Ecological effects of triclosan and triclosan monophosphate on defined mixed cultures of oral species grown in continuous culture

K. A. Saunders, J. Greenman* and C. McKenzie

Bristol Oral Microbiology Unit, Faculty of Applied Sciences, University of the West of England, Frenchay Campus, Coldharbour Lane, Bristol BS16 1QY, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The effects of triclosan and its phosphorylated derivative, triclosan monophosphate were studied using a continuous culture microcosm model. Two conditions were simulated, a caries-like state (pH 5.5 with artificial saliva plus glucose as growth medium) and a periodontal disease-like state (pH 7.5 with BHI plus yeast extract, haemin and cysteine as growth medium). Both cultures were maintained anaerobically at 37°C at a growth rate of 0.1/h. Steady-state chemostats were pulsed with triclosan or triclosan monophosphate (initial concentrations between 20 and 40 mg/L) and changes in the ecological composition noted after 6 h. The caries-like microcosm steady state was dominated by streptococci, Lactobacillus and Veillonella sp. with low but detectable levels of Neisseria, Actinomyces and Fusobacterium sp. No significant ecological shifts occurred following pulses of either antimicrobial agent; all species were affected to approximately the same degree. The periodontal disease-like microcosm steady state was dominated by streptococci, Fusobacterium, Veillonella, Actinomyces, Prevotella and Porphyromonas sp. with low numbers of Neisseria and Lactobacillus sp. Significant ecological shifts were apparent following pulses of triclosan. The streptococci became the dominant group followed by Fusobacterium sp. For triclosan monophosphate, the streptococci again became dominant although Lactobacillus and Actinomyces were now the main sub-dominant species and Gram-negative anaerobes including Fusobacterium sp. were markedly inhibited. It is concluded that in the periodontal disease state, both triclosan and triclosan monophosphate affected the Gram-negative anaerobes to a greater extent than the Gram-positive groups and that this effect was more marked for triclosan monophosphate.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Dental plaque develops naturally on teeth as part of the body's normal flora and serves as a barrier to colonization by exogenous species. However, unless removed by diligent oral hygiene, it can predispose a site to either caries or periodontal diseases. In caries, acidogenic and aciduric species become predominant at the expense of many acid-sensitive species, whilst in periodontal diseases there is an increase in the total plaque mass as well as the proportions of many obligate anaerobes. For many people, the customary hygiene of tooth brushing alone is insufficient to provide a level of plaque control sufficient to ensure oral health. As a consequence, the incorporation of various antimicrobial compounds into dental products as a means of plaque control has been commonplace for many years.1,2

Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) is widely used in a number of oral hygiene products. It has been shown to be capable of inhibiting the growth of a wide range of microorganisms, including Gram-positive and Gram-negative bacteria and fungi, with MICs usually ranging from 0.1 to 30 mg/L.3,4 The molecular mechanism of action of triclosan appears to involve the inhibition of the enzyme enoyl-acyl carrier protein reductase. This enzyme is crucial to the final regulatory step in the fatty acid synthase cycle and the ultimate effect of triclosan is to block lipid biosynthesis leading to growth inhibition or lysis depending on the concentration employed.5,6 Owing to its hydrophobic and lipophilic nature, triclosan adsorbs to the lipid portion of the bacterial cell membrane.7 Triclosan monophosphate is a phosphorylated derivative of triclosan, which in comparison is highly soluble in aqueous solutions. In vitro triclosan monophosphate (which itself may be devoid of antimicrobial activity) is hydrolysed into triclosan by the action of microbial phosphatases.8 Thus, sensitivity to triclosan monophosphate depends in part on the total phosphatase activity present in the microenvironment of the exposure. Since different species of microorganism differ in their sensitivities to either triclosan or triclosan monophosphate it might be expected that considerable ecological shifts would occur when mixed communities of oral species were exposed to these compounds at inhibitory concentrations. This study compared the effects of triclosan and triclosan monophosphate following their pulsed addition to defined mixed cultures of oral species growing in continuous culture as microcosm models to represent ecological states (caries and periodontal disease) occurring in vivo.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

The organisms used in these studies were Streptococcus oralis EF186, Streptococcus gordonii Blackburn, Streptococcus mutans ATCC 2-27351, Streptococcus mitis I SK137, Lactobacillus casei AC 413, Actinomyces viscosus WVU 627, Veillonella dispar ATCC 17745, Fusobacterium nucleatum ATCC 10953, Prevotella nigrescens T 588, Porphyromonas gingivalis W50 and Neisseria subflava A 1078. With the exception of S. gordonii, S. mitis I and P. gingivalis, all cultures were obtained from D. Bradshaw and P. D. Marsh, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, UK. S. gordonii and S. mitis I were obtained from Procter and Gamble, Cincinnati, OH, USA. P. gingivalis was part of a culture collection maintained at the University of the West of England. The species were chosen for their ease of isolation and identification and their known prevalence and importance in dental plaque in both health and disease.

Cultures were maintained on Fastidious Anaerobe Agar (Lab M, Bury, UK) supplemented with 7% defibrinated horse blood (TCS Biologicals, Buckingham, UK) and sub-cultured weekly. With the exception of N. subflava, all strains were incubated anaerobically at 37°C in an anaerobic workstation (MK3, Don Whitley Scientific, Shipley, UK) providing an atmosphere of 10% CO2, 10% H2 in 80% N2. N. subflava was incubated aerobically at 30°C (Leec MK II proportional temperature controller, Leec Ltd, UK).

Chemostat conditions

The mixed cultures were grown in an LH 500 series II modular chemostat (LH Engineering, Stokes Poges, UK) with a working volume of 750 mL, operated at 37°C, at a dilution rate of 0.1/h and under a gas sparge of 10% CO2 in nitrogen. The pH was maintained at pH 5.5 ± 0.05 (caries-like state) or at pH 7.5 ± 0.05 (periodontal disease-like state) by the automatic addition of 2 M NaOH and 2 M H2SO4. Chemostats were inoculated by syringe injection of a 10 mL volume of cell suspensions in broth to give final chemostat concentrations of approximately 5.0 x 107 viable cells (cfu) per mL.

Growth medium and inocula for caries-like microcosm (synthetic saliva)

To ensure reproducibility, a synthetic saliva based on that developed by Shellis9 and supplemented with glucose was adopted as the growth medium.10 The final composition was as follows: thiamine (7 µg/L); riboflavin (50 µg/L); folic acid (0.1 µg/L); nicotinic acid (30 µg/L); pyroxidine (600 µg/L); pantothenic acid, Ca salt (80 µg/L); biotin (0.8 µg/L); B12 (3 µg/L); K3 (15 µg/L); alanine (3.3 mg/L); arginine (1.9 mg/L); aspartic acid (1.6 mg/L); glutamic acid (3.9 mg/L); glycine (8.9 mg/L); histidine (1 mg/L); leucine (3.4 mg/L); isoleucine (1.9 mg/L); lysine (2.7 mg/L); methionine (30 µg/L); phenylalanine (3.4 mg/L); proline (200 µg/L); serine (2.1 mg/L); threonine (2.9 mg/L); tyrosine (2.1 mg/L); valine (1.8 mg/L); potassium di-hydrogen orthophosphate (355 mg/L); sodium citrate (15 mg/L); sodium hydrogen carbonate (535 mg/L); di-sodium hydrogen orthophosphate (375 mg/L); albumin (25 mg/L); ammonium chloride (235 mg/L); calcium chloride (210 mg/L); magnesium chloride (45 mg/L); potassium chloride (1.16 g/L); potassium thiocyanate (220 mg/L); mucin (2.0 g/L); urea (175 mg/L); uric acid (10 mg/L); creatinine (100 µg/L); choline (15 mg/L) and glucose (2.0 g/L). The chemostat inoculant consisted of N. subflava, L. casei, A. viscosus, all the streptococci, F. nucleatum and V. dispar.

Growth medium for periodontal disease-like microcosm

For the periodontal disease-like conditions, half-strength brain–heart infusion (BHI), yeast extract plus glucose basal medium was used. The final concentrations (g/L) were as follows: BHI (Difco, East Molesey, UK) (18.5), yeast extract (5), glucose (2), haemin (0.01) and cysteine (0.5). The chemostat inoculant contained P. gingivalis and P. intermedia in addition to the other groups used for the caries-like state.

Identification and enumeration of the bacterial species

A system of identification and enumeration was used for determination of the chemostat microbial composition. Samples from the chemostat were vortexed for 10 s and used to prepare a 10-fold dilution series (10–1 to 10–6) in Fastidious Anaerobe Broth (Lab M). Aliquots of each dilution were plated on to the following selective and non-selective media: 7% v/v horse blood agar supplemented with 2.5 mg/L vancomycin (Sigma) for enumeration of F. nucleatum, P. intermedia, P. gingivalis and N. subflava; Ritz medium11 for streptococci; Man, Rogosa, Sharpe medium12 for L. casei; Zylber and Jordan medium13 for A. viscosus; Rogosa medium14 for V. dispar. Plates were incubated at 37°C in an anaerobic atmosphere of 10% CO2, 10% H2 in 80% N2 with the exception of N. subflava plates which were incubated aerobically. After 5 days a differential count was made based on the examination of colony morphology and verified with stains using Jensen's modification of Gram's method.15 Colonies of P. gingivalis were tested for N-{alpha}-benzoyl-l-arginine p-nitroaniline (BAPNA) hydrolysis which distinguishes them from P. intermedia (negative reaction). The streptococci were counted as a whole group since the selective medium did not allow for their full differentiation.

Triclosan and triclosan monophosphate pulsing

For each starting condition the chemostat mix was grown to steady state at D = 0.1/h and samples taken to determine the pre-pulse conditions. At time zero, a relatively rapid pulse (5.0 mL/min) of triclosan or triclosan monophosphate was made by addition of sterile stock solutions of the appropriate antimicrobial to give a final concentration of 10 mg/L. Further pulses of triclosan/triclosan monophosphate compounds were made at 6 h intervals.

Statistical analysis

A statistical analysis of all results was performed. Means and standard errors were calculated for all experiments and one/two way analysis of variance tests were carried out where appropriate to determine the true significance of the results. A value of P < 0.05 was taken as the level of significance.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Caries-like state

The pre-pulse conditions were not significantly different for the caries-like states (Tables I and IIGoGo). L. caseii was the dominant species (41–46%) followed by the streptococci (27–29%) and V. dispar (26–28%) with minor proportions of F. nucleatum (<0.3%), A. viscosus (<0.1%) and N. subflava (<0.001%). Following three triclosan and triclosan monophosphate pulses, reductions were apparent for all groups. However, there was no significant difference in the relative proportions of the microorganisms.


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Table I. Effects of triclosan pulsing on caries-type mixed culture (results expressed as mean log cfu/mL (± S.E.) and approximate percentage of total)
 

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Table II. Effects of triclosan monophosphate (TMP) pulsing on caries-type mixed culture (results expressed as mean log cfu/mL (± S.E.) and approximate percentage of total)
 
Periodontal disease-like state

The periodontal disease-like mixed culture (Tables III and IVGoGo) was characterized by F. nucleatum as the dominant species (49–56%) with the streptococci (16–18%) and V. dispar (11–13%) being the main sub-dominant species. There were low proportions of P. gingivalis (7–8%), P. intermedia (4–5%), A. viscosus (5–8%), L. casei (0.4–1%) and N. subflava (<0.001%). The pre-pulse conditions were not significantly different. Following triclosan pulses (Table IIIGo), reductions were apparent both in total numbers and in the ecological proportions of the different species. The mixed culture changed to one dominated by the streptococci with A. viscosus and F. nucleatum being the main sub-dominant types. Proportions of L. casei increased whilst proportions of P. gingivalis and P. intermedia decreased. N. subflava were close to their minimal detectable levels throughout. Following pulses of triclosan monophosphate (Table IVGo) a similar trend of reduction in total count and dominance of the streptococci was observed. However, following the final triclosan monophosphate pulse, L. casei proportions increased significantly whilst proportions of F. nucleatum, P. gingivalis, P. intermedia and V. dispar decreased. For the periodontal disease-like microcosm, the decreases in the proportions of all Gram-negative species following pulsing were more marked following the triclosan monophosphate pulses than they were for triclosan.


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Table III. Effects of triclosan pulsing on periodontal-type mixed culture (results expressed as mean log cfu/mL (± S.E.) and approximate percentage of total)
 

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Table IV. Effects of TMP pulsing on periodontal-type mixed culture (results expressed as mean log cfu/mL (± S.E.) and approximate percentage of total)
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In order to improve the evaluation of antimicrobial agents in the laboratory, more complex models have been devised which are able to reflect the complexity of the oral microflora. The chemostat, while not attempting to model physical aspects of a mouth, does allow studies to be undertaken which examine specific interactions among members of the normal flora, both in the presence and absence of antimicrobial compounds under defined and controllable conditions.1 Studies such as these allow even the smallest inhibitory effects to be amplified, and, in addition, both direct and indirect inhibitory effects can be detected. Such mixed-culture systems have been used to study the effects of many different inhibitors on the stability of the resident oral microflora.16,17 These studies have served to show the effectiveness of the chemostat models in predicting more accurately the in vivo effects of prospective agents.

In this study, caries-like and periodontal disease-like microcosm models were used to compare the effects of triclosan and triclosan monophosphate. Comparison of the pre-pulse steady-state conditions showed that the two types of microcosm developed to produce two distinctive and representative mixed cultures that were consistent for the control conditions (pre-pulse state). The low recovery of N. subflava throughout is an indication that the states were highly anaerobic.

In the caries-like microcosm, all the microbial groups appeared to be equally sensitive to the antimicrobial pulses and the proportions of species were very similar throughout. Triclosan is considered to be a broad-spectrum antimicrobial3,4 and the results show that all species within the population were affected. Work conducted testing triclosan in systems where the pH was not controlled has shown that it may inhibit the production of acids by the microflora and consequently provide an additional protective function against caries in the oral cavity.18,19 This result could not be observed using the chemostat caries-like microcosm model as pH was strictly controlled throughout the pulses.

On observation of the effects of the antimicrobial pulses in the periodontal disease-like microcosm, it was apparent that the streptococci were the least susceptible to the triclosan/triclosan monophosphate and therefore dominated the community. Moreover, the Gram-negative species as a whole were in general most affected by pulsing and this effect was more marked using triclosan monophosphate. This effect was particularly evident following the first pulse where concentrations were equivalent. This marked effect of triclosan on Gram-negative bacteria has been reported previously, both in vitro7 and in vivo,20,21 where it is thought that differences in susceptibility between Gram-positive and Gram-negative species may relate to differences in cell wall structure and lipid composition and the penetration of triclosan to the proposed site of action, the cytoplasmic membrane.

Previous work by Bradshaw et al.17 using a similar combination of species in a supragingival microcosm model (pH 6.5, D = 0.1/h) showed that triclosan, after a single pulse (10 mg/L final concentration), had little or no effect with the exception that proportions of F. nucleatum and A. viscosus were significantly reduced. Triclosan may be more lipophilic at a pH of 7.5 and able to target the oral Gram-negative species more effectively than at the lower pH employed in the caries-like conditions. In addition, triclosan monophosphate may be able to penetrate the outer cell membrane of Gram-negative species even more efficiently than can triclosan, giving rise to its more marked ecological effects.22

For the periodontal disease-like ecological state, it is difficult to explain why pulsing with triclosan or triclosan monophosphate should give rise to slightly different effects in each case with regard to the response of the sub-dominant species. In an ecological mix it is probable that different groups of organisms are interacting through a wide variety of mechanisms including competition and inhibition on the one hand, and cross-feeding and cross-protection on the other. Cause and effect are thus difficult to ascribe. For these same reasons it is impossible to predict the behaviour of individual species based on conventional MIC test data (obtained using pure culture, closed systems); such data would not be valid when applied to mixed-culture open systems.

The lack of ecological changes observed following triclosan/triclosan monophosphate pulsing of the caries-like model suggests that if these trends occurred following use of these antimicrobial agents in vivo, the caries-promoting species (Streptococcus, Lactobacillus and Actinomyces) would not become any more prominent. Moreover, if ecological shifts occurred in vivo similar to those observed following triclosan/triclosan monophosphate pulsing of the periodontal disease-like microcosm (where streptococci were enriched in favour of the Gram-negative species) it would result in a flora associated more readily with periodontal health than with periodontal disease.23


    Acknowledgments
 
The authors thank Mr Steven Jones for his assistance in preparation of the manuscript. This investigation was supported by a grant from Procter and Gamble, Cincinnati, OH, USA.


    Notes
 
* Corresponding author. Tel: +44-117-9763836; Fax: +44-117-9763871; E-mail: John.Greenman{at}uwe.ac.uk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Marsh, P. D. (1993). The role of chemostats in the evaluation of antimicrobial agents for use in dental products. Microbial Ecology in Health and Disease 6, 147–9.[ISI]

2 . Marsh, P. D. (1993). Antimicrobial strategies in the prevention of dental caries. Caries Research 27, Suppl. 1, 72–6.[ISI][Medline]

3 . Regös, J. & Hitz, H. R. (1974). Investigations on the mode of action of Triclosan, a broad spectrum antimicrobial agent. Zentralblatt Für Bakteriologie, Parasitenkunde, Infektionskrankheiten Und Hygiene-Erste Abteilung Originale-Reihe A: Medizinische Mikrobiologie Und Parasitologie 226, 390–401.[Medline]

4 . Vischer, W. A. & Regös, J. (1974). Antimicrobial spectrum of Triclosan, a broad-spectrum antimicrobial agent for topical application Zentralblatt Fur Bakteriologie, Parasitenkunde, Infektionskrankheiten Und Hygiene-Erste Abteilung Originale-Reihe A: Medizinische Mikrobiologie Und Parasitologie 226, 376–89.[Medline]

5 . McMurry, L. M., Oethinger, M. & Levy, S. B. (1998). Triclosan targets lipid synthesis. Nature 394, 531–2.[ISI][Medline]

6 . Levy, C. W., Roujeinikova, A., Sedelnikova, S., Baker, P. J., Stuitje, A. R., Slabas, A. R. et al. (1999). Molecular basis of triclosan activity. Nature 398, 383–4.[ISI][Medline]

7 . Meincke, B. E., Kranz, R. G. & Lynch, D. L. (1980). Effect of irgasan on bacterial growth and its adsorption into the cell wall. Microbios 28, 133–47.[ISI][Medline]

8 . Greenman, J. & Nelson, D. G. (1996). Hydrolysis of triclosan monophosphate by dental plaque and selected species of oral micro-organisms. Journal of Dental Research 75, 1578–84.[Abstract]

9 . Shellis, R. P. (1978). A synthetic saliva for cultural studies of dental plaque. Archives of Oral Biology 23, 485–9[ISI][Medline]

10 . Saunders, K. A. (1995). Modelling dental plaque interactions of defined communities of oral organisms. Ph.D. thesis, University of the West of England, Bristol, UK.

11 . Ritz, H. L. (1967). Microbial population shifts in developing human dental plaque. Archives of Oral Biology 12, 1561–8.[ISI][Medline]

12 . Man, De J. C., Rogosa, M. & Sharpe, M. E. (1960). A medium for the cultivation of lactobacilli. Journal of Applied Bacteriology 23, 130.

13 . Zylber, L. J. & Jordan, H. V. (1982). Development of a selective medium for detection and enumeration of Actinomyces viscosus and Actinomyces naeslundii in dental plaque. Journal of Clinical Microbiology 15, 253–9.[ISI][Medline]

14 . Rogosa, M. L., Fitzgerald, R. J., Mackintosh, M. E. & Beaman, A. J. (1958). Improved medium for selective isolation of Veillonella. Journal of Bacteriology 76, 455–9.[ISI][Medline]

15 . Cruickshank, R. (1968). Medical Microbiology, pp. 650–1. E & S Livingstone, London.

16 . McDermid, A. S., McKee, A. S. & Marsh, P. D. (1987). A mixed-culture chemostat system to predict the effect of anti-microbial agents on the oral flora: preliminary studies using chlorhexidine. Journal of Dental Research 66, 1315–20.[Abstract]

17 . Bradshaw, D. J., Marsh, P. D., Watson, G. K. & Cummins, D. (1993). The effects of triclosan and zinc citrate, alone and in combination, on a community of oral bacteria grown in vitro. Journal of Dental Research 72, 25–30.[Abstract]

18 . Tahmassebi, J., Duggal, M. S. & Curzon, M. E. (1994). Effect of a calcium carbonate-based toothpaste with 0.3% triclosan on pH changes in dental plaque in vivo. Caries Research 28, 272–6.[ISI][Medline]

19 . van der Hoeven, J. S., Cummins, D., Schaeken, M. J. & van der Ouderaa, F. J. (1993). The effect of chlorhexidine and zinc/triclosan mouthrinses on the production of acids in dental plaque. Caries Research 27, 298–302.[ISI][Medline]

20 . Stephen, K. W., Saxton, C. A., Jones, C. L., Ritchie, J. A. & Morrison, T. (1990). Control of gingivitis and calculus by a dentifrice containing a zinc salt and triclosan. Journal of Periodontology 61, 674–9.[ISI][Medline]

21 . Svatun, B., Saxton, C. A. & Rolla, G. (1990). Six-month study of the effect of a dentifrice containing zinc citrate and triclosan on plaque, gingival health and calculus. Scandinavian Journal of Dental Research 98, 301–4.[ISI][Medline]

22 . Greenman, J., McKenzie, C. & Nelson, D. G. (1997). Effects of triclosan and triclosan monophosphate on maximum specific growth rates, biomass and hydrolytic enzyme production of Streptococcus sanguis and Capnocytophaga gingivalis in continuous culture. Journal of Antimicrobial Chemotherapy 40, 659–66.[Abstract]

23 . Marsh, P. D. & Martin, M. V. (1999). Oral Microbiology, 4th edn, pp. 104–26. Wright, Oxford.

Received 27 July 1999; returned 2 October 1999; revised 11 November 1999; accepted 1 December 1999





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