Ecological Control: In Vitro Inhibition of Anaerobic Bacteria by Oral Streptococci

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1 æoriginal ARTICLE æ Ecological Control: In Vitro Inhibition of Anaerobic Bacteria by Oral Streptococci A. Doran 1, S. Kneist 2 and J. Verran 1 From the 1 Department of Biological Sciences, Manchester Metropolitan University, Manchester, UK and 2 Department of Preventive Dentistry, Friedrich-Schiller University of Jena, Germany Correspondence to: Joanna Verran, Department of Biological Sciences, Manchester Metropolitan University, Chester Street, Manchester M1 5GD, UK. Tel.: / ; Fax: / ; j.verran@mmu.ac.uk Microbial Ecology in Health and Disease 2004; 16: 23/27 It has been proposed that oral malodour occurs at neutral and alkaline ph, and is inhibited by an acid environment. Thus the production of lactic and acetic acids from carbohydrate fermentation by oral streptococci generates a low ph that can interfere with the growth of other microorganisms. Additionally, some streptococci may produce hydrogen peroxide (H 2 O 2 ) and/or bacteriocins. The purpose of this study was to investigate whether products of streptococcal growth and carbohydrate metabolism had any effect in vitro on the growth of selected anaerobes associated with oral malodour. Oral streptococci were compared for their ability to ferment glucose or sucrose using acid indicator media and ph measurement, and for their ability to produce H 2 O 2. The inhibition of anaerobes by end products of streptococcal growth in glucose-containing medium was evaluated using both an agar overlay technique and a cross-streak technique. Anaerobic bacteria were inhibited by products of growth of oral streptococci, particularly strains of Streptococcus mutans and Streptococcus salivarius in the presence, but not in the absence, of glucose. It is most likely that this inhibition results from the production of lactic acid rather than H 2 O 2 or bacteriocins, because the pattern of inhibition agreed more with acidogenicity than with any other variable. Oral streptococci may play an important role in the ecology of the oral flora and in the control of oral malodour. Key words: oral malodour, anaerobic bacteria, oral streptococci. INTRODUCTION The resident flora of the oral cavity comprises over 350 species (1), many of which produce odours under in vitro conditions (2). Metabolic studies on bacterial groups isolated from the oral cavity suggest that those with greatest odour potential are Gram-negative anaerobes including Porphyromonas gingivalis, Prevotella intermedia, Bacteroides spp. and Fusobacterium nucleatum (2/4). In particular, the metabolism of salivary glycoproteins by anaerobes found on the tongue results in the evolution of volatile sulphur compounds, hydrogen sulphide and methyl mercaptan that contribute to the distinctive odour (5, 6). Oral malodour is a generic descriptor term for foul smells emanating from the mouth (7). Few Gram-positive species (e.g. Peptostreptococcus) have been directly associated with malodour, although a role in the initial breakdown of the substrates, and hence an indirect contributory function, has been indicated (8). Salivary incubation studies have demonstrated that oral malodour occurs at neutral and alkaline ph, and is inhibited by acidity when a fermentable sugar such as glucose is added to whole saliva before it is incubated (9, 10). Malodour cessation (below ph 6.5) was measured by the human nose, and it was concluded that ph is a key malodour variable. It was also observed that a shift of the initial resident microflora to a more Gram-negative bacterial composition accompanied odour formation (9). However, the mode of inhibition of this odour generation was not examined. Previous studies have shown that acids produced during streptococcal carbohydrate metabolism were inhibitory to anaerobes, especially P. gingivalis (11). Microbial homeostasis results from a dynamic balance of microbial interactions, which include synergism and antagonism (12). Antagonism is a major mechanism in maintaining microbial homeostasis of an oral biofilm. The production of lactic and acetic acids from carbohydrate fermentation by oral streptococci renders a low ph that can interfere with the growth of surrounding microorganisms (13). Additionally some lactic acid bacteria (certain catalase-negative streptococci and lactobacilli) produce hydrogen peroxide (H 2 O 2 ) (13) and/or bacteriocins. Bacteriocins are proteins that show bactericidal activity towards closely related species or other bacteria (14); for example, the bacteriocin nicin from Lactococcus lactis is used for food preservation (15) owing to its inhibitory effect on many bacteria and bacterial endospores. An inhibitory effect of Streptococcus viridans isolated from the throat of a healthy individual on several pathogens of the respiratory and gastrointestinal tracts has been demonstrated (16). The inhibitory mechanism was not characterized but it was # Taylor & Francis ISSN X Microbial Ecology in Health and Disease DOI: /

2 24 A. Doran et al. suggested that ph, peroxide-mediated antagonism or bacteriocins might play a key role. The purpose of this study was to evaluate whether products of streptococcal growth and carbohydrate metabolism had any effect on the growth of selected anaerobes associated with oral malodour in vitro. MATERIALS AND METHODS Microorganisms Oral streptococci (Table I) were screened for their ability to ferment carbohydrates to produce acid, and to produce H 2 O 2, using indicator media. Four strains of S. mutans isolated from plaque of cariesfree children (aged 11 /12 years) were included in the study. Previous work (17, 18) had determined the acidogenicity of these strains, and the production of bacteriocins against other streptococcal species. Inhibition of S. sanguis growth by the production of bacteriocins has been demonstrated with strains R254 and R854. The acidogenicity of these two strains differed: ph 6.02 (9/0.02) for strain R254 and ph 5.48 (9/0.11) for R854. Acid production on solid indicator media The medium (termed BCP) used to observe acid production (Table II) was adapted from a medium previously used for differentiating acidogenic bacteria (19, 20). Bromocresol purple was used as an indicator of acid production; it has a ph range of 5.2 /6.8 with a corresponding colour change / yellow to purple (the ph of the medium was adjusted to 7.4 before autoclaving). Thus a bacterial colony that appears with a yellow halo in the purple medium is an indication of acid production. An inoculum was created in an X-shaped pattern on the medium using the streptococci listed in Table I and plates were incubated anaerobically or in 5% CO 2 for 24 h at 378C. Composition Table II Composition of agar used to indicate acid production Yeast extract (Sigma) 5 Tryptone (Sigma) 5 Potassium dihydrogen phosphate (BDH) 1.2 Di-potassium hydrogen phosphate (BDH) 1.2 Bacteriological Agar, High Clarity No. 1 (Difco) 15 Carbohydrate (glucose/sucrose) (Sigma) 10 *Bromocresol purple 0.4% (BDH) 8 ml/l *Bromocresol purple (BCP) was filter-sterilized and added to the molten medium after autoclaving. Acid production in liquid media Overnight cultures of streptococcal strains grown in Todd Hewitt Broth (THB, Difco, USA) were used to inoculate wells of a microtitre plate, which contained 200 ml of a basal growth medium (ph 7.5) (21). This medium was composed of thioglycollate medium without added glucose or indicator (Difco; 24 g l 1 ) and purple broth base (Difco; 16 g l 1 ) that contained bromocresol purple as the ph indicator, with the required carbohydrate supplement at 0.5% w/v. The microtitre plates were incubated overnight (18 h) in 5% CO 2 at 378C. The end ph was recorded by pipetting 20-ml samples onto the ion-sensitive field effect transistor (ISFET) solid state sensor of a ph Boy-P2 meter (Camlab, UK). Screen for production of H 2 O 2 by oral streptococci The production of H 2 O 2 by streptococci was determined using a modified medium (12, 22). The cultures were inoculated onto brain heart infusion agar (LAB M, Bury, Manchester, UK) containing 0.25 g l 1 trimethylbenzidine (Sigma, Poole, UK) and 0.01 g l 1 horseradish peroxidase (Sigma) and incubated anaerobically and in 5% CO 2 for 48 hat378c. Isolated colonies with blue haloes were deemed to produce H 2 O 2. g/l Table I Microorganisms used in the study Microorganism Reference Source Streptococcus mutans NCO10449 National Collection of Type Cultures, PHLS, London, UK Streptococcus salivarius NCOO8618 Streptococcus sanguis NCO10904 Streptococcus mitis NCO12261 Streptococcus mutans R916 Strains kindly provided by Suzanne Kneist, Department of Preventive Dentistry, Streptococcus mutans R1476 University of Jena, Germany (17) Streptococcus mutans R854 Streptococcus mutans R254 Prevotella intermedia BH Clinical isolates kindly provided by John Greenman, University of West England, Fusobacterium nucleatum NCTC Bristol, UK Veillonella dispar ATCC Porphyromonas gingivalis W50 Peptostreptococcus micros ATCC 33270

3 Inhibition of anaerobic bacteria by oral streptococci 25 Inhibition of anaerobes by products of streptococcal growth Two methods were used to compare inhibition patterns in the presence and absence of a fermentable carbohydrate, with and without a ph indicator. Agar overlay technique. Carbohydrate peptone agar (CPA) was prepared by dissolving 10 g glucose, 10 g peptone (Sigma), 5 g sodium chloride (BDH, Poole, UK) and 20 g High Clarity agar (Lab M) in 1 litre of distilled water. The ph was adjusted to 7.2 with 1 M NaOH before the medium was autoclaved. Negative control plates did not contain glucose. CPA was inoculated with streptococcal strains in an X-shaped pattern and incubated in CO 2 and anaerobic conditions at 378C for 48 h. Overnight cultures (0.3 ml) of anaerobic bacteria (Table I) in fastidious anaerobe broth (Lab M) incubated at 378C for 48 h (10% H 2, 5% CO 2, 85% N 2 ) were inoculated into fastidious anaerobe agar (Lab M), which had been prepared in 10-ml universal bottles, autoclaved, and cooled to 508C. This was mixed gently and poured over the peptone agar containing carbohydrate (1%), which had previously been inoculated with the streptococcal stock cultures. All plates were incubated anaerobically at 378C for 2/5 days. A zone of inhibition of the anaerobe around the X-shaped streptococcal inoculum was indicative of the production of an inhibitory agent by streptococci. Cross-streaking technique. This is a modified cross-streak method for the detection of bacteriocins (23). It utilizes both sides of the agar contained in a petri dish. The producer strains (streptococci) were inoculated on the surface of BCP (9/glucose), using a single streak across the plate. The plates were then incubated for 24 h in 5% CO 2 at 378C. After incubation, the agar was detached from the edges of the petri dish with a sterile spatula and inverted so that the agar disc fell into the lid. The sterile surface (previously at the bottom of the dish) was then uppermost in the lid and was inoculated with the indicator strain (anaerobic bacteria). All plates were incubated anaerobically at 378C for 2/5 days. The incorporation of bromocresol purple into the underlying medium enabled both acid production and inhibition zones to be observed simultaneously. RESULTS Acid and H 2 O 2 production by oral streptococci In liquid media, all streptococci produced acid when incubated in the presence of sucrose or glucose. In general the ph fell from ph 7.2 (control) to ph 4.2/5.3, depending on the strain and the carbohydrate source. In terms of ranking acidogenicity, S. mutans was most acidogenic when incubated with sucrose or glucose (ph 4.2 and ph 4.3, respectively). The final ph attained by other streptococci varied, with lower ph values usually obtained when incubated with sucrose. On solid media, comparable findings were obtained for most strains under anaerobic or CO 2 incubation conditions. All streptococcal strains produced acid (yellow coloration of agar) in the presence of glucose or sucrose (11). H 2 O 2 was produced only by S. sanguis and S. mitis, only when incubated in 5% CO 2. Inhibition of anaerobes Using the agar overlay technique, growth of streptococci was observed on all plates, in the presence or absence of glucose (Table III). Inhibition of anaerobic bacteria was observed in several circumstances (Fig. 1). S. mutans inhibited anaerobic bacteria on media with added glucose (in 21 of 25 test combinations) more often than on media without glucose (6/25). S. salivarius tended to inhibit anaerobes on media with glucose (5/5) but not on media without glucose (1/5). S. sanguis and S. mitis inhibited most anaerobes (9/10) on media with glucose but not without (0/ 10). Under CO 2 conditions less inhibition was observed Table III Inhibition of anaerobes by streptococci using the agar overlay technique (n/3) incubated under anaerobic conditions Prevotella intermedia Fusobacterium nucleatum Veillonella dispar Porphyromonas gingivalis Peptostreptococcus micros G N G N G N G N G N S. mutans / / / / / / / / / / S. salivarius / / / / / / / / / / S. sanguis a / / / / / / a / / a / / S. mitis / / a / / / / / / / / S. mutans R916 / / / / / / / / / / R1476 / / / / / / / / / / R854 / / / / / / / / / / R254 / / / / / / / / / / G, glucose; N, no glucose;/, inhibition of anaerobic growth observed;/, no inhibition of anaerobic growth observed. a Result was negative when streptococci were incubated in 5% CO 2.

4 26 A. Doran et al. Cross-streak technique If present, inhibition of anaerobic bacteria by streptococci using the cross-streak technique was apparent on the surface of the inverted agar above the producer strain inoculum (Table IV). The presence of acid was indicated by colour changes in the BCP agar, and agreed with findings in previous experiments. It was therefore possible to determine whether inhibition occurred in the absence of apparent low ph. Patterns of inhibition varied in the presence of glucose, but in its absence there was growth inhibition in only one instance. The S. mutans and S. salivarius strains showed the widest spectrum of antimicrobial activity, causing inhibition in 26 of 30 test combinations. Veillonella dispar and Peptostreptococcus micros were most sensitive to the end products of streptococcal metabolism in the presence of glucose. Fig. 1. Inhibition of anaerobic bacteria by streptococci using the agar overlay technique. using S. sanguis and S. mitis, with inhibition being lost in four of nine instances. DISCUSSION Anaerobic bacteria were inhibited by metabolic end products of glucose fermentation by a range of oral streptococci, especially strains of S. mutans and S. salivarius. A role for H 2 O 2 in the inhibition (12, 13) is refuted, since of the strains tested, only S. sanguis and S. mitis produced H 2 O 2. This finding agrees with other studies (24, 25) where S. sanguis I and II and S. mitior inhibited the growth of plaque bacteria. Inhibition in the absence of acid was observed in a minority of cases, and may presumably be due to some bacteriocin-like element. Fewer inhibition events in the absence of glucose are apparent using the cross-streak method. The difference in inhibition patterns observed between the agar overlay and the cross-streak methods may be due to several factors. The cross-streak method eliminates direct contact between the producer and indicator strains, so that phage particles cannot reach the indicator bacteria. Therefore, an inhibition area due to phages cannot be formed. This is often the case with other methods where it is not easy to decipher whether a zone of confluent lysis is due to phages or bacteriocins (23). Other differences between the two methods include the inoculum size / an X pattern, or a single streak. More acid might diffuse into the medium from the former inoculum, producing more inhibition of indicator strains. Previous work (11) on acid production ranked the inhibition using the agar overlay method, and indicated that S. mutans and S. salivarius were the most effective strains. With a lower inoculum, as for the reverse agar method, the effect may well have been reduced to below the level of detection provided by the BCP indicator. End ph data were used to rank the acidogenicity of the test species, showing that S. mutans and S. salivarius were the most acidogenic of the strains tested. Table IV Inhibition of anaerobes by streptococci using cross-streak method (n/3) Prevotella intermedia Fusobacterium nucleatum Veillonella dispar Porphyromonas gingivalis Peptostreptococcus micros G N G N G N G N G N S. mutans / / / / / / / / / / S. salivarius / / / / / / / / / / S. sanguis / / / / / / / / / / S. mitis / / / / / / / / / / S. mutans R916 / / / / / / / / / / R1476 / / / / / / / / / / R854 / / / / / / / / / / R254 / / / / / / / / / / G, glucose; N, no glucose;/, inhibition of anaerobic growth observed;/, no inhibition of anaerobic growth observed.

5 Inhibition of anaerobic bacteria by oral streptococci 27 The use of S. mutans strains R254 and R854 with known bacteriocin activity against S. sanguis did not show inhibition of the anaerobes when glucose was absent. It is generally acknowledged that most bacteriocins show bactericidal activity to closely related species (14). Future work could investigate inhibition patterns, if any, between oral streptococci of the same species. However, a wider spectrum of activity has been demonstrated for bacteriocins produced by S. viridans (16). It certainly appears that, in a minority of cases, there may be a role for bacteriocin-like substances in the inhibition of oral anaerobes by oral streptococci. In conclusion, it is most likely that most of the inhibition of anaerobes results from the production of lactic acid by streptococci, rather than H 2 O 2 or bacteriocins, because the pattern of inhibition agreed more with acidogenicity than with any other variable. The results indicate that oral streptococci play an important role in the ecology of the oral flora and, potentially, in the control of oral malodour (11). REFERENCES 1. Marsh P, Martin M. Oral Microbiology, 4th edn. Oxford: Wright, Hartley G, El-Maaytah M, McKenzie C, Greenman J. The tongue microbiota of low odorous and malodorous individuals. Microb Ecol Health Dis 1996; 9: 215/ Hartley G, McKenzie C, Greenman J, El-Maaytah MA, Scully C, Porter S. Tongue microbiota and malodour, effects of metronizadole mouthrinse on tongue microbiota and breath odour. Microb Ecol Health Dis 1999; 11: 226/ Persson S, Edlund M-B, Claesson R, Carlsson J. The formation of hydrogen sulfide and methyl meracaptan by oral bacteria. Oral Microbiol Immunol 1990; 5: 195/ Schmidt NF, Missan S, Tarbet WJ, Cooper AD. The correlation between organoleptic mouth-odour ratings and levels of volatile sulphur compounds. Oral Surg 1978; 45: 560/7. 6. De Boever EH, Loesche WJ. Assessing the contribution of anaerobic microflora of the tongue to oral malodour. J Am Dent Assoc 1995; 126: 1384/ Kleinberg I, Westbay G. Oral malodour. Crit Rev Oral Biol Med 1990; 1: 247/ Sterer N, Rosenberg M. Effect of deglycosylation of salivary glycoproteins on oral malodour production. Int Dent J 2002; 52: 229/ McNamara TF, Alexander JF, Lee M. The role of microorganisms in the production of oral malodour. Oral Surg 1972; 34: 41/ Kleinberg I, Westbay G. Salivary and metabolic factors involved in oral malodour formation. J Periodontol 1992; 63: 768/ Verran J, Doran AL, Smith C. Denture stomatitis, oral malodour and oral biofilm ecology. In: Gilbert P, Alison D, Brading M, Verran J, Walker J, eds. Biofilm Community Interactions: Chance or Necessity. Cardiff: BioLine, 2001: 131/ Marsh PD, Bradshaw DJ. Physiological approaches to the control of oral biofilms. Adv Dent Res 1997; 11: 176/ Sookkhee S, Chulasiri M, Prachyabrued W. Lactic acid bacteria from healthy oral cavity of Thai volunteers: inhibition of oral pathogens. J Appl Microbiol 2001; 90: 172/ Tagg JR, Dajani AS, Wannamaker LW. Bacteriocins of gram-positive bacteria. Bacteriological Reviews 1976; 40: 722/ Clarke S. Lactic acid bacteria and human health. Biomedical Scientist 1996; April: 175/ Jinhua D, Yixiu G, Moore JE, Zhikun Z, Zongda M, Murphy PG. The inhibitory activity of Streptococcus viridans on several pathogens of the respiratory and gastrointestinal tracts and central nervous system. Microb Ecol Health Dis 2002; 14: 1 / Kneist S, Bergholz T, Stosser L. Acid production by human strains of mutans streptococci. Caries Res 1999; 33: Kneist S, Scharff S, de Soet JJ, van Loveren C, Stosser L. Bacteriocin production by human strains of mutans and oral streptococci. Caries Res 2000; 34: Keele BB Jr, Reeves MS, Navia JM. An improved medium differentiating acidogenic bacteria. J Dent Res 1973; 52: 1054/ Handleman SL, Mills JR, Meggo L. A medium for differentiating acidogenic bacteria. Arch Oral Biol 1968; 13: 1187/ Beighton D, Hardie JM, Whiley RA. A scheme for the identification of viridans streptococci. J Med Microbiol 1991; 35: 367/ Eschenbach DA, Davick PR, William BL, Klebanoff SJ, Young-Smith K, Critchlow CM, Holmes KK. Prevalence of hydrogen peroxide-producing Lactobacillus species in normal women and women with bacterial vaginosis. J Clin Microbiol 1989; 27: 251/ Kekessy DA, Piguet JD. New method for detecting bacteriocin production. J Appl Microbiol 1970; 20: 282/ LeBien WT, Bromel MC. Antibacterial properties of a peroxidogenic strain of Streptococcus mitior (mitis). Can J Microbiol 1975; 21: 101/ Holmberg K, Hallander HO. Production of bactericidal concentrations of hydrogen peroxide by Streptococcus sanguis. Arch Oral Biol 1973; 18: 423/34.

Topographic distribution of bacteria associated with oral malodour on the tongue

archives of oral biology 53, Suppl. 1 (28) S8 S12 journal homepage: www.intl.elsevierhealth.com/journals/arob Topographic distribution of bacteria associated with oral malodour on the tongue Robert P.