A Mixed-culture Chemostat System to Predict the Effect of Anti-microbial Agents on the Oral Flora: Preliminary Studies using Chlorhexidine

Abstract

A mixed-culture chemostat system, composed of nine bacterial species representative of plaque in health and disease, has been assessed as an improved laboratory method of evaluating the likely in vivo effects of antimicrobial agents used in dentistry. The advantages of the system include reproducibility, the long-term stable cultivation of bacteria under controllable conditions, and repeated sampling, for bacteriological and biochemical studies, without disrupting the stability of the community. The effects of (i) the continuous provision of chlorhexidine (CHX) and (ii) three pulses of CHX (final concentration in both experiments = 0.24 mmol/L) on the composition of the chemostat communities were monitored. Only L. casei survived the continuous provision of CHX; the other bacteria were killed and were lost at different rates which generally corresponded to their known sensitivities to CHX. After each CHX pulse, the numbers of bacteria fell markedly. Again, L. casei was least affected, while A. viscosus, B. intermedius, and F. nucleatum were temporarily undetectable but returned to their original levels within 2-4 generation times. Counts of S. mutans were affected more by CHX than those of S. sanguis or S. mitior. The effect of successive pulses of CHX on the viability of some bacteria and on acid production (as measured by pH-fall experiments) decreased, suggesting that adaptation to CHX had occurred. The fact that the in vitro observations paralleled previous clinical findings suggests that the mixed-culture system could be used as a predictive model of the probable effect on the oral flora of new anti-microbial agents prior to expensive trials in animals or human volunteers.

Effects of a Chlorhexidine Gluconate-Containing Mouthwash on the Vitality and Antimicrobial Susceptibility of In Vitro Oral Bacterial Ecosystems

“…Addition of CHX caused the loss of A. naeslundii from in vitro CDFF plaques, while Porphyromonas gingivalis was markedly inhibited (19). Similarly, defined community chemostat studies confirmed the non- (29), suggesting that hierarchies of susceptibility in pure culture can be extrapolated to community effects. In a previous study, large variations in the composition of microcosm plaques in replicate fermenters were demonstrated using the same pooled saliva as inocula (28).…”

Levels of infection and colonization of some oral bacteria after use of naf, chlorhexidine and a combined chlorhexidine with naf mouthrinses

“…Despite reduction of mutans streptococci levels, oral streptococci were not total, suggesting that the site and function of mutans streptococci in oral ecosystem may be occupied by other bacteria, probably some strain of streptococci not reduced by this treatment. This hypothesis is supported by the fact that some oral streptococci strains show variable susceptibility to chlorhexidine and fluoride (17,19,21). Concerning oral lactobacilli, levels were not reduced by fluoride and chlorhexidine probably because these microorganisms were hidden in habitats not affected by these products as observed among irradiated patients (12).…”

“…action of chlorhexidine (19,20) followed its bacteriostatic action that in association with fluoride would inhibit bacterial metabolism in different sites. Bacterial recolonization is observed after some days (26,29) or months based on adopted treatment, infection levels, sucrose ingestion or dental restorations.…”

“…Oral streptococci and lactobacilli were not affected by treatment with chlorhexidine mouthrinses. Oral streptococci shows varied susceptibilities to chlorhexidine (19,21) from 4 to 32 µg / ml (27). Oral lactobacilli frequently are not affected by chlorhexidine because of difficulty to achieve niches of lactobacilli into mouth (4,17).…”

Are dental diseases examples of ecological catastrophes?

“…chlorhexidine, Triclosan) can lead to unexpected favourably selective antimicrobial effects. Target species such as S. mutans and Gram-negative anaerobes remain sensitive while many species associated with dental health are relatively unaffected by these short contact times with inhibitors (Bradshaw et al, 1993;Kinniment et al, 1996b;McDermid et al, 1987). In addition, these agents at sub-MIC levels reduce metabolism by inhibiting glycolysis, sugar transport and proteases, which again will help to stabilize microbial communities and maintain homeostasis (Cummins, 1991;Marsh et al, 1983b).…”