Effects of a chlorhexidine gluconate-containing mouthwash on the vitality and antimicrobial susceptibility of in vitro oral bacterial ecosystems

Abstract

Oral bacterial microcosms, established using saliva inocula from three individuals, were maintained under a feast-famine regime within constant-depth film fermenters. Steady-state communities were exposed four times daily, postfeeding, to a chlorhexidine (CHX) gluconate-containing mouthwash (CHXM) diluted to 0.06% (wt/vol) antimicrobial content. The microcosms were characterized by heterotrophic plate counts and PCR-denaturing gradient gel electrophoresis (DGGE). CHXM caused significant decreases in both total anaerobe and total aerobe/facultative anaerobe counts (P < 0.05), together with lesser decreases in gram-negative anaerobes. The degree of streptococcal and actinomycete inhibition varied considerably among individuals. DGGE showed that CHXM exposure caused considerable decreases in microbial diversity, including marked reductions in Prevotella sp. and Selenomonas infelix. Pure-culture studies of 10 oral bacteria (eight genera) showed that Actinomyces naeslundii, Veillonella dispar, Prevotella nigrescens, and the streptococci were highly susceptible to CHX, while Lactobacillus rhamnosus, Fusobacterium nucleatum, and Neisseria subflava were the least susceptible. Determination of the MICs of triclosan, CHX, erythromycin, penicillin V, vancomycin, and metronidazole for microcosm isolates, before and after 5 days of CHXM exposure, showed that CHXM exposure altered the distribution of isolates toward those that were less susceptible to CHX (P < 0.05). Changes in susceptibility distributions for the other test agents were not statistically significant. In conclusion, population changes in plaque microcosms following repeated exposure to CHXM represented an inhibition of the most susceptible flora with a clonal expansion of less susceptible species.

FIG. 1.

Effect of 48-h exposure to pulsed CHXM (Corsodyl), adjusted to 0.06% (wt/vol) antimicrobial content, on groups of oral bacteria in dental-plaque microcosms. (A) Total strict anaerobes; (B) total aerobes and facultative anaerobes; (C) gram-negative anaerobes; (D) total streptococci; (E) actinomycetes; (F) total lactobacilli. The boxes represent distributions for three separate experiments (open, baseline samples; shaded, treated samples), each utilizing saliva from a different individual (volunteers A to C) as the inoculum. The horizontal bars within the boxes represent median values; the error bars indicate standard deviations. *, P < 0.05 (Student’s t test).

FIG. 2.

Negative image of a parallel DGGE gel showing microcosm samples removed on days 3 (A), 5 (B), and 11 (C) (baseline), together with days 15 (E) and 17 (F) (after 3 and 5 days of CHXM addition, respectively). Bands B1 to B5 and CH1 are indicated.