Ó 2013 Eur J Oral Sci
Eur J Oral Sci 2014; 122: 57–61 DOI: 10.1111/eos.12102 Printed in Singapore. All rights reserved
European Journal of Oral Sciences
Influence of pH on inhibition of Streptococcus mutans by Streptococcus oligofermentans Liu Y, Chu L, Wu F, Guo L, Li M, Wang Y, Wu L. Influence of pH on inhibition of Streptococcus mutans by Streptococcus oligofermentans. Eur J Oral Sci 2014; 122: 57–61. © 2013 Eur J Oral Sci Streptococcus oligofermentans is a novel strain of oral streptococcus that can specifically inhibit the growth of Streptococcus mutans. The aims of this study were to assess the growth of S. oligofermentans and the ability of S. oligofermentans to inhibit growth of Streptococcus mutans at different pH values. Growth inhibition was investigated in vitro using an interspecies competition assay. The 4-aminoantipyine method was used to measure the initial production rate and the total yield of hydrogen peroxide in S. oligofermentans. S. oligofermentans grew best at pH 7.0 and showed the most pronounced inhibitory effect when it was inoculated earlier than S. mutans. In terms of the total yield and the initial production rate of hydrogen peroxide by S. oligofermentans, the effects of the different culture pH values were as follows: pH 7.0 > 6.5 > 6.0 > 7.5 > 5.5 = 8.0 (i.e. there was no significant difference between pH 5.5 and pH 8.0). Environmental pH and the sequence of inoculation significantly affected the ability of S. oligofermentans to inhibit the growth of S. mutans. The degree of inhibition may be attributed to the amount of hydrogen peroxide produced.
Dental biofilm is a complicated multispecies community composed of a large number of microbes, with more than 700 species identified (1–3). These microbes maintain their ecological homeostasis by coexistence and competition. Dental caries is the result of destruction of the oral micro-ecological balance among different species (4). Streptococcus mutans is still regarded as a primary pathogen in dental caries, although other microorganisms may also be involved (5). Some beneficial bacteria, such as Streptococcus sanguinis and Streptococcus gordonii (6, 7) are able to inhibit the growth of S. mutans, as well as other caries-causing pathogens, by producing hydrogen peroxide (H2O2). Streptococcus oligofermentans is a novel strain of streptococcus that was first isolated from the dental plaque of caries-free human subjects by TONG et al. in 2003 (8). An in-vitro study demonstrated that S. oligofermentans inhibits the growth of S. mutans by producing a much higher level of H2O2 compared with S. sanguinis and S. gordonii. In addition, S. oligofermentans can use its lactate oxidase and pyruvate oxidase activities to convert lactic acid and pyruvate (metabolic intermediates generated by cariescausing pathogens, such as S. mutans) into H2O2 (9, 10). It was also reported that the H2O2 produced by S. oligofermentans could be derived from L-amino acids, present in the saliva, through L-amino acid oxidase (11).
Ying Liu1, Lei Chu2, Fei Wu3, Lili Guo4, Mengci Li1, Yinghui Wang1, Ligeng Wu1 1
Department of Endodontics, School of Stomatology, Tianjin Medical University, Tianjin; 2Department of Paediatrics, Yantai Stomatological Hospital, Yantai; 3Department of Endodontics, Yantai Stomatological Hospital, Yantai; 4Department of Stomatology, The First Affiliated Hospital of Henan University of TCM, Henan, China
Ligeng Wu, Department of Endodontics, School of Stomatology, Tianjin Medical University, No.12 Qixiangtai Road, Heping District, Tianjin, China E-mail: [email protected] Key words: hydrogen peroxide; interaction; pH; Streptococcus mutans; Streptococcus oligofermentans Accepted for publication October 2013
The oral environment is a habitat in which factors including nutrients, pH, and oxygen undergo constant change (12). Environmental factors, such as pH, significantly influence the growth and interaction of bacteria (13). Previous studies have shown that S. mutans can withstand relatively low-pH conditions and can maintain cariogenicity even at pH 5.5 (14). In contrast, large numbers of bacterial species, such as S. sanguinis, either cannot survive or lose bioactivity under low-pH conditions (15). It has been reported that the inhibition of S. mutans by S. sanguinis is significantly reduced under low-pH conditions (15, 16). Moreover, the presence of carbohydrate and oxygen has been shown to significantly enhance the ability of S. oligofermentans to inhibit S. mutans (17). However, whether S. oligofermentans is aciduric and how pH affects the interaction between S. oligofermentans and S. mutans have not yet been explored. Therefore, in this study we aimed to investigate the coexistence and competition between S. oligofermentans and S. mutans by varying the pH to simulate the dental biofilm environment.
Material and methods Bacterial strains and media Streptococcus mutans ATCC10449 was obtained from the Institute of Microbiology of the Chinese Academy of
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Sciences. S. oligofermentans was kindly provided by Huichun Tong of the Institute of Microbiology of the Chinese Academy of Sciences. S. oligofermentans and S. mutans were inoculated [5%; volume by volume (v/v)] into tryptone–peptone–yeast (TPY) medium and grown under an atmosphere of 5% CO2 at 37°C. Growth curves at different pH Growth curves at different pH values were generated according to the protocol of LIU et al. (17), with the modification that TPY broth was buffered to different pH values (pH 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0) by the addition of hydrochloric acid or sodium hydroxide. S. oligofermentans and S. mutans were inoculated separately at a volume representing 5% of the total culture volume. Aliquots of the culture were taken after inoculation and every 2 h thereafter up to 24 h of culture; the optical density at 600 nm (OD600) of these aliquots was used to generate growth curves. Competition assays Overnight cultures of S. oligofermentans and S. mutans were adjusted to the same optical density (OD600 1.0). Then, 6-ll aliquots of each strain were inoculated adjacent to each other on TPY agar plates of different pH values (pH 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0) as follows: (i) both bacteria were inoculated at the same time, (ii) S. oligofermentans was inoculated 20 h before S. mutans, and (iii) S. mutans was inoculated 20 h before S. oligofermentans. The plates were cultured for another 24 h at 37°C under an atmosphere of 5% CO2 and the growth was monitored. Growth inhibition was assessed by the presence and area of the proximal zone of inhibition at the intersection of the two bacterial colonies. Assays of the total H2O2 yield First of all, the H2O2 regression equation was created by measuring the absorbance at 510 nm (A510) using the protocol of SEKI et al. (18). S. oligofermentans was grown on TPY agar plates of different pH values (pH 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0). In order to investigate the total H2O2 yield during the logarithmic phase, aliquots of the culture were taken at 4, 5, 6, 7, 8, 9, and 10 h after inoculation. Then, the yield of H2O2 was determined using the 4-aminoantipyine method, as described in LIU et al. (17). At each time-point, three replicates were made and the H2O2 yield was calculated.
measured. Then, the initial rate of H2O2 production was calculated. The measurement was repeated three times. Statistical analysis The mean maximum absorbance values of bacteria were compared using the Student’s t-test and one-way ANOVA. Linear correlation analysis was used to generate the standard H2O2 curve, and, based on the linear regression equation, the initial rate of H2O2 production was calculated. The total yield and initial rate of H2O2 production under different pH conditions were compared using-way ANOVA via SPSS software (version 16.0; SPSS, Chicago, IL, USA; a = 0.05).
Results Growth of S. oligofermentans and S. mutans
The 24-h growth of S. oligofermentans and S. mutans under different pH conditions was investigated. At pH 7.0, growth of S. oligofermentans and S. mutans was optimal; both entered the logarithmic and plateau phases steadily and achieved similar OD values after 24 h of growth. However, when the culture pH was lower or higher than pH 7.0, the growth of both strains was reduced by different degrees. As shown in Fig. 1, the difference in mean maximum absorbance between S. oligofermentans and S. mutans at pH 7.0 was not statistically significant, whereas S. mutans achieved a higher cell density than S. oligofermentans during growth under other pH conditions (P < 0.05). Coexistence between S. oligofermentans and S. mutans under different pH conditions
The in-vitro inhibitory effect of S. oligofermentans on S. mutans growth was observed following the use of different inoculation sequences under different pH conditions. Our results showed that the sequence of inoculation and pH variation resulted in different inhibitory
Initial H2O2 production rate The initial H2O2 production rate of S. oligofermentans was studied using the methods provided by YU et al. (19) and LIU et al. (17), involving some modifications. Briefly, S. oligofermentans grown in TPY broth (pH 7.0) was collected by centrifugation. The pellet was washed three times, resuspended in PBS of the same pH as the TPY to be used, then reinoculated into TPY broth of different pH (pH5.5, 6.0, 6.5, 7.0, 7.5, and 8.0). Inoculates were then cultured under an atmosphere of 5% CO2 at 37°C. At 10, 20, and 30 min after inoculation, aliquots of culture were taken and the supernatant was collected after centrifugation. Phenol and 4-aminoantipyine (0.5 mg ml 1) were added to initiate the reaction. After 4 min, 1 ml of horseradish peroxidase (HRP) was added, and the A510 was
Fig. 1. Mean maximum absorbance (at 600 nm) of Streptococcus oligofermentans (black bars) and Streptococcus mutans (grey bars) at different pH values (5.5, 6.0, 6.5, 7.0, 7.5, and 8.0). Different letters above the bars indicate statistically significant differences (P < 0.05).
pH influences interspecies interaction
effects. At pH values of 5.5 and 8.0, S. oligofermentans and S. mutans exhibited poor growth and no interaction was observed regardless of the inoculation sequence. At pH values of 6.0, 6.5, 7.0, and 7.5, S. oligofermentans and S. mutans showed interactions that were significantly influenced by the inoculation sequence. When S. mutans was inoculated first, the growth of S. oligofermentans was completely inhibited. When the two species were inoculated at the same time, or when S. oligofermentans was inoculated first, an inhibitory effect of S. oligofermentans on S. mutans was observed (Fig. 2). Under different pH conditions, the inhibitory effects were as follows: pH 7.0 > pH 6.5 > pH 6.0 = 7.5 (i.e. there was no statistically significant difference between pH 6.0 and pH 7.5). Our results indicated that the inhibition of S. mutans by S. oligofermentans was also influenced by the inoculation sequence. Under an environment of identical pH, when S. oligofermentans was inoculated first, the inhibition areas of S. mutans were larger than those obtained when both bacteria were inoculated simultaneously. As shown in Fig. 2, when the two species were inoculated simultaneously, the inhibition zones of the S. mutans grown on the plates with pH values of 7.0, 6.5, 6.0, and 7.5 were one-third, one-fifth, one-eighth, and oneeighth of the entire culture area, respectively. When S. oligofermentans was inoculated before S. mutans, the inhibition zones of S. mutans were three-fifths, twofifths, one-fifth, and one-fifth of the entire culture area, respectively. H2O2 production by S. oligofermentans
On the basis of the A510 measurements, a standard curve for H2O2 was generated and the linear correlation equation y = 273.089x 59.879 (r2 = 0.996) was established. The production of H2O2 by S. oligofermentans was pH-dependent. For S. oligofermentans, the relative effects of pH on the initial rate and the total yield of H2O2 during the logarithmic phase were observed to occur in the following order: pH 7.0 > pH 6.5 > pH 6.0 > pH 7.5 > pH 5.5 = pH 8.0 (i.e. there
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Fig. 3. Hydrogen peroxide (H2O2) production by Streptococcus oligofermentans during the logarithmic growth phase in culture at different pH values (5.5, 6.0, 6.5, 7.0, 7.5, and 8.0). The data represent three independent experiments. The differences in H2O2 yield at different pH values were statistically significant, except at pH 5.5 and pH 8.0 (a = 0.05).
was no statistically significant difference between pH 5.5 and 8.0; P > 0.05) (Fig. 3).
Discussion In this study, we investigated the impact of environmental pH on the growth of S. oligofermentans and the interspecies interaction between S. oligofermentans and S. mutans under different pH conditions. Our study indicated that the optimal pH for S. oligofermentans growth is pH7.0. At pH 5.5 and pH 8.0, growth of S. oligofermentans was much reduced. Based on the growth curves, we conclude that S. oligofermentans possesses both acid-resistant and alkaline-resistant abilities but is less acid and alkaline tolerant compared with S. mutans. To shed new light on how these environmental pH factors affect the interaction between S. oligofermentans and S. mutans, an in-vitro monospecies biofilm model and interspecies interaction assay were conducted. For
Fig. 2. Inhibition of growth of Streptococcus oligofermentans by Streptococcus mutans at different pH values. S. mutans was inoculated on the right and S. oligofermentans on the left. The first row represents inoculation of S. oligofermentans and S. mutans simultaneously. The second row represents inoculation of S. oligofermentans 20 h before inoculation of S. mutans. The third row represents inoculation of S. mutans 20 h before inoculation of S. oligofermentans. Sm, Streptococcus mutans; So, Streptococcus oligofermentans.
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these experiments, the order of inoculation was varied as follows: (i) the two species of bacteria were inoculated simultaneously, (ii) S. oligofermentans was inoculated before S. mutans, and (iii) S. mutans was inoculated before S. oligofermentans. At pH 5.5 and pH 8.0, both S. oligofermentans and S. mutans lost their ability to compete with each other, which may be a result of the poor growth of both strains. At the various other pH values studied, if S. mutans was inoculated first, then the growth of S. oligofermentans was significantly inhibited regardless of pH, which suggests that pH has no effect on the inhibition of S. oligofermentans by S. mutans. It has been reported that earlier inoculation of S. mutans inhibited 11 common oral microbial species, including S. sanguinis, which was inoculated later (15). S. mutans is known to produce mutacins that disrupt the attachment and/or growth of other bacterial strains in plaque (20). Therefore, the inhibition of S. oligofermentans by S. mutans could be attributed to the production of mutacins. This proposed model will need further investigation. When both S. oligofermentans and S. mutans were inoculated simultaneously, or S. oligofermentans was inoculated before S. mutans, the inhibitory effect of S. oligofermentans on S. mutans was observed. The effects of pH on the inhibitory effect were as follows: pH 7.0 > pH 6.5 > pH 6.0 = pH 7.5 (Fig. 2; i.e. there was no statistically significant difference between pH 6.0 and pH 7.5). Given that the growth of S. mutans was better than that of S. oligofermentans at pH 6.0, 6.5, and 7.5, and similar to that of S. oligofermentans at pH 7.0 (as shown in Fig. 1), we propose that the inhibition of S. mutans by S. oligofermentans may be a result of the production of inhibitory factors, such as H2O2, rather than cell density. In-vitro studies have indicated that S. oligofermentans suppresses the growth of S. mutans by producing large amounts of H2O2 (9). Therefore, we propose a model in which variability in pH affects the inhibitory capacity of S. oligofermentans based on H2O2 production. Thus, we investigated the H2O2-production capacity of S. oligofermentans under different pH conditions. Our results showed that the relative effects of pH on the initial rate and the total yield of H2O2 production during the logarithmic growth phase of S. oligofermentans at different pH values were as follows: pH 7.0 > pH 6.5 > pH 6.0 > pH 7.5, and the effect of pH on the inhibition of S. mutans by S. oligofermentans was almost the same (pH 7.0 > pH 6.5 > pH 6.0 and pH 7.5). Therefore, our results support the model in which the effect of pH on the inhibitory capability of S. oligofermentans correlates with H2O2 production. H2O2 production was about 1.2–1.5 fold higher at pH 6.0 than at pH 7.5, but no difference was observed between the inhibitory effect of the two species at pH 6.0 and pH 7.5. There are two possible explanations for this observation: (i) the difference in H2O2 production, although statistically significant, was below the threshold level required to induce inhibition by S. oligofermentans, or (ii) the production of H2O2 by
S. oligofermentans at pH 7.5 was less than that at pH 6.0; however, the antagonism of S. mutans on S. oligofermentans growth was more potent at pH 7.5. The capability of S. oligofermentans to produce H2O2 may be affected by the growth conditions and the environmental pH. Our results are also consistent with those of CHEN et al. (21), indicating that the amount of H2O2 produced by S. oligofermentans was closely related to the growth phase and the proliferation rate. In the present study, we showed that pH 7.0 was the optimal pH for growth and H2O2 production of S. oligofermentans. In contrast, growth and H2O2 production of S. oligofermentans were significantly reduced under ‘stress’ conditions. In addition, the change in environmental pH may up- or down-regulate peroxidogenic enzymes, leading to a variation in H2O2 production; however, the exact mechanism involved in regulating these enzymes needs further investigation. Streptococcus sanguinis is widely known to play an important role in antagonizing S. mutans growth and is recognized as a beneficial bacterium with regard to dental caries (15, 22). Yet, early studies have indicated that S. sanguinis lost its inhibitory effect on S. mutans under more caries-causing conditions in the presence of carbohydrates and that S. oligofermentans exhibited much higher potential to inhibit S. mutans growth (17). Therefore, we suggest that S. oligofermentans has the potential to be a novel and beneficial oral bacterium owing to its stronger inhibition of S. mutans. However, it is also true that when S. mutans (not a pioneer colonizer) was inoculated earlier, S. oligofermentans was inhibited by S. mutans. Moreover, only one strain of S. mutans was tested in this study, and the inhibitory effect of S. oligofermentans on other strains or clinical isolates of S. mutans has not been explored. Therefore, more in-depth studies on S. oligofermentans are needed to support this inhibitory model. In summary, the optimal pH for S. oligofermentans is pH 7.0, and S. oligofermentans possesses both acidresistant and alkaline-resistant abilities. The interaction between S. oligofermentans and S. mutans was affected by the sequence of inoculation and the environmental pH. Hence, based on the results of this study, S. oligofermentans should be considered as a potential probiotic bacterium. Acknowledgements – We are grateful to Professor Huichun Tong of the Institute of Microbiology, Chinese Academy of Sciences, for providing us with S. oligofermentans. We also thank research assistant Tao Chen and research associate George Adams (Department of Immunology, University of Texas Southwestern Medical Center) for revising the manuscript. Conflicts of interest – The authors report no conflicts of interest.
References 1. KULIK EM, SANDMEIER H, HINNI K, MEYER J. Identification of archaeal rDNA from subgingival dental plaque by PCR amplification and sequence analysis. FEMS Microbiol Lett 2001; 196: 129–133.
pH influences interspecies interaction 2. AAS JA, PASTER BJ, STOKES LN, OLSEN I, DEWHIRST FE. Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 2005; 43: 5721–5732. 3. PENNISI E. A mouthful of microbes. Science 2005; 307: 1899– 1901. 4. SBORDONE L, BORTOLAIA C. Oral microbial biofilms and plaquerelated diseases: microbial communities and their role in the shift from oral health to disease. Clin Oral Investig 2003; 7: 181–188. 5. LOESCHE WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev 1986; 50: 353–380. 6. HILLMAN JD, SHIVERS M. Interaction between wild-type, mutant and revertant forms of the bacterium Streptococcus sanguis and the bacterium Actinobacillus actinomycetemcomitans in vitro and in the gnotobiotic rat. Arch Oral Biol 1988; 33: 395–401. 7. ROBERTS FA, DARVEAU RP. Beneficial bacteria of the periodontium. Periodontol 2000 2002; 30: 40–50. 8. TONG H, GAO X, DONG X. Streptococcus oligofermentans sp. nov., a novel oral isolate from caries-free humans. Int J Syst Evol Microbiol 2003; 53: 1101–1104. 9. TONG H, CHEN W, MERRITT J, QI F, SHI W, DONG X. Streptococcus oligofermentans inhibits Streptococcus mutans through conversion of lactic acid into inhibitory H2O2: a possible counteroffensive strategy for interspecies competition. Mol Microbiol 2007; 63: 872–880. 10. LIU L, TONG H, DONG X. Function of the pyruvate oxidaselactate oxidase cascade in interspecies competition between Streptococcus oligofermentans and Streptococcus mutans. Appl Environ Microbiol 2012; 78: 2120–2127. 11. TONG H, CHEN W, SHI W, QI F, DONG X. SO-LAAO, a novel L-amino acid oxidase that enables Streptococcus oligofermentans to outcompete Streptococcus mutans by generating H2O2 from peptone. J Bacteriol 2008; 190: 4716–4721. 12. KRETH J, MERRITT J, QI F. Bacterial and host interactions of oral streptococci. DNA Cell Biol 2009; 28: 397–403. 13. KOLENBRANDER PE, PALMER RJ Jr, RICKARD AH, JAKUBOVICS NS, CHALMERS NI, DIAZ PI. Bacterial interactions and succes-
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sions during plaque development. Periodontol 2000 2006; 42: 47–79. DONG YM, PEARCE EI, YUE L, LARSEN MJ, GAO XJ, WANG JD. Plaque pH and associated parameters in relation to caries. Caries Res 1999; 33: 428–436. KRETH J, MERRITT J, SHI W, QI F. Competition and coexistence between Streptococcus mutans and Streptococcus sanguinis in the dental biofilm. J Bacteriol 2005; 187: 7193– 7203. NOBBS AH, ZHANG Y, KHAMMANIVONG A, HERZBERG MC. Streptococcus gordonii has environmentally constrains competitive binding by Streptococcus sanguinis to saliva-coated hydroxyapatite. J Bacteriol 2007; 189: 3106–3114. LIU Y, WU L, WU F, CHU L, LIU X, XIA K, WANG Y. Interspecies competition and inhibition within the oral microbial flora: environmental factors influence the inhibition of Streptococcus mutans by Streptococcus oligofermentans. Eur J Oral Sci 2012; 120: 179–184. SEKI M, IIDA K, SAITO M, NAKAYAMA H, YOSHIDA S. Hydrogen peroxide production in Streptococcus pyogenes: involvement of lactate oxidase and coupling with aerobic utilization of lactate. J Bacteriol 2004; 186: 2046–2051. YU S, ZHANG J, ZHANG Y, ZHANG JLIU H. [Influence of environmental factors on synthesis rate of hydrogen peroxide by Streptococcus oralis]. Zhonghua Kou Qiang Yi Xue Za Zhi 2005; 40: 481–484. HOSSAIN MS, BISWAS I. Mutacins from Streptococcus mutans UA159 are active against multiple streptococcal species. Appl Environ Microbiol 2011; 77: 2428–2434. CHEN W, TONG HC, DONG XZ. [Characterization of hydrogen peroxide production by a novel oral streptococci, S. oligofermentans isolated from human oral cavity]. Wei Sheng Wu Xue Bao 2006; 46: 820–822. BECKER MR, PASTER BJ, LEYS EJ, MOESCHBERGER ML, KENYON SG, GALVIN JL, BOCHES SK, DEWHIRST FE, GRIFFEN AL. Molecular analysis of bacterial species associated with childhood caries. J Clin Microbiol 2002; 40: 1001–1009.
Eur J Oral Sci 2014; 122: 57–61 DOI: 10.1111/eos.12102 Printed in Singapore. All rights reserved
European Journal of Oral Sciences
Influence of pH on inhibition of Streptococcus mutans by Streptococcus oligofermentans Liu Y, Chu L, Wu F, Guo L, Li M, Wang Y, Wu L. Influence of pH on inhibition of Streptococcus mutans by Streptococcus oligofermentans. Eur J Oral Sci 2014; 122: 57–61. © 2013 Eur J Oral Sci Streptococcus oligofermentans is a novel strain of oral streptococcus that can specifically inhibit the growth of Streptococcus mutans. The aims of this study were to assess the growth of S. oligofermentans and the ability of S. oligofermentans to inhibit growth of Streptococcus mutans at different pH values. Growth inhibition was investigated in vitro using an interspecies competition assay. The 4-aminoantipyine method was used to measure the initial production rate and the total yield of hydrogen peroxide in S. oligofermentans. S. oligofermentans grew best at pH 7.0 and showed the most pronounced inhibitory effect when it was inoculated earlier than S. mutans. In terms of the total yield and the initial production rate of hydrogen peroxide by S. oligofermentans, the effects of the different culture pH values were as follows: pH 7.0 > 6.5 > 6.0 > 7.5 > 5.5 = 8.0 (i.e. there was no significant difference between pH 5.5 and pH 8.0). Environmental pH and the sequence of inoculation significantly affected the ability of S. oligofermentans to inhibit the growth of S. mutans. The degree of inhibition may be attributed to the amount of hydrogen peroxide produced.
Dental biofilm is a complicated multispecies community composed of a large number of microbes, with more than 700 species identified (1–3). These microbes maintain their ecological homeostasis by coexistence and competition. Dental caries is the result of destruction of the oral micro-ecological balance among different species (4). Streptococcus mutans is still regarded as a primary pathogen in dental caries, although other microorganisms may also be involved (5). Some beneficial bacteria, such as Streptococcus sanguinis and Streptococcus gordonii (6, 7) are able to inhibit the growth of S. mutans, as well as other caries-causing pathogens, by producing hydrogen peroxide (H2O2). Streptococcus oligofermentans is a novel strain of streptococcus that was first isolated from the dental plaque of caries-free human subjects by TONG et al. in 2003 (8). An in-vitro study demonstrated that S. oligofermentans inhibits the growth of S. mutans by producing a much higher level of H2O2 compared with S. sanguinis and S. gordonii. In addition, S. oligofermentans can use its lactate oxidase and pyruvate oxidase activities to convert lactic acid and pyruvate (metabolic intermediates generated by cariescausing pathogens, such as S. mutans) into H2O2 (9, 10). It was also reported that the H2O2 produced by S. oligofermentans could be derived from L-amino acids, present in the saliva, through L-amino acid oxidase (11).
Ying Liu1, Lei Chu2, Fei Wu3, Lili Guo4, Mengci Li1, Yinghui Wang1, Ligeng Wu1 1
Department of Endodontics, School of Stomatology, Tianjin Medical University, Tianjin; 2Department of Paediatrics, Yantai Stomatological Hospital, Yantai; 3Department of Endodontics, Yantai Stomatological Hospital, Yantai; 4Department of Stomatology, The First Affiliated Hospital of Henan University of TCM, Henan, China
Ligeng Wu, Department of Endodontics, School of Stomatology, Tianjin Medical University, No.12 Qixiangtai Road, Heping District, Tianjin, China E-mail: [email protected] Key words: hydrogen peroxide; interaction; pH; Streptococcus mutans; Streptococcus oligofermentans Accepted for publication October 2013
The oral environment is a habitat in which factors including nutrients, pH, and oxygen undergo constant change (12). Environmental factors, such as pH, significantly influence the growth and interaction of bacteria (13). Previous studies have shown that S. mutans can withstand relatively low-pH conditions and can maintain cariogenicity even at pH 5.5 (14). In contrast, large numbers of bacterial species, such as S. sanguinis, either cannot survive or lose bioactivity under low-pH conditions (15). It has been reported that the inhibition of S. mutans by S. sanguinis is significantly reduced under low-pH conditions (15, 16). Moreover, the presence of carbohydrate and oxygen has been shown to significantly enhance the ability of S. oligofermentans to inhibit S. mutans (17). However, whether S. oligofermentans is aciduric and how pH affects the interaction between S. oligofermentans and S. mutans have not yet been explored. Therefore, in this study we aimed to investigate the coexistence and competition between S. oligofermentans and S. mutans by varying the pH to simulate the dental biofilm environment.
Material and methods Bacterial strains and media Streptococcus mutans ATCC10449 was obtained from the Institute of Microbiology of the Chinese Academy of
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Sciences. S. oligofermentans was kindly provided by Huichun Tong of the Institute of Microbiology of the Chinese Academy of Sciences. S. oligofermentans and S. mutans were inoculated [5%; volume by volume (v/v)] into tryptone–peptone–yeast (TPY) medium and grown under an atmosphere of 5% CO2 at 37°C. Growth curves at different pH Growth curves at different pH values were generated according to the protocol of LIU et al. (17), with the modification that TPY broth was buffered to different pH values (pH 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0) by the addition of hydrochloric acid or sodium hydroxide. S. oligofermentans and S. mutans were inoculated separately at a volume representing 5% of the total culture volume. Aliquots of the culture were taken after inoculation and every 2 h thereafter up to 24 h of culture; the optical density at 600 nm (OD600) of these aliquots was used to generate growth curves. Competition assays Overnight cultures of S. oligofermentans and S. mutans were adjusted to the same optical density (OD600 1.0). Then, 6-ll aliquots of each strain were inoculated adjacent to each other on TPY agar plates of different pH values (pH 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0) as follows: (i) both bacteria were inoculated at the same time, (ii) S. oligofermentans was inoculated 20 h before S. mutans, and (iii) S. mutans was inoculated 20 h before S. oligofermentans. The plates were cultured for another 24 h at 37°C under an atmosphere of 5% CO2 and the growth was monitored. Growth inhibition was assessed by the presence and area of the proximal zone of inhibition at the intersection of the two bacterial colonies. Assays of the total H2O2 yield First of all, the H2O2 regression equation was created by measuring the absorbance at 510 nm (A510) using the protocol of SEKI et al. (18). S. oligofermentans was grown on TPY agar plates of different pH values (pH 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0). In order to investigate the total H2O2 yield during the logarithmic phase, aliquots of the culture were taken at 4, 5, 6, 7, 8, 9, and 10 h after inoculation. Then, the yield of H2O2 was determined using the 4-aminoantipyine method, as described in LIU et al. (17). At each time-point, three replicates were made and the H2O2 yield was calculated.
measured. Then, the initial rate of H2O2 production was calculated. The measurement was repeated three times. Statistical analysis The mean maximum absorbance values of bacteria were compared using the Student’s t-test and one-way ANOVA. Linear correlation analysis was used to generate the standard H2O2 curve, and, based on the linear regression equation, the initial rate of H2O2 production was calculated. The total yield and initial rate of H2O2 production under different pH conditions were compared using-way ANOVA via SPSS software (version 16.0; SPSS, Chicago, IL, USA; a = 0.05).
Results Growth of S. oligofermentans and S. mutans
The 24-h growth of S. oligofermentans and S. mutans under different pH conditions was investigated. At pH 7.0, growth of S. oligofermentans and S. mutans was optimal; both entered the logarithmic and plateau phases steadily and achieved similar OD values after 24 h of growth. However, when the culture pH was lower or higher than pH 7.0, the growth of both strains was reduced by different degrees. As shown in Fig. 1, the difference in mean maximum absorbance between S. oligofermentans and S. mutans at pH 7.0 was not statistically significant, whereas S. mutans achieved a higher cell density than S. oligofermentans during growth under other pH conditions (P < 0.05). Coexistence between S. oligofermentans and S. mutans under different pH conditions
The in-vitro inhibitory effect of S. oligofermentans on S. mutans growth was observed following the use of different inoculation sequences under different pH conditions. Our results showed that the sequence of inoculation and pH variation resulted in different inhibitory
Initial H2O2 production rate The initial H2O2 production rate of S. oligofermentans was studied using the methods provided by YU et al. (19) and LIU et al. (17), involving some modifications. Briefly, S. oligofermentans grown in TPY broth (pH 7.0) was collected by centrifugation. The pellet was washed three times, resuspended in PBS of the same pH as the TPY to be used, then reinoculated into TPY broth of different pH (pH5.5, 6.0, 6.5, 7.0, 7.5, and 8.0). Inoculates were then cultured under an atmosphere of 5% CO2 at 37°C. At 10, 20, and 30 min after inoculation, aliquots of culture were taken and the supernatant was collected after centrifugation. Phenol and 4-aminoantipyine (0.5 mg ml 1) were added to initiate the reaction. After 4 min, 1 ml of horseradish peroxidase (HRP) was added, and the A510 was
Fig. 1. Mean maximum absorbance (at 600 nm) of Streptococcus oligofermentans (black bars) and Streptococcus mutans (grey bars) at different pH values (5.5, 6.0, 6.5, 7.0, 7.5, and 8.0). Different letters above the bars indicate statistically significant differences (P < 0.05).
pH influences interspecies interaction
effects. At pH values of 5.5 and 8.0, S. oligofermentans and S. mutans exhibited poor growth and no interaction was observed regardless of the inoculation sequence. At pH values of 6.0, 6.5, 7.0, and 7.5, S. oligofermentans and S. mutans showed interactions that were significantly influenced by the inoculation sequence. When S. mutans was inoculated first, the growth of S. oligofermentans was completely inhibited. When the two species were inoculated at the same time, or when S. oligofermentans was inoculated first, an inhibitory effect of S. oligofermentans on S. mutans was observed (Fig. 2). Under different pH conditions, the inhibitory effects were as follows: pH 7.0 > pH 6.5 > pH 6.0 = 7.5 (i.e. there was no statistically significant difference between pH 6.0 and pH 7.5). Our results indicated that the inhibition of S. mutans by S. oligofermentans was also influenced by the inoculation sequence. Under an environment of identical pH, when S. oligofermentans was inoculated first, the inhibition areas of S. mutans were larger than those obtained when both bacteria were inoculated simultaneously. As shown in Fig. 2, when the two species were inoculated simultaneously, the inhibition zones of the S. mutans grown on the plates with pH values of 7.0, 6.5, 6.0, and 7.5 were one-third, one-fifth, one-eighth, and oneeighth of the entire culture area, respectively. When S. oligofermentans was inoculated before S. mutans, the inhibition zones of S. mutans were three-fifths, twofifths, one-fifth, and one-fifth of the entire culture area, respectively. H2O2 production by S. oligofermentans
On the basis of the A510 measurements, a standard curve for H2O2 was generated and the linear correlation equation y = 273.089x 59.879 (r2 = 0.996) was established. The production of H2O2 by S. oligofermentans was pH-dependent. For S. oligofermentans, the relative effects of pH on the initial rate and the total yield of H2O2 during the logarithmic phase were observed to occur in the following order: pH 7.0 > pH 6.5 > pH 6.0 > pH 7.5 > pH 5.5 = pH 8.0 (i.e. there
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Fig. 3. Hydrogen peroxide (H2O2) production by Streptococcus oligofermentans during the logarithmic growth phase in culture at different pH values (5.5, 6.0, 6.5, 7.0, 7.5, and 8.0). The data represent three independent experiments. The differences in H2O2 yield at different pH values were statistically significant, except at pH 5.5 and pH 8.0 (a = 0.05).
was no statistically significant difference between pH 5.5 and 8.0; P > 0.05) (Fig. 3).
Discussion In this study, we investigated the impact of environmental pH on the growth of S. oligofermentans and the interspecies interaction between S. oligofermentans and S. mutans under different pH conditions. Our study indicated that the optimal pH for S. oligofermentans growth is pH7.0. At pH 5.5 and pH 8.0, growth of S. oligofermentans was much reduced. Based on the growth curves, we conclude that S. oligofermentans possesses both acid-resistant and alkaline-resistant abilities but is less acid and alkaline tolerant compared with S. mutans. To shed new light on how these environmental pH factors affect the interaction between S. oligofermentans and S. mutans, an in-vitro monospecies biofilm model and interspecies interaction assay were conducted. For
Fig. 2. Inhibition of growth of Streptococcus oligofermentans by Streptococcus mutans at different pH values. S. mutans was inoculated on the right and S. oligofermentans on the left. The first row represents inoculation of S. oligofermentans and S. mutans simultaneously. The second row represents inoculation of S. oligofermentans 20 h before inoculation of S. mutans. The third row represents inoculation of S. mutans 20 h before inoculation of S. oligofermentans. Sm, Streptococcus mutans; So, Streptococcus oligofermentans.
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these experiments, the order of inoculation was varied as follows: (i) the two species of bacteria were inoculated simultaneously, (ii) S. oligofermentans was inoculated before S. mutans, and (iii) S. mutans was inoculated before S. oligofermentans. At pH 5.5 and pH 8.0, both S. oligofermentans and S. mutans lost their ability to compete with each other, which may be a result of the poor growth of both strains. At the various other pH values studied, if S. mutans was inoculated first, then the growth of S. oligofermentans was significantly inhibited regardless of pH, which suggests that pH has no effect on the inhibition of S. oligofermentans by S. mutans. It has been reported that earlier inoculation of S. mutans inhibited 11 common oral microbial species, including S. sanguinis, which was inoculated later (15). S. mutans is known to produce mutacins that disrupt the attachment and/or growth of other bacterial strains in plaque (20). Therefore, the inhibition of S. oligofermentans by S. mutans could be attributed to the production of mutacins. This proposed model will need further investigation. When both S. oligofermentans and S. mutans were inoculated simultaneously, or S. oligofermentans was inoculated before S. mutans, the inhibitory effect of S. oligofermentans on S. mutans was observed. The effects of pH on the inhibitory effect were as follows: pH 7.0 > pH 6.5 > pH 6.0 = pH 7.5 (Fig. 2; i.e. there was no statistically significant difference between pH 6.0 and pH 7.5). Given that the growth of S. mutans was better than that of S. oligofermentans at pH 6.0, 6.5, and 7.5, and similar to that of S. oligofermentans at pH 7.0 (as shown in Fig. 1), we propose that the inhibition of S. mutans by S. oligofermentans may be a result of the production of inhibitory factors, such as H2O2, rather than cell density. In-vitro studies have indicated that S. oligofermentans suppresses the growth of S. mutans by producing large amounts of H2O2 (9). Therefore, we propose a model in which variability in pH affects the inhibitory capacity of S. oligofermentans based on H2O2 production. Thus, we investigated the H2O2-production capacity of S. oligofermentans under different pH conditions. Our results showed that the relative effects of pH on the initial rate and the total yield of H2O2 production during the logarithmic growth phase of S. oligofermentans at different pH values were as follows: pH 7.0 > pH 6.5 > pH 6.0 > pH 7.5, and the effect of pH on the inhibition of S. mutans by S. oligofermentans was almost the same (pH 7.0 > pH 6.5 > pH 6.0 and pH 7.5). Therefore, our results support the model in which the effect of pH on the inhibitory capability of S. oligofermentans correlates with H2O2 production. H2O2 production was about 1.2–1.5 fold higher at pH 6.0 than at pH 7.5, but no difference was observed between the inhibitory effect of the two species at pH 6.0 and pH 7.5. There are two possible explanations for this observation: (i) the difference in H2O2 production, although statistically significant, was below the threshold level required to induce inhibition by S. oligofermentans, or (ii) the production of H2O2 by
S. oligofermentans at pH 7.5 was less than that at pH 6.0; however, the antagonism of S. mutans on S. oligofermentans growth was more potent at pH 7.5. The capability of S. oligofermentans to produce H2O2 may be affected by the growth conditions and the environmental pH. Our results are also consistent with those of CHEN et al. (21), indicating that the amount of H2O2 produced by S. oligofermentans was closely related to the growth phase and the proliferation rate. In the present study, we showed that pH 7.0 was the optimal pH for growth and H2O2 production of S. oligofermentans. In contrast, growth and H2O2 production of S. oligofermentans were significantly reduced under ‘stress’ conditions. In addition, the change in environmental pH may up- or down-regulate peroxidogenic enzymes, leading to a variation in H2O2 production; however, the exact mechanism involved in regulating these enzymes needs further investigation. Streptococcus sanguinis is widely known to play an important role in antagonizing S. mutans growth and is recognized as a beneficial bacterium with regard to dental caries (15, 22). Yet, early studies have indicated that S. sanguinis lost its inhibitory effect on S. mutans under more caries-causing conditions in the presence of carbohydrates and that S. oligofermentans exhibited much higher potential to inhibit S. mutans growth (17). Therefore, we suggest that S. oligofermentans has the potential to be a novel and beneficial oral bacterium owing to its stronger inhibition of S. mutans. However, it is also true that when S. mutans (not a pioneer colonizer) was inoculated earlier, S. oligofermentans was inhibited by S. mutans. Moreover, only one strain of S. mutans was tested in this study, and the inhibitory effect of S. oligofermentans on other strains or clinical isolates of S. mutans has not been explored. Therefore, more in-depth studies on S. oligofermentans are needed to support this inhibitory model. In summary, the optimal pH for S. oligofermentans is pH 7.0, and S. oligofermentans possesses both acidresistant and alkaline-resistant abilities. The interaction between S. oligofermentans and S. mutans was affected by the sequence of inoculation and the environmental pH. Hence, based on the results of this study, S. oligofermentans should be considered as a potential probiotic bacterium. Acknowledgements – We are grateful to Professor Huichun Tong of the Institute of Microbiology, Chinese Academy of Sciences, for providing us with S. oligofermentans. We also thank research assistant Tao Chen and research associate George Adams (Department of Immunology, University of Texas Southwestern Medical Center) for revising the manuscript. Conflicts of interest – The authors report no conflicts of interest.
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