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Microbiological Aspects of the Chemical Control of Plaque and Gingivitis P.D. Marsh J DENT RES 1992 71: 1431 DOI: 10.1177/00220345920710071501 The online version of this article can be found at: http://jdr.sagepub.com/content/71/7/1431

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Microbiological Aspects of the Chemical Control of Plaque and Gingivitis P.D. MARSH Pathology Division, PHLS Centre for Applied Microbiology and Research, Salisbury, SP4 OJG, England Antimicrobial agents, delivered either by mouthrinse or toothpaste, vehicle for the delivery of these agents, their incorporation into can be used to maintain plaque at levels compatible with oral health toothpastes has proved more difficult because of formulation probby (a) reducing existing plaque, (b) preventing the formation ofnew lems. Plaque control could be achieved by agents that remove plaque, (c) selectively inhibiting those particular bacteria that are attached organisms or prevent colonization of the enamel, i.e., associated with disease, and (d) inhibiting the expression of viru- agents need not have antimicrobial activityperse. However, most of lence determinants. Although many antimicrobial agents would the successful agents for plaque control in current use do have appear to be suitable for plaque control, few have been found to antimicrobial activity, and hence the majority of this paper will be possess clinical efficacy. This is because ofinherent problems in the devoted to this category of compound. Most of these compounds are mode of action of agents in the mouth, and with difficulties with also directed against supra-gingival plaque to either prevent distheir formulation into dental products. Currently formulated anti- ease or to treat early gingivitis where there is no deepening of the microbial agents include metal salts (e.g., zinc, stannous, copper), gingival crevice. Treatment of established disease by the local or phenols (triclosan), plant extracts (sanguinarine), enzymes (e.g., systemic delivery ofantimicrobial agents is beyond the scope ofthis glucanase, amyloglucosidase/glucose oxidase), "essential oils" (e.g., paper, but this topic has been reviewed recently by Addy (1990). thymol, menthol), and bisbiguanides (chlorhexidine). Although Antimicrobial agents delivered by toothpaste or mouthrinse are many of these agents exhibit a broad spectrum of antimicrobial intended for non-supervised, regular usage. A careful balance has activity in the laboratory, they may display valuable selective to be achieved, therefore, between clinical benefit derived from the properties on plaque. The effect of an agent will be concentration- use of such agents and the potential for adverse effects on the dependent. Initially, the inhibitor may be briefly at levels above its natural ecology of dental plaque. MIC, but thereafter, it will be desorbed offoral surfaces and operate at sub-lethal concentrations. At these latter levels, agents can be effective by inhibiting metabolism (e.g., acid production, protease Microbial composition of dental plaque in health. activity), and slowing bacterial growth. Agents with complemen- As soon as a tooth has been cleaned, salivary proteins and glycoprotary modes of action are being combined to increase their antibac- teins are adsorbed to enamel to form the acquired pellicle. Bacteria terial effectiveness. The long-term use of dental products contain- interact with this pellicle via a number of specific molecular intering antimicrobial agents should not (a) disrupt the natural balance actions between adhesins on the bacterial cell surface and receptors of the oral microflora, (b) lead to colonization by exogenous organ- (ligands) in pellicle (Gibbons, 1989). Subsequent bacteria of the isms, or (c) lead to the development of microbial resistance. Several same or different species are able to adhere not only to pellicle but products are now available that satisfy these criteria, and are also to the pre-attached cells (co-aggregation) (Kolenbrander, 1988; clinically effective in helping to control plaque and gingivitis. Kolenbrander et al., 1990). Once attached, cells grow on the tooth surface, and micro-colonies have been observed. Eventually, and J Dent Res 71 (7):1431-1438, July, 1992 especially at stagnant sites, the combination ofbacterial multiplication with further adhesion and co-aggregation produces confluent Introduction. growth. Thus, plaque builds up in an orderly manner with an increase in species diversity with time. The final composition of Dental plaque is the film of micro-organisms found on the tooth plaque at a site is influenced by local environmental conditions, surface embedded in a matrix of polymers of salivary and bacterial especially the redox potential and the provision of essential nutriorigin. Dental plaque develops naturally on the tooth surface and ents-for example, by gingival crevicular fluid. Experimental forms part of the host defenses of the mouth by acting as a barrier studies of plaque development have shown that the early colonizers to colonization by exogenous micro-organisms (Marsh, 1989). This are streptococci, particularly S. sanguis, S. oralis, and S. mitis; barrier effect has been termed colonization resistance, and can be Actinomyces spp., haemophili, and Neisseria spp. are also isolated broken by, for example, the long-term use of broad-spectrum anti- during early plaque build-up (Liljemark et al., 1986; Nyvad and biotics. Under such circumstances, there can be overgrowth by non- Kilian, 1987). It is conceivable that the subsequent development of resident micro-organisms (especially yeasts and enteric bacteria) plaque could be affected if the attachment and growth ofthese early and the emergence of antibiotic-resistant strains (Sanders and colonizers were inhibited by antimicrobial agents. Sanders, 1984). Once developed, the microbial composition of plaque is relatively Although dental plaque forms naturally on teeth, in the absence stable, although variations occur at different sites around the of adequate oral hygiene, it can accumulate beyond levels that are mouth. In general terms, the fissures are colonized predominantly compatible with dental health, and at a susceptible site, dental by streptococci, whereas streptococci and actinomyces are most caries or periodontal disease can occur. In many individuals, the numerous at approximal sites. The microflora of the healthy customary oral hygiene method of toothbrushing is, by itself, usu- gingival crevice is relatively sparse, and is also composed mainly of ally insufficient over long periods to provide a level of plaque control Gram-positive species, especially streptococci (Slots, 1977). The consistent with oral health. Consequently, the incorporation of crevice has a lower redox potential than other sites, and has a pH chemical agents with antiplaque or antimicrobial activity into around neutrality. These conditions are more conducive than those dental products has been proposed as a potential prophylactic at other oral sites to the growth of obligately anaerobic species; in method ofreducing plaque-mediated disease (Hull, 1980; Kornman, one study, approximately 25% of the total microflora of the crevice 1986; Addy, 1990). Whereas mouthrinses have proved a successful was obligately anaerobic (Slots, 1977). Organisms that have been implicated in periodontal diseases-such as spirochetes, Eikenella Presented at the symposium on "New Agents in the Chemical Control of corrodens, Actinobacillus actinomycetemcomitans, and some blackPlaque and Gingivitis", during the IADR's 69th General Session, April 17, pigmented anaerobes, mainly Prevotella intermedia and P. 1991, Acapulco, Mexico melaninogenica-can also be recovered on occasions in low numbers Downloaded from jdr.sagepub.com at Universitats-Landesbibliothek on December 20, 2013 For personal use only. No other uses without permission.

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Concentration of An Antimicrobial Agent With Time

PREDOMINANT PLAQUE MICROFLORA IN HEALTH AND DISEASE

HEALTH S. mitis S. oralis S. sangui * ENAMEL

~~~Actinomycea Spp Neisseria spp.

Haemopholus spp. CARIES S. mutans / S. sobrinu/ Lactobacillus spp. ROOT CARIES S. mutana A. viscosua Lactobacillus spp.

c 0

N

PERIODONTAL

DISEAESU

Actinomycea spp. P. gingiNas P. intermedia

W. recta A. actinomycetemcomitans E. corrode "B. forsyhus" F. nubeatum

Treponema spp.

Fig. 1-Transitions in the composition of the predominant plaque microflora in health and disease.

MIC

C

E N T R A T I

SUB-MIC

0

N

TIME Fig. 2-Diagrammatic representation of the change in concentration of an antimicrobial agent, delivered by mouthwash or dentifrice, with time (MIC = minimum inhibitory concentration).

from the healthy gingival crevice (Frisken et al., 1987; Ashley et al., 1988). The resident plaque microflora of a site in health remains relatively stable with time. This stability (termed microbial homeostasis) is not due to indifference among the component species but rather results from a dynamic balance of microbial interactions, including both synergism and antagonism (Sanders and Sanders, 1984; Marsh, 1989). Care has to be taken to ensure that the regular use of antimicrobial agents in dental products does not increase the tendency for microbial homeostasis to break down.

al., 1987). Although it is still not clear whether gingivitis is a necessary stage for the development of more advanced forms of periodontal disease, it has been found in clinical studies that some species that predominate in chronic periodontitis, but which are not detectable in the healthy gingival crevice, are also found as a small percentage of the microflora in gingivitis (Moore et al., 1987). Bacteria that are associated with periodontal diseases are often proteolytic, and produce enzymes that can damage tissues directly or interfere with the activity of the host defenses (Slots and Genco, 1984). They also produce metabolites that are cytotoxic, e.g., acids, ammonia, and sulfur-containing compounds. The host amines, Microbial composition of dental plaque in disease. mounts an immunological response to the bacterial challenge, and Plaque preferentially accumulates at stagnant or retentive sites the resulting inflammation can also lead to tissue damage due to the unless it is removed by diligent oral hygiene. As the plaque mass release of proteolytic enzymes from phagocytic and other host cells increases, the buffering and antimicrobial properties of saliva are (Genco and Slots, 1984; Meikle et al., 1986). less able to penetrate plaque and protect the enamel and gingival tissues. There is a shift in the balance ofthe predominant bacteria Microbiological aspects of chemical plaque control. in plaque away from those associated with health (Fig. 1), and microbial homeostasis breaks down. From the comments made above, it is clear that gingivitis (and dental Gingivitis is associated with the accumulation of plaque around caries), unlike some classical medical infections, is not caused by overt the gingival margin. This leads to a shift in the composition of microbial pathogens that have their primary habitat outside of the plaque away from a streptococci-dominated microflora toward higher mouth. Both of these common dental diseases are associated with levels of Actinomyces spp. and an increase in the isolation of shifts in the balance of the resident plaque microflora (Fig. 1), capnophilic (C02-requiring) and obligately anaerobic bacteria. The although it is possible that periodontopathogens might be acquired microflora increases in diversity during the development of gingivi- from reservoirs at mucosal sites within the oral cavity (van der Velden tis, but no particular group ofbacteria is uniquely associated with et al., 1986; Frisken et al., 1987). Since this resident microflora has disease. Several studies have reported increased proportions of functions that are beneficial to the host, the aim is to use chemical Fusobacterium nucleatum, P. intermedia, Capnocytophaga spp., agents to control plaque accumulation rather than to eliminate it. In Eubacterium spp., and spirochetes in gingivitis (Savitt and this way, the agents are generally considered to function prophylactiSocransky, 1984; Moore et al., 1987). The precise mechanisms as to cally to prevent plaque-mediated disease, although some can treat how these changes are brought about are still uncertain, but they gingivitis. There are four main mechanisms whereby plaque accumulation may parallel the impact of a changing environment on plaque composition which is now well-documented for caries. In caries, the can be controlled and disease prevented by the use of antimicrobial regular consumption of dietary carbohydrates leads to frequent agents deliveredfrom dental products. First, the rate ofaccumulation conditions oflow pH in plaque which inhibits many of the predomi- of new plaque can be reduced, keeping the relative balance of indinant species but favors the growth and metabolism of mutans vidual species compatible with that associated with health. Second, streptococci and lactobacilli (Bradshaw et al., 1989). Bacteria existing plaque can be reduced or removed. Third, the growth ofthose implicated in gingivitis and more advanced forms of periodontal species associated with disease can be selectively suppressed, and disease may also be present in the gingival crevice in health, but fourth, the production ofvirulence factors (e.g., proteases, cytotoxins) only in low numbers, and as such are not clinically significant. can be inhibited. However, as stressed earlier, itis also essential that However, during inflammation the local environment is changed by these agents do not disrupt the ecology of dental plaque to such an an increased flow of gingival crevicular fluid-this provides not only extent as to induce undesirable changes in the composition of the components of the host defenses but also novel nutrients-and this resident oral microflora. These constraints limit the choice of antimimay favor the growth ofputative periodontopathogens (ter Steeg et crobial agents that can be considered for unsupervised, regular use. Downloaded from jdr.sagepub.com at Universitats-Landesbibliothek on December 20, 2013 For personal use only. No other uses without permission.

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TABLE 1 CLASSES OF SOME COMMONLY AVAILABLE ANTIMICROBIAL AGENTS, WITH EXAMPLES, USED FOR PLAQUE CONTROL

Class of Agent

Example

Main Delivery Vehicle

Bisbiguanide

Chlorhexidine

Mouthwash

Detergent Enzyme

Sodium lauryl sulfate Mutanase/glucanase;

Zinc; copper; stannous Sanguinarine

Mouthwash and toothpaste Toothpaste Toothpaste Mouthwash Toothpaste and mouthwash Mouthwash and toothpaste

Triclosan

Toothpaste and mouthwash

Cetylpyridinium chloride

Mouthwash

Hexetidine

Mouthwash

Amyloglucosidase/glucose oxidase Thymol; eucalyptol

"Essential oils" Metal ions Plant extracts Phenols Quaternary ammonium compounds

A wide range of antimicrobials with apparently relevant properties for use as plaque control agents exists. However, relatively few of these agents have beenfound to be suitable for use (Table 1) because of (a) a lack ofcompatibility with dental product formulations (Cummins and Creeth, 1992), or(b) a lack ofclinical efficacy. Reasons forthis lack of activity in vivo of agents that appear to be efficacious in the laboratory are related to the limitations of their evaluation in vitro (Marsh, 1992), andtothemechanismsofactionoftheseantimicrobials in the mouth (Goodson, 1989). For example, some agents are inactivated either when adsorbed to a surface or when bound to host proteins, while the mouth provides unfavorable pharmacokinetics for other agents.

Mechanism of action of plaque control agents. The effects of antimicrobial on dental plaque will be dependent on the concentration ofthe agent delivered to the site (Table 2). Agents may be present initially at a relatively high concentration (at or above the minimum concentration, MIC, known to inhibit microorganisms in the laboratory) but for only short periods of time (perhaps only a few minutes). After this time, a large proportion of the agent will be lost by expectoration and swallowing. Subsequent effects are dependent on the concentration of the agent that is retained on oral surfaces; this may be at a level below the MIC (subMIC, Fig. 2). At high concentrations, an agent may be bactericidal and reduce the level of an organism in plaque or saliva. Agents that desorb bacteria that are already attached to oral surfaces would produce the same apparent effect on plaque. Ifthe agent is only bacteriostatic, then organisms would remain viable but multiplication wouldbe prevented, and their numbers would remain static. At lower (sublethal) concentrations, an agent may still be effective by reducing the production of virulence determinants that contribute to the pathogenicity of an organism, e.g., by inhibiting acid (cytotoxin) production or protease activity. Indeed, the mere slowing of the rate of division of bacteria could be significant in reducing plaque accumulation (Table 2).

Laboratory evaluation of antimicrobial agents for plaque control: Limitations and implications. Ofnecessity, antimicrobial agents for plaque control are evaluated in the laboratory. Many of the methods used for their assessment are based on those that have been successfully developed for applications in medicine. Infections in medicine are due mainly to

colonization of sites by exogenous or opportunistic pathogens. In this situation, treatment involves getting as high a concentration of an antibiotic as possible to the infected site for as long as possible. Ideally, such a concentration should kill rather than just inhibit the growth of the pathogen. Consequently, the minimum bactericidal concentration (MBC: the concentration at which 99.9% of test bacteria are killed within a given time period) and the minimum inhibitory concentration (MIC: the lowest concentration that prevents bacterial growth) are determined routinely. The MIC is also usually quoted as the primary indicator of potential efficacy of plaque control agents, even though the contact time in the body between high concentrations of antibiotics and target organisms is usually measured in hours rather than in minutes, as is the case with antimicrobial agents from dental products and plaque. Compounds such as chlorhexidine (Emilson, 1977; Stanley et al., 1989), cetylpyridinium chloride (Gjermo et al., 1970), sanguinarine (Dzink and Socransky, 1985), and triclosan (Ritchie and Jones, 1988; Gaffar et al., 1990) have been shown to have a broad spectrum of antimicrobial activity against relevant oral Gram-positive and Gram-negative bacteria. However, the MIC is not necessarily a reliable indicator ofclinical efficacy. Agents with closely similar spectra of activity and MIC values against oral micro-organisms can vary markedly in their clinical performance. For example, chlorhexidine has been shown to be the most effective agent in vivo, while cetylpyridinium chloride, which has equivalent antimicrobial activity in the laboratory, produces little antiplaque benefit in humans (Gjermo et al., 1970), possibly because it is inactivated when adsorbed to surfaces (Moran and Addy, 1984). Likewise, organisms with similar MIC values to a particular agent could be affected quite differently in plaque depending on (a) whether the inhibitor has a bactericidal or bacteriostatic effect on the organisms, (b) the kinetics of inhibition, (c) the penetration of the agent into a biofilm such as dental plaque, and (d) the environmental conditions within plaque (Marsh, 1992). For example, some organisms might be affected rapidly, while others may require much longer exposure times to the antimicrobial agent (Wilson et al., 1990), possibly in excess of those likely to occur in the mouth. For these reasons, it can be difficult to interpret MIC data unless relevant assay conditions and exposure times between antimicrobial agent and micro-organism are used (Guggenheim and Schmid, 1989; Marsh, 1992). The implications of this are that the full potential of the agent may not be expressed under its routine conditions of use in the mouth, and that properties other than the mere killing of microorganisms can become significant. Consequently, in order for the evaluation of potential antimicrobial agents in the laboratory to be

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TABLE 2

EFFECT OF CONCENTRATION ON THE PRINCIPAL MODES OF ACTION OF ANTIMICROBIAL AGENTS USED FOR PLAQUE CONTROL High Concentration (> MIC) Bactericidal

Low Concentration* (< MIC) Reduced adherence

Membrane disruption Perturbation of ion gradients Inhibition of sugar metabolism (transport; glycolysis; glucan formation) Inhibition of proteases Inhibition of amino acid metabolism Reduction of bacterial growth rate 'Many of these effects are interrelated, e.g., membrane disruption can lead to perturbations ofion gradients and inhibition of enzymes, both of which may result in reduced bacterial growth rates.

Bacteriostatic Reduced plaque accumulation

improved, more complex in vitro models of the oral microflora have been devised. Several approaches have been based on the cultivation of complex mixed cultures of oral bacteria by use of continuous culture techniques (McDermid et al., 1987), in the presence and absence ofrelevant surfaces (Gaffar et al., 1990; Keevil et al., 1987). These approaches have a number of advantages, including (a) the long-term cultivation ofdiverse mixed cultures under conditions that more closely reflect their growth in vivo, and (b) the detection ofboth direct andindirect inhibition ofbacteria. In a diverse microflora such as that found in the gingival crevice in disease, the persistence of many species is dependent on the metabolism of other organisms. The removal of one of these key organisms from a food web (direct inhibition) can lead to secondary inhibitory effects on dependent species (indirect inhibition). Furthermore, when the system is used to dose antimicrobial agents continuously, because the model is a continuous flow-through system, even a small inhibition of an organism is amplified over a relatively short period of time (McDermid et al., 1987). Data from these types of studies have shown, for example, that organisms with a similar MIC value for triclosan can vary enormously in their degree ofinhibition in mixed continuous cultures (Table 3), and also that agents may have a hitherto unsuspected selectivity of action (Bradshaw et al., 1991). In this latter study, Streptococcus oralis and S. gordonii were relatively unaffected by triclosan, whereas a number of species associated with plaquemediated disease were severelyinhibited (S. mutans, A. viscosus, and P. intermedia). Combining triclosan with zinc citrate enhanced the inhibition of some of the Gram-negative periodontopathogens (F. nucleatum and P. gingivalis), while still leaving the growth of streptococci associated with health (Fig. 1) relatively unaffected (Bradshaw et al., 1991). Similar studies that used a chemostat model system in conjunction with flow cells containing hydroxyapatite disks to facilitate biofilm formation in vitro have shown that the antiplaque activity oftriclosan can be enhanced ifit is combined with acopolymertoboostits retention at surfaces (Gaffaretal., 1990).

Microbiological properties of currently-used antimicrobial agents for plaque control. As stated earlier, out of the wide range of antimicrobial agents that have been screened, relatively few have been formulated into proven products for plaque control (Table 1). The successful agents include bisbiguanides, plant extracts, enzymes, metal ions, and phenols. Some ofthe microbiological properties ofthese agents will now be described briefly. Other agents of potential use were reviewed recently by Scheie (1989). A number of bisbiguanides have anti-plaque properties, but

chlorhexidine is the agent that has been studied in most detail and which serves as the 'gold standard" when comparisons with other products are made. Chlorhexidine can reduce dental plaque, caries, and gingivitis in humans (Gjermo, 1989; Addy, 1990). It is a broadspectrum antimicrobial agent with activity against a wide range of Gram-positive and Gram-negative supragingival and subgingival plaque bacteria (Emilson, 1977; Stanley et al., 1989) and fungi (including yeasts). At high concentrations, chlorhexidine is bactericidal, and acts as a detergent by damaging the cell membrane (Gjermo, 1989). The benefits of chlorhexidine are not limited to its initial bactericidal effects, since other agents with similar MIC values and spectrum of activity against oral bacteria do not have the same anti-plaque properties (Gjermo et al., 1970). Chlorhexidine adsorbs to oral surfaces and is retained for many hours at sub-lethal concentrations. At these levels, chlorhexidine appears to have a number of properties whereby it can interfere with the metabolism of oral bacteria. It can inhibit acid production by plaque in vivo (Oppermann, 1979), supporting laboratory studies that have shown that sub-MIC levels of chlorhexidine can (a) abolish the activity of the phosphoenolpyruvate-phosphotransferase sugar transport system, and thereby markedly inhibit acid production, in oral streptococci (Marshet al., 1983), (b) inhibit arginine uptake and catabolism in S. sanguis (Rogers et al., 1987), (c) inhibit proteolysis (including the trypsin-like protease) of P. gingivalis (Minhas and Greenman, 1989; Millward and Wilson, 1990), and (d) affect other membrane functions, including ATP synthase activity and the maintenance of ion gradients in streptococci (Harold et al., 1969). Mutans streptococci are more sensitive to chlorhexidine than other oral streptococci (Emilson, 1977; Stanleyet al., 1989), and this property has been exploited successfully to reduce the levels in plaque and saliva ofthese cariogenic bacteria in subjects with a high risk of developing caries (Schiott et al., 1976a; Mikkelsen et al., 1981), and to reduce transmission of mutans streptococci from mother to infants (Kohler et al., 1983). There has been less research carried out to determine whether chlorhexidine has any analogous selective effects on periodontopathic bacteria. However, a significant reduction in levels of Actinomyces spp. was found after six months' daily use of a chlorhexidine mouthrinse (Briner et al., 1986a). This may be of significance for prevention of periodontal diseases, since these organisms are associated with gingivitis, and other periodontopathogens can co-aggregate withActinomyces spp. (Kolenbrander, 1988). Chlorhexidine is generally reserved for short-term use because of local side-effects (Lang and Brecx, 1986; Addy, 1990). It is also usually deliveredonly by mouthrinse because it is incompatible with conventional dentifrice formulations. Other bisbiguanides with anti-plaque activity include alexidine and

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MICROBIOLOGICAL ASPECTS OF PLAQUE CONTROL

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TABLE 3 EFFECTS OF PULSES OF TRICLOSAN (10 mg/L) AND ZINC CITRATE (25mg/L) ON A MIXED CULTURE OF ORAL BACTERIA GROWN IN CONTINUOUS CULTURE Triclosan* MIC (mglL)

Bacterium Streptococcus oralis

Triclosan 0

Percentage Inhibition Triclosan + zinc citrate 0

Streptococcus gordonii

10

4.5

6.7

Streptococcus mutans

10

95.5

89.8

Actinomyces viscosus

20

83.0

93.8

Lactobacillus casei

30.8

71.2

Neisseria subflava

73.7

99.7

Veillonella dispar

20

0

79.1

Fusobacterium nucleatum

50

45.0

94.5

Prevotella intermedia

10

92.8

99.9

99.9 46.9 Porphyromonas gingivalis 50 Percentage inhibition is defined as the reduction in viable count of each species 24 h after a pulse of inhibitor (data taken from Bradshaw et al., 1991). *Data taken from Ritchie and Jones (1988). **-, not determined.

octenidine; there are few microbiological data on these compounds, but they probably function in a similar manner to chlorhexidine (Scheie, 1989; Addy, 1990). Other agents that have been formulated into mouthrinses include hexetidine, sanguinarine, and "essential oils". Hexetidine has moderate anti-plaque and antimicrobial activity but little effect on gingivitis compared with chlorhexidine; it is retained in the mouth for longer than cetylpyridinium chloride but less than chlorhexidine (Roberts and Addy, 1981). The antibacterial activity of hexetidine has been enhanced by combining it with metal ions such as zinc or copper (Scheie, 1989). Sanguinarine is an alkaloid extract from the rhizome of the plant Sanguinaria canadensis. It has a broad spectrum of activity against Gram-positive and Gram-negative oral bacteria, especially species associated with gingivitis and more advanced forms of periodontal diseases (Dzink and Socransky, 1985). It has also been shown to inhibit enzyme activity and reduce glycolysis; the adherence of oral bacteria to hydroxyapatite is also inhibited (Scheie, 1989). Commercial mouthrinses contain sanguinarine in combination with zinc ions, so that it is not always possible to discern which properties are due to the plant extract and which are due to the metal salt. The clinical anti-plaque findings with sanguinarine are equivocal at present (Scheie, 1989; Addy, 1990). A mixture of "essential oils" has been in use as a mouthrinse for over a century. These "oils" (thymol, eucalyptol, methyl salicylate, menthol) can reduce pre-formed plaque and retard the development of existing plaque and gingivitis (Gordon et al., 1985). The long-term rinsing with these "oils" has also been reported to reduce the levels of endotoxin in plaque (Fine et al., 1985), which could be of significance in preventing inflammation. Detergents are present in mouthrinses and dentifrices; they can be bactericidal, but the duration of their inhibitory activity is uncertain at present. Detergents such as sodium lauryl sulfate (SLS) can inactivate bacterial enzymes, including those associated with sugar transport (Schachtele, 1975) andextracellularpolysaccharide synthesis (Ciardi et al., 1978). These properties of SLS have hampered the formulation of

enzymes withtherapeutic potential into dentifrices. In one product, in order for enzyme activity to be maintained, SLS was replaced by a non-ionic detergent. Nevertheless, products are available containing amyloglucosidase and glucose oxidase to boost the activity of the salivary peroxidase host-defense system, and dextranase or mutanase to reduce plaque accumulation. The amyloglucosidase/ glucose oxidase dentifrice has been reported to reduce plaque and gingivitis (Midda and Cooksey, 1986), although others have found variable clinical results (Etemadzadeh et al., 1985). A substantial anti-plaque activity has been claimed for a dentifrice containing dextranase (Kitamura et al., 1980), while others have reported inconclusive results (Scheie, 1989; Addy, 1990). Metal salts have anti-plaque and antimicrobial activity (Scheie, 1989; Addy, 1990). At high concentrations they can be bactericidal, while at sub-lethal levels they can inhibit a number of enzyme systems relating to carbohydrate metabolism (sugar transport, glycolysis, glucan formation) (Scheie, 1989) and proteolysis. Zinc salts have been incorporated into a number of dental products, including dentifrices. Zinc citrate has moderate plaque-inhibitory activity (Saxton, 1986), but its ability to control gingivitis has not been fully investigated. In the laboratory, zinc salts can inhibit acid production by oral streptococci (including S. mutans) (Cummins, 1991) and the trypsin-like protease of P. gingivalis (Marsh, 1991). Zinc salts can be complexed by protein (Ritchie and Jones, 1990), and so their efficacy in the mouth may be reduced. Metal salts, and especially zinc, are used in combination with a number of other antimicrobial agents in dental products (Scheie, 1989). Phenols are broad-spectrum antimicrobial agents, and triclosan (2,4,4' trichloro-2'-hydroxydiphenyl ether) has been found to be retained in the mouth and to have clinical efficacy without side-effects. Triclosan has a broad spectrum of antimicrobial activity (Ritchie and Jones, 1988; Gaffar et al., 1990; Table 3) and moderate anti-plaque properties (Saxton, 1986). At sub-MIC levels, it can interfere with bacterial metabolism, including acid production by oral streptococci (Cummins, 1991) and the trypsinlike protease of P. gingivalis (Marsh, 1991). Triclosan has been

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MARSH

incorporated into dentifrices by several manufacturers. In order to boost its anti-plaque activity, two strategies have been used: Triclosan has been combined with (a) a co-polymer (polyvinyl methyl ether maleic acid), which increases its oral retention, or with (b) another antimicrobial agent, zinc citrate. The triclosan/co-polymer dentifrice can reduce plaque formation, supragingival calculus, and prevent gingivitis in clinical trials in humans (Garcia-Godoy et al., 1990; Lobene et al., 1990). These agents also exhibited anti-plaque activity when used as a mouthrinse (Singh et al., 1990). The triclosan/co-polymer toothpaste has been shown to have increased activity in the reduction of salivary bacteria when compared with triclosan alone (Addy et al., 1989). The triclosan/zinc citrate dentifrice combination is based on the premise that additive or synergistic interactions can be obtained from relatively low concentrations ofboth agents. Additive anti-plaque activities have been obtainedin clinical studies (Saxton, 1986). The two agents appear to have complementary modes of action, in that zinc citrate was more effective on existing plaque, whereas triclosan inhibited plaque formation on clean surfaces (Saxton et al., 1988). Triclosan/zinc citrate can also prevent gingivitis (Jones et al., 1990), reduce supragingival plaque and calculus formation (Stephen et al., 1990), and suppress the enrichment in plaque of anaerobic bacteria and Actinomyces species (Jones et al., 1990). These clinical findings are supported by laboratory data. Additive or synergistic inhibitory effects on the growth of relevant plaque bacteria (Bradshaw et al., 1991; Table 3), on glycolysis by oral streptococci (Cummins, 1991), and on protease activity by P. gingivalis (Marsh, 1991) have been obtained with combinations of triclosan and zinc citrate.

The long-term effects of antimicrobial agents the ecology of dental plaque.

on

As stressed earlier, care has to be taken with the long-term unsupervised use of dental products containing antimicrobial agents to ensure that adverse alterations to the ecology of dental plaque do not occur. Several studies have been performed to monitor the composition of the oral microflora during the routine use of such products by human volunteers, and these studies are necessary if a product is to receive ADA approval (Page, 1989). Undesirable alterations to the composition of plaque would include the suppression of the predominant organisms leading to overgrowth by either (a) exogenous species (e.g., yeasts, enteric bacteria), or (b) organisms associated with oral diseases. Likewise, the development of resistance or tolerance to these agents would also be undesirable. During the six months' use of a chlorhexidine mouthrinse, although the total numbers of bacteria in plaque were reduced, there were no detectable shifts in the balance of individual microorganisms (Briner et al., 1986a). At the end of the study, the overall sensitivity of the plaque microflora to chlorhexidine was unchanged, although occasional less-sensitive streptococci and Actinomyces strains were isolated (Briner et al., 1986b). Similar findings were reported after two years' use of a chlorhexidine mouthrinse (Schiott et al., 1976b) and three months' use of a gel (Emilson et al., 1976). Recent findings have shown that the long-term (six-month) use of triclosan-containing products led neither to the disruption of the ecology of plaque nor to the development of microbial resistance (Jones et al., 1988; Stephen et al., 1990; Zambon et al., 1990). Similarly, no adverse shifts in the oral microflora were found after six months' use of a mouthrinse containing various essential oils (Minah et al., 1989). These types of oral ecology studies are labor-intensive and difficult to execute. The inevitable wide inter-subject variations in plaque composition mean that relatively large shifts in the balance of the resident microflora

would have to occur for them to be discerned. As future improvements in the formulation and delivery of these agents are made, studies must continue to ensure that the microbial ecology of plaque is preserved.

Requirements for plaque control and plaque ecology: A microbiological paradox? There are now a number of dentifrices and mouthrinses available commercially that contain antimicrobial agents to control plaque and gingivitis. These products have to be developed with two conflicting microbiological requirements: Agents have to be delivered at concentrations sufficient for clinical benefit to be achieved, but without disruption to the natural ecology of the site. This apparent paradox may seem impossible to reconcile, particularly since most ofthe successful agents are described as having a broad spectrum of antimicrobial activity. Clues to the possible solution of this dilemma can be found in the results of some of the more stringent laboratory tests on successful agents, where (a) a degree of specificity (hitherto unsuspected) is apparent in their spectrum of antimicrobial activity that cannot be predicted from MIC data, and (b) studies with sub-MIC doses, which suggest that successful agents have a multifunctional mode of action, particularly against the production of virulence factors (Table 2). For example, triclosan produced a markedly selective inhibitory effect when pulsed into a mixed-culture chemostat system at or below the MIC of the component species (Bradshaw et al., 1991; Table 3). Viable counts of S. gordonii were relatively unaffected, whereas those ofS. mutans and P. intermedia were reduced by >90%, despite all three species having apparently similar MIC values (Ritchie and Jones, 1988; Gaffar et al., 1990). Likewise, counts of some species with a high MIC (F. nucleatum) were reduced to a greater extent than organisms with apparently lower MIC values (Veillonella dispar, S. oralis). Triclosan and zinc citrate/triclosan were also more selective against Gram-negative periodontopathic anaerobic species in both the chemostat (Bradshawet al., 1991; Table 3) and the experimental gingivitis models (Jones et al., 1990), while leaving streptococci that predominate in health (S. oralis, S. gordonii) relatively unaffected. Agents functioning in this way may help stabilize the balance of the plaque microflora under conditions that may otherwise predispose a site to gingivitis, in the same way that fluoride has been proposed to help maintain homeostasis in plaque to prevent caries (Bowden, 1990; Bradshaw et al., 1990). A primary mechanism of action of these antimicrobial agents could include, therefore, the suppression of potentially pathogenic species under conditions in the mouth under which they would normally flourish. As new agents, and combinations of agents, are developed with improved anti-plaque and antimicrobial properties, the challenge will be to increase clinical efficacy while preserving microbial homeostasis in the mouth. REFERENCES

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