The effects of hyperosmosis or high pH on a dual-species biofilm of Enterococcus faecalis and Pseudomonas aeruginosa: an in vitro study

Abstract

Aim: To investigate the effect of hyperosmotic hyperosmosis or alkaline stress on a dual-species biofilm of Enterococcus faecalis and Pseudomonas aeruginosa.

Methodology: Biofilms were grown on glass cover slips suspended in bacterial inoculate for 96 h, after which the cover slips with attached biofilms were immersed in brain heart infusion broth (BHI-broth) with 6 mol L(-1) sodium chloride (NaCl) representing the hyperosmotic group or Ca(OH)(2), pH 12.1, representing the alkaline group. Two per cent sodium hypochlorite and BHI- broth served as positive and negative controls, respectively. After treatment, the biofilms were washed, harvested and plated on blood-agar plates after serial dilution. The bactericidal effect was assessed by determining the colony-forming units (CFU). The effect on the biofilm mass was imaged with confocal laser scanning microscopy (CLSM).

Results: Hyperosmosis reduced the CFU of both species significantly after 72 h (P < 0.0001). After 168 h, P. aeruginosa was eradicated and the E. faecalis reduction was more than 99%. High pH could not induce a significant bacterial reduction. CLSM revealed dense flocculation of the biofilms incubated in alkaline broth.

Conclusion: Hyperosmosis effectively reduced a dual-species biofilm of E. faecalis and P. aeruginosa, whilst high pH had limited bactericidal effect in this model.