in the resistant biofilms that developed in the presence of AZM. Interestingly, the MexAB-OprM and MexCD-OprJ efflux pumps, but not type III secretion, appear to be integral to biofilm formation in the presence of AZM, as evidenced by the fact that a mutant deleted in both mexAB-oprM and mexCD-oprJ was unable to form a biofilm in the presence of AZM. A mutant deleted in type III secretion was still able to form biofilms in the presence of drug. Furthermore, single mexAB-oprM- and mexCD-oprJ-null mutants were able to form a biofilm in the presence of drug, indicating that either of the pumps can confer resistance to AZM during biofilm development. In contrast to planktonically grown cells, where no mexC expression was detectable regardless of the presence of AZM, biofilms exhibited induction of mexC expression from the outset of their formation, but only in the presence of AZM. mexA, which is constitutively expressed in planktonic cells, was uniformly expressed in biofilms regardless of the presence of AZM. These data indicate that the MexCD-OprJ pump acts as a biofilm-specific mechanism for AZM resistance.

Figures

FIG. 1.

Differential expression of efflux pump transcripts in P. aeruginosa biofilms developed in the presence of azithromycin (2 μg/ml) (PAO1-BV) versus control PAO1 biofilms not exposed to AZM.

FIG. 2.

Differential expression of known type III secretion operons and genes that were up- or downregulated more than fivefold in P. aeruginosa growing in biofilms in response to azithromycin (2 μg/ml) (PAO1-BV) versus control PAO1 biofilms not exposed to AZM. Type III secretion genes were assigned to operons according to Frank (13). Operons are as follows: operon A, pcrGVHpopBD; operon B, popNpcr1234DR; operon C, pscUT1RQPO; operon D, pscBCDEFGHIJKL; operon E, exsCBA. Transcript levels of genes within each operon were first summed and then divided by the number of genes in that operon to determine the average transcript level of the operon. Actual transcript levels of type III secretion genes are shown in Table S1 in the supplemental material. *, >20-fold change.

FIG. 3.

Validation of microarray data by RT-PCR. Lanes: M, 1 kb Plus marker; 1, RNA from biofilm not exposed to AZM; 2, RNA from biofilm developed in the presence of 2 μg/ml AZM; 3, master mix without template; 4; PAO1 DNA.

FIG. 4.

Scanning confocal laser micrographs of representative image areas of biofilms cultivated in flow cells in the presence and absence of 2 μg/ml AZM. (A) PAO1; (B) PAO238 [Δ(mexAB-oprM)Δ(mexCD-oprJ)]; (C) PAO438 [Δ(mexAB-oprM)Δ(mexCD-oprJ) nfxB::mini-Tn7-nfxB+-mexC+D+-oprJ+]; (D) PAO200 [Δ(mexAB-oprM)]; (E) PAO461 [Δ(mexCD-oprJ)]. Magnification, ×400.

FIG. 5.

Biofilm characteristics of wild-type PAO1 and PAO1 mutants formed in the presence (dashed lines) or absence (solid lines) of 2 μg/ml AZM. (A) Total biomass (μm3/μm2); (B) average thickness (μm). Each data point is the average of five image stacks collected from randomly selected areas ± 1 standard deviation.

FIG. 6.

Scanning confocal micrographs of biofilms formed by PAO1 (mexC-gfp) with or without continuous exposure to 2 μg/ml AZM. (A) Biofilms were examined to identify cells expressing mexC-gfp (green signal) compared to total cells (red signal). (B) The percentage of cells expressing mexC was plotted versus biofilm height. Quantitative analysis was done using the colocalization function in Metamorph Imaging software.

FIG. 7.

Scanning confocal micrographs of PAO1-BV (mexC-gfp) biofilms after 4 days of cultivation in the absence or presence of 2 μg/ml AZM. (A) Biofilms were examined to identify cells expressing mexC-gfp (green signal) compared to total cells (red signal). (B) The percentage of cells expressing mexC-gfp was plotted versus biofilm height. Quantitative image analysis was done using the colocalization feature in Metamorph Imaging Software. □, cells grown with AZM; ♦, cells grown without AZM.