Clinical Biofilm Ring Test® Reveals the Potential Role of β-Lactams in the Induction of Biofilm Formation by P. aeruginosa in Cystic Fibrosis Patients

Abstract

Biofilms are characterized by high tolerance to antimicrobials. However, conventional antibiograms are performed on planktonic microorganisms. Through the clinical Biofilm Ring Test^® (cBRT), initially aimed to measure the adhesion propensity of bacteria, we discerned a variable distribution of biofilm-producer strains among P. aeruginosa samples isolated from expectorations of cystic fibrosis (CF) patients. Despite a majority of spontaneous adherent isolates, few strains remained planktonic after 5 h of incubation. Their analysis by an adapted protocol of the cBRT revealed an induction of the biofilm early formation by sub-inhibitory doses of β-lactams. Microscopic observations of bacterial cultures stained with Syto 9/Propidium Iodide (PI) confirmed the ability of antimicrobials to increase either the bacterial biomass or the biovolume occupied by induced sessile cells. Finally, the cBRT and its derivatives enabled to highlight in a few hours the potential inducer property of antibiotics on bacterial adhesion. This phenomenon should be considered carefully in the context of CF since patients are constantly under fluctuating antimicrobial treatments. To conclude, assays derived from the Biofilm Ring Test^® (BRT) device, not only define efficient doses preventing biofilm formation, but could be useful for the antimicrobial selection in CF, to avoid inducer molecules of the early biofilm initiation.

Keywords: Pseudomonas aeruginosa; antibiotics; biofilm; clinical Biofilm Ring Test®; cystic fibrosis; early bacterial adhesion induction.

Figure 1

Biofilm profiling of P. aeruginosa clinical isolates according to their ability to block the migration of magnetic microbeads in clinical Biofilm Ring Test (cBRT) assay. Biofilm formation in 96-well polystyrene plates was shown after 5 h of incubation at 37 °C for representative strains of three adhesion profiles: strain #22 (no-biofilm producer), strain #2 (weak-biofilm producer) and strain #21 (moderate-biofilm producer). A gradual blocking of beads was noticed depending on the microbial serial dilutions and therefore reflecting the strength of the biofilm produced by bacteria. Images were obtained after magnetization of the plates on the block test and scanning with the plate reader. Negative controls with only Brain Heart Infusion (BHI) medium and magnetic microparticles were displayed in the last line of microplates.

Figure 2

Distribution of adhesion profiles among 38 clinical strains of P. aeruginosa. The cBRT assay allows for the categorization of bacteria according to their biofilm strength. Through the calculation of Biofilm-forming Potential (BP) values, clinical isolates were classified as no-, weak-, moderate- or high-biofilm producer. No high-biofilm producer strains were found in this panel.

Figure 3

Representative images of adherent P. aeruginosa cells after 5 h of incubation, in the absence or presence of β-lactam molecules. Spontaneous adhesion of the clinical strains #6, #20, #22, and #31 was assessed by epifluorescence microscopy. Green fluorescence resulted from the staining of viable cells with Syto9 dye. Propidium iodide was also used to stain in red bacteria with compromised membranes but no dead cell was detected. Pictures are an overlay of both fluorescence channels. The addition of 0.1× Minimal Inhibitory Concentration (MIC) of antimicrobials to cultures could lead to either increased biomass of adherent bacteria or an alteration of the cell shape. Scale bar, 3 µm.