Enzimas Y Control De Placa

l dJournal of Medical Microbiology (2009), 58, 482–491

DOI 10.1099/jmm.0.006601-0

An in vitro evaluation of hydrolytic enzymes as dental plaque control agents
Ruth G. Ledder,1 Tejal Madhwani,1 Prem K. Sreenivasan,2 William De Vizio2 and Andrew J. McBain1
Correspondence Ruth G. Ledder ruth.ledder@manchester.ac.uk
1

School of Pharmacy and Pharmaceutical Sciences, University ofManchester, Manchester M13 9PT, UK Colgate–Palmolive Company, 909 River Road, Piscataway, NJ 08855, USA

2

Received 16 September 2008 Accepted 25 December 2008

The plaque-control potential of commercially available amylase, lipase and protease was evaluated by observing their effects on coaggregation and on bacterial viability within various plaque microcosms. A quantitative coaggregation assayindicated that protease significantly inhibited the extent of coaggregation of Actinomyces naeslundii and Streptococcus oralis (P ,0.05) and of Porphyromonas gingivalis and S. oralis. Amylase significantly (P ,0.05) increased the coaggregation of A. naeslundii versus Fusobacterium nucleatum and A. naeslundii versus P. gingivalis. Concomitant challenge of constant-depth film fermenter-grown plaqueswith the enzymes did not result in detectable ecological perturbations (assessed by differential culture and denaturing gradient gel electrophoresis). Similar dosing and analysis of multiple Sorbarod devices did not reveal increases in bacterial dispersion which could result from disaggregation of extant plaques. A short-term hydroxyapatite colonization model was therefore used to investigatepossible enzyme effects on early-stage plaque development. Whilst culture did not indicate significant reductions in adhesion or plaque accumulation, a vital visual assay revealed significantly increased aggregation frequency following enzyme exposure. In summary, although hydrolytic enzymes negatively influenced binary coaggregation, they did not cause statistically significant changes in bacterialviability within plaque microcosms. In contrast, enzyme exposure increased aggregation within extant plaques.

INTRODUCTION
Hydrolytic enzymes with specificity for molecules involved in coaggregation and biofilm architecture have previously been evaluated for use as anti-plaque agents (Marsh, 1992). Whilst a small number of publications have focused on enzymic cleaning of dentures(Budtz-Jorgensen, 1977; Odman, 1992), other studies have assessed the potential of enzymic formulations as adjuncts for in situ oral hygiene (Hull, 1980; Kelstrup et al., 1973, 1978; Robinson et al., 1975). Few investigations have, however, systematically evaluated hydrolytic enzymes against complex, multispecies plaques or tested the effects at the cellular level against coaggregation, which has been defined ascell–cell adherence of genetically distinct bacteria (Kolenbrander, 2000) and is regarded as an important process in the development of multi-species biofilms (Hughes et al., 1988). Coaggregation was first observed in bacteria associated with dental plaque (Childs & Gibbons, 1990; Gibbons &
Abbreviations: CDFF, constant-depth film fermenter; DGGE, denaturing gradient gel electrophoresis; HA,hydroxyapatite; MSD, multiple Sorbarod devices.

Nygaard, 1970), and research activity related to the phenomenon remains largely focused on oral bacteria (Foster & Kolenbrander, 2004; Kigure et al., 1995; Shen et al., 2005). The importance of coaggregation in the formation of oral biofilms can be inferred from two types of evidence: (i) the incidence of coaggregative bacteria in the mouth ismarkedly higher than in most other environments (Rickard et al., 2003); and (ii) structures indicative of coaggregative interaction can be visualized in situ (Bolstad et al., 1996; Kolenbrander, 2000). An example of the latter is the characteristic corncob formations that are formed by Fusobacterium nucleatum and Streptococcus sanguinis (DiRienzo et al., 1985). Coaggregation has been defined as the...