Effects of chlorhexidine on a tongue-flora microcosm and VSC production using an in vitro biofilm perfusion model

An in vitro perfusion biofilm model, derived from tongue-scrape microflora removed from one individual, was employed to study sulfide biogenesis and the effects of repeated exposure to chlorhexidine (CHX). Volatile sulfur compounds (VSC) were measured using a carbon veil electrode within the biofilm and a halimeter for liquid and gas phase levels, respectively. The microflora of the perfusate and the biofilm were assessed by microbiological techniques and polymerase chain reaction (PCR) to estimate diversity. Biofilms treated with a 1 mL pulse of 0.1% CHX twice a day for three days showed (1) a large reduction in viable count (>90% kill), (2) a (slow) reduction in the VSC production rate, consistent with the reduction in microbes rather than direct inhibitory effects on the biotransformation steps, and (3) a preferential reduction of strict anaerobes. Treated biofilms allowed to recover over 3-5 days showed a nominal amount of regrowth in some experiments, although population numbers were still well below those found in untreated controls. The microbiological composition of biofilms treated but allowed to recover was markedly different from the controls, with proportionally fewer strict anaerobes. Thus, CHX treatment caused detectable ecological shifts with consequent long-term effects on the response of the biofilm in terms of VSC generation, consistent with clinical observations. The model appears highly suited for testing the efficacy of putative anti-malodour or antimicrobial agents.