Pseudomonas aeruginosa biofilms exposed to imipenem exhibit changes in global gene expression and beta-lactamase and alginate production

Abstract

The lungs of cystic fibrosis (CF) patients are commonly colonized with Pseudomonas aeruginosa biofilms. Chronic endobronchial P. aeruginosa infections are impossible to eradicate with antibiotics, but intensive suppressive antibiotic therapy is essential to maintain the lung function of CF patients. The treatment often includes beta-lactam antibiotics. How these antibiotics influence gene expression in the surviving biofilm population of P. aeruginosa is not clear. Thus, we used the microarray technology to study the effects of subinhibitory concentrations of a beta-lactam antibiotic, imipenem, on gene expression in biofilm populations. Many genes showed small but statistically significant differential expression in response to imipenem. We identified 34 genes that were induced or repressed in biofilms exposed to imipenem more than fivefold compared to the levels of induction or repression for the controls. As expected, the most strongly induced gene was ampC, which codes for chromosomal beta-lactamase. We also found that genes coding for alginate biosynthesis were induced by exposure to imipenem. Alginate production is correlated to the development of impaired lung function, and P. aeruginosa strains isolated from chronically colonized lungs of CF patients are nearly always mucoid due to the overproduction of alginate. Exposure to subinhibitory concentrations of imipenem caused structural changes in the biofilm, e.g., an increased biofilm volume. Increased levels of alginate production may be an unintended adverse consequence of imipenem treatment in CF patients.

FIG. 1.

Genes of P. aeruginosa growing in biofilms which were up- or downregulated more than fivefold in response to imipenem (1.0 μg/ml). Only genes of assigned function are shown.

FIG. 2.

Verification of GeneChip microarray results by real-time PCR (RT-PCR). The average fold changes in gene expression due to exposure to imipenem on days 1, 2, and 3 are shown.

FIG. 3.

Expression of algU, algD, ampC, and creD genes in the presence of increasing concentrations of imipenem measured by real-time PCR. Each experiment was done in duplicate. The data are presented as means, and error bars represent 95% confidence intervals. In all four charts, the expression levels are normalized to the level of expression of the noninduced biofilms.

FIG. 4.

Ethanol precipitation of exopolysaccharides from a P. aeruginosa biofilm exposed to imipenem (1.0 μg/ml) (left) compared to that for a nonexposed biofilm (right). The biofilm was exposed to imipenem for 24 h. Uronic acid assay measurements verified the increased levels of production of uronic acids from the imipenem-exposed P. aeruginosa biofilm, showing an approximately 20-fold higher level of uronic acids.

FIG. 5.

Scanning confocal photomicrographs of P. aeruginosa PAO1 biofilms established in flow cells. The effects of exposure to imipenem (0.5 μg/ml) for 37 h (C) or 18 h (B) or no exposure (A) were examined on day 5. For each condition (A to C) a top view and two side views of the biofilms are shown. Biofilms were stained with the cell-permeant nucleic acid stain SYTO 62.

FIG. 6.

Scanning confocal photomicrographs of P. aeruginosa biofilms established in flow cells. Select microcolonies stained with ConA-FITC, which reveals polysaccharides such as alginate, are shown from the top. (A) PAO1 biofilm not exposed to antibiotics; (B) PDO300 (a derivate of PAO1 that constitutively overproduces alginate) not exposed to antibiotics; (C) PAO1 exposed to imipenem for 18 h; (D) PAO1 exposed to imipenem for 37 h.