AI-2/LuxS is involved in increased biofilm formation by Streptococcus intermedius in the presence of antibiotics

Abstract

Bacteria utilize quorum-sensing communication to organize their behavior by monitoring the concentration of bacterial signals, referred to as autoinducers (AIs). The widespread detection of AI-2 signals and its enzymatic synthase (LuxS) in bacteria suggests that AI-2 is an inter- and intraspecies communication signal. We have previously shown that antibiotic susceptibility is affected by AI-2 signaling in Streptococcus anginosus. Since chronic infections involve persistent biofilms resilient to antibiotic treatment, we explored the role of AI-2/LuxS in Streptococcus intermedius biofilm formation and cell viability when the organism was exposed to sub-MICs of ampicillin, ciprofloxacin, or tetracycline. The S. intermedius wild type (WT) and its isogenic luxS mutant, strain SI006, were exposed to sub-MICs of ampicillin, ciprofloxacin, or tetracycline. Biofilms were formed on polystyrene discs in microtiter plates. To assess planktonic cell viability, the ATP microbial viability assay was performed and the numbers of CFU were determined. For complementation assays, the AI-2 precursor dihydroxy pentanedione (DPD) was used as a supplement for SI006. Relative luxS expression was quantified by real-time PCR. The sub-MICs of all three antibiotics increased biofilm formation in S. intermedius WT. However, biofilm formation by SI006 was either unaffected or reduced (P < or = 0.05). Bacterial viability tests of biofilm and planktonic cell cultures indicated that SI006 was more susceptible to antibiotics than the WT. DPD complemented the luxS mutant phenotype. Real-time PCR revealed modest yet significant changes in luxS expression in the presence of antibiotic concentrations that increased biofilm formation. In conclusion, in S. intermedius, AI-2/LuxS was involved in antibiotic susceptibility and increased biofilm formation at sub-MICs of antibiotic.

FIG. 1.

Biofilm formation by S. intermedius WT (continuous line), luxS mutant SI006 (dotted line), and luxS mutant SI006 supplemented with 0.8 nM DPD (interrupted line) following 12 h of incubation in the presence of sub-MICs of ampicillin (A), ciprofloxacin (B), and tetracycline (C). The data points represent mean values (n = 9) with the standard errors of the means. *, significant increase (P ≤ 0.05) in biofilm formation by S. intermedius WT (continuous line) but not luxS mutant SI006 (dotted line); OD₅₃₀, optical density at 530 nm.

FIG. 2.

Scanning electron microscopy images of biofilms of the S. intermedius WT (A), luxS mutant SI006 (B), and luxS mutant SI006 supplemented with 0.8 nM DPD (C) following 12 h of incubation in the absence of antibiotics (control) (panels 1) and in the presence of 0.04 μg/ml ampicillin (panels 2), 0.15 μg/ml ciprofloxacin (panels 3), or 0.08 μg/ml tetracycline (panels 4).

FIG. 3.

Luminescence (ATP level) as a measure of metabolic activity in planktonic cell cultures determined by the BacTiter-Glo viability assay following 12 h of incubation of the S. intermedius WT (continuous line), luxS mutant SI006 (dotted line), and luxS mutant SI006 supplemented with 0.8 nM AI-2 (interrupted line) in the presence of sub-MICs of ampicillin (A), ciprofloxacin (B), and tetracycline (C). The data points represent mean values (n = 12) with the standard errors of the means. *, significantly higher (P ≤ 0.05) ATP levels in the S. intermedius WT (continuous line) than in the luxS mutant SI006 (dotted line).