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Streptococcus salivarius Promotes Mucin Putrefaction and Malodor Production by Porphyromonas gingivalis
1 Department of Prosthodontics, the Hebrew University-Hadassah School of Dental Medicine, POB 12272, Jerusalem 91120, Israel; and Correspondence: * corresponding author, sterer{at}hadassah.org.il
Although the contribution of the oral microbiota to oral malodor is well-documented, the potential role of Gram-positive micro-organisms is unclear. In the current study, we tested the hypothesis that Gram-positive micro-organisms contribute to malodor production by deglycosylating oral glycoproteins, rendering them susceptible to subsequent proteolysis. To this end, we examined the effect of Streptococcus salivarius on Porphyromonas gingivalis-mediated putrefaction of a model glycoprotein (pig gastric mucin). Malodor was scored by two odor judges, and volatile sulfides were determined with the use of a sulfide monitor. Mucin degradation was followed by electrophoresis on SDS-PAGE. Results showed that the addition of S. salivarius or β-galactosidase promoted mucin degradation and concomitant malodor production. Addition of glycosidic inhibitors (p-APTG and glucose) inhibited this process. These results suggest that Gram-positive micro-organisms such as S. salivarius contribute to oral malodor production by deglycosylating salivary glycoproteins, thus exposing their protein core to further degradation by Gram-negative micro-organisms.
Key Words: β-Galactosidase • Streptococcus salivarius • Porphyromonas gingivalis • oral malodor • mucin
Oral malodor, a common condition, usually originates in the mouth itself, primarily on the posterior portion of the tongue dorsum (Grapp, 1933; Tonzetich, 1977; De Boever and Loesche, 1995). The observations that antibacterial agents can reduce malodor (Morris and Read, 1949), and that various oral micro-organisms produce malodor when incubated in vitro (Berg and Fosdick, 1946; McNamara et al., 1972; Kleinberg and Codipilly, 1997), support the premise that oral malodor is bacterial in origin. This is considered to result from proteolysis, followed by subsequent breakdown of particular amino acids (e.g., methionine, cysteine, tryptophan, and lysine) to yield malodorous volatile products (e.g., methylmercaptan, hydrogen sulfide, indole, skatole, and cadaverine) (Fosdick and Piez, 1953; Hayes and Hyatt, 1974; Kostelc et al., 1981; Claesson et al., 1990; Goldberg et al., 1994). It has previously been assumed that oral malodor is caused exclusively by Gram-negative, rather than Gram-positive, species. Only Gram-negative species tend to produce foul odors in vitro following growth in the presence of various amino acids (Kleinberg and Codipilly, 1997; Persson et al., 1990). When saliva is incubated and allowed to putrefy, Gram-negative species predominate (McNamara et al., 1972). However, Gram-positive bacteria, mainly streptococci, constitute a high proportion of the micro-organisms on the tongue dorsum (Gordon and Gibbons, 1966; Aas et al., 2005). Furthermore, antibiotics active against Gram-positive microbiota can effectively reduce oral malodor in an in vitro model (Goldberg et al., 1997). Significantly, many of the available proteins in the mouth are glycoproteins (Levine et al., 1987; Kleinberg and Westbay, 1992), which require prior removal of their carbohydrate side-chains before the protein core can be degraded (Gottschalk and Fazekas De St Groth, 1960). Gram-positive oral bacteria are able to grow in saliva utilizing carbohydrate side-chains of salivary glycoproteins cleaved by various glycosidases, such as β-galactosidase (De Jong et al., 1984; De Jong and Van Der Hoeven, 1987). In view of this, we postulated that Gram-positive oral micro-organisms play a critical initial role in oral malodor formation by removing carbohydrate side-chains from oral glycoproteins, thus facilitating subsequent proteolysis of their protein core by Gram-negative micro-organisms. In the present study, we tested this hypothesis using a model malodor system in which a defined glycoprotein (pig gastric mucin) was pre-incubated in the presence of Streptococcus salivarius, a common oral Gram-positive micro-organism, and subsequently inoculated with Porphyromonas gingivalis, a putrefactive Gram-negative micro-organism.
Bacterial Strains, Isolation, and Growth Conditions Porphyromonas gingivalis PK 1924 (Kolenbrander and Andersen, 1989) was subcultured weekly on tryptic soy agar sheep blood agar plates (Hy-Labs, Rehovot, Israel). The bacteria were incubated anaerobically at 37°C in anaerobic jars with an AnaeroGenTM anaerobic kit (Oxoid, Hampshire, England). Streptococcus salivarius NS1, first identified based on its ability to produce β-galactosidase, was isolated by the inoculation of tongue-coating samples on Brain Heart Infusion Agar plates (Hy Labs, Rehovot, Israel) supplemented with 0.05 mL of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal, Sigma, St. Louis, MO, USA) (20 mg/mL), and 0.05 mL isopropyl-β-D-galactopyranoside (IPTG, Sigma, St. Louis, MO, USA) (50 mg/mL). The colonies, which stained a clear blue, indicative of β-galactosidase activity (Gossrau, 1977), were subsequently identified by use of the rapid ID 32 STREP kit (BioMerieux, Marcy L’Etoile, France).
Experimental Protocols Test tubes containing 1 mL of the dissolved mucin, with or without glucose (5 mg/mL) or p-aminophenyl-β-D-thiogalactopyranoside (p-APTG) (5 mg/mL), were inoculated with 0.1 mL of Streptococcus salivarius suspended in PBS (pH 7.4) to an initial concentration of 1 OD (405 nm). Test tubes were incubated, with shaking (150 rpm), at 37°C for 24 hrs. After the first incubation, test tubes were inoculated with 0.1 mL of P. gingivalis suspended in PBS to an initial concentration of 1 OD (405 nm), and incubated anaerobically at 37°C for an additional 48 hrs. Following a second incubation, malodor production and volatile sulfide levels were assessed, and ethanol-precipitated mucin samples were analyzed to demonstrate mucin degradation, as described below. The experiment was replicated 6 times.
Pre-incubation of Mucin in the Presence of β-galactosidase
Volatile Sulfide Compounds (VSC) Measurements
Organoleptic Measurements
Mucin Degradation Analyzed by SDS-PAGE Densitometry
Statistical Analysis
Pre-incubation of Mucin in the Presence of Streptococcus salivarius When S. salivarius was pre-incubated in the presence of mucin, odor judge scores and volatile sulfide levels were similar to those of the mucin controls (Figs. 1A, 1B  ). Whereas P. gingivalis alone produced substantial levels of odor and volatile sulfides, pre-incubation in the presence of S. salivarius resulted in significant increases in odor judge scores (p = 0.026 and p = 0.015 for judges 1 and 2, respectively, as compared with mucin incubation in the presence of P. gingivalis alone; Fig. 1A), and a concomitant non-significant (p = 0.065) increase in volatile sulfides (Fig. 1B). Furthermore, addition of a β-galactosidase inhibitor (p-APTG) to the S. salivarius-P. gingivalis combined incubation significantly lowered both odor judge scores (Fig. 1A) and volatile sulfide levels (Fig. 1B) (p = 0.006 and p = 0.02, respectively, as compared with S. salivarius + P. gingivalis + mucin mixture). In the presence of glucose, both malodor and volatile sulfide production were negligible.
Following the various incubations, mucin samples were analyzed by SDS-PAGE (Fig. 2 ). Incubation in the presence of P. gingivalis alone resulted in 40% mucin degradation (lane 3), whereas, following pre-incubation in the presence of S. salivarius, 90% of the mucin was degradated (lane 4), as compared with the mucin control. In the presence of β-galactosidase inhibitor p-APTG (lane 5), mucin degradation was reduced to 30%. In the presence of glucose (lane 6), only 12% mucin degradation was observed. As expected, degradation of mucin in the presence of S. salivarius alone was not significant (lane 2).
Pre-incubation of Mucin in the Presence of β-galactosidase When mucin was incubated in the presence of β-galactosidase alone, odor judge scores and volatile sulfide levels were similar to those of the mucin control (Figs. 3A, 3B ). Whereas P. gingivalis alone produced substantial levels of odor and volatile sulfides, pre-incubation in the presence of β-galactosidase resulted in a significant increase in volatile sulfide production (p = 0.009; Fig. 3B), as compared with mucin incubation in the presence of P. gingivalis alone, and a concomitant non-significant increase in odor judge scores (p = 0.132 and p = 0.065 for judges 1 and 2, respectively; Fig. 3A). The addition of p-APTG to the β-galactosidase-P. gingivalis combined incubation mixture significantly reduced both odor judge scores (p = 0.008 and p = 0.01 for judges 1 and 2, respectively; Fig. 3A) and volatile sulfide levels (p = 0.005; Fig. 3B, as compared with β-galactosidase + P. gingivalis + mucin mixture). In the presence of glucose, both malodor and volatile sulfide production were negligible.
Following the various incubations, mucin samples were analyzed by SDS-PAGE (Fig. 4 ). Mucin degradation of 40% was observed in the presence of P. gingivalis alone (lane 3), whereas, following pre-incubation in the presence of β-galactosidase, 60% of the mucin was degraded (lane 4), as compared with the mucin control. In the presence of p-APTG (lane 5), mucin degradation was reduced to 30%. In the presence of glucose (lane 6), only 15% mucin degradation was observed. A limited non-significant degradation of mucin in the presence of β-galactosidase alone was observed (lane 2).
Previously, we have shown that pre-incubation of filtered saliva in the presence of β-galactosidase leads to higher levels of subsequent malodor production by P. gingivalis, and that this process is mitigated by inhibitors of deglycosylation (Sterer and Rosenberg, 2002). Furthermore, a significant association between levels of β-galactosidase in saliva and oral malodor levels was found in a population of 64 subjects (Sterer et al., 2002). We proposed that (i) deglycosylation of oral glycoproteins is an important first step toward their subsequent degradation and malodor formation by Gram-negative bacteria; and (ii) this initial process may be carried out primarily by Gram-positive micro-organisms, followed by subsequent proteolysis and malodor elaboration by Gram-negative species. These initial studies, however, were carried out with saliva, which contains, in addition to glycoproteins, myriad potential nutrients, enzymes, and other components that might affect the results obtained. Analysis of the present data adds further support to the hypothesis, with the use of a better-defined model system that involves mucin, rather than whole saliva. Although porcine mucin differs slightly from salivary mucin, it has been commonly used as a commercially available substrate in oral microbiology studies, due to its considerable overall similarity to human mucins’ O-linked carbohydrate side-chains (Rafay et al., 1996). Results shown here demonstrate that pre-incubation of mucin alone with S. salivarius helps promote its subsequent putrefaction by P. gingivalis. To test the hypothesis that this is the result of deglycosylation, we also incubated the mucin in the presence of β-galactosidase alone. Here too, incubation in the presence of the enzyme itself promoted subsequent mucin putrefaction by P. gingivalis. Interestingly, some breakdown of mucin was observed in the presence of P. gingivalis alone. This may be due to glycosidic enzymes, such as β-N-acetylglucosaminidase, produced by this bacterium (Homer et al., 1992). Nevertheless, in the presence of S. salivarius, or β-galactosidase alone, both mucin degradation and malodor elaboration were significantly increased. These processes were inhibited by the addition of glucose, a putative glycosidic inhibitor (Rafay et al., 1996), as well as the specific β-galactosidase inhibitor, p-APTG. Since Gram-positive micro-organisms are highly adapted to the cleaving and utilizing of carbohydrate side-chains from salivary mucins (De Jong et al., 1984; De Jong and Van Der Hoeven, 1987), it is likely that these micro-organisms carry out this initial process in vivo. This premise is supported by the findings that streptococci, including S. salivarius, are predominant residents of the tongue dorsum (Gordon and Gibbons, 1966; Aas et al., 2005). We have previously postulated that post-nasal drip, rich in mucins, can accumulate on the tongue dorsum and constitute a major substrate for putrefaction and odor production by indigenous microbiota on the tongue (Rosenberg, 1996). Our results, taken together, show that Gram-negative and Gram-positive micro-organisms work together to degrade salivary glycoproteins and produce oral malodor.
This research was performed in the Alpha Omega Research Laboratories, Goldschleger School of Dental Medicine, Tel Aviv University, and in the Ronald E. Goldstein Center for Esthetic Dentistry and Dental Materials Research, Hebrew University. The research was funded by departmental research budgets. Received for publication October 20, 2005. Revision received June 18, 2006. Accepted for publication June 19, 2006.
Journal of Dental Research, Vol. 85, No. 10,
910-914 (2006)
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ABSTRACT
INTRODUCTION
-inch-diameter disposable plastic straw was inserted into the air inlet of the monitor. We measured the volatile sulfide levels at the test tube headspace by inserting the plastic straw 2 cm into each test tube, immediately after opening it, and recording the maximal reading (
0.05 was considered statistically significant. 
) and judge 2 (
) (with results presented on a semi-integer scale of 0–5), and (B) volatile sulfide levels measured with the use of a sulfide monitor (HalimeterTM), as ppb sulfide equivalents. * indicates high significance (p = 0.026 and p = 0.015 for judges 1 and 2, respectively, N = 6).
