Inhibitory Effect of Enterococcus faecium WB2000 on Volatile Sulfur Compound Production by Porphyromonas gingivalis

Volatile sulfur compounds (VSCs) produced by oral anaerobes are the major compounds responsible for oral malodor. Enterococcus faecium WB2000 is recognized as an antiplaque probiotic bacterium. In this study, the effect of E. faecium WB2000 on VSC production by Porphyromonas gingivalis was evaluated, and the mechanism of inhibition of oral malodor was investigated. P. gingivalis ATCC 33277 was cultured in the presence of four lactic acid bacteria, including E. faecium WB2000. Subsequently, P. gingivalis ATCC 33277, W50, W83, and two clinical isolates were cultured in the presence or absence of E. faecium WB2000, and the emission of VSCs from spent culture medium was measured by gas chromatography. The number of P. gingivalis ATCC 33277 in mixed culture with E. faecium WB2000 decreased at 6 h, and the rate of decrease was higher than that in mixed cultures with the other lactic acid bacteria. The numbers of five P. gingivalis strains decreased at similar rates in mixed culture with E. faecium WB2000. The concentration of methyl mercaptan was lower in spent culture medium from P. gingivalis and E. faecium WB2000 cultures compared with that from P. gingivalis alone. Therefore, E. faecium WB2000 may reduce oral malodor by inhibiting the growth of P. gingivalis and neutralizing methyl mercaptan.


Introduction
Oral malodor is caused mainly by the metabolism of sulfur amino acids by anaerobic bacteria inhabiting the oral cavity [1]. The main compounds responsible for oral malodor are volatile sulfur compounds (VSCs), such as hydrogen sulfide (H 2 S), methyl mercaptan (CH 3 SH), and dimethyl sulfide; these compounds are produced by some periodontopathic bacteria. Indeed, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Prevotella intermedia generate considerable amounts of H 2 S and CH 3 SH [2].
Probiotic bacteria, defined as live microorganisms that benefit the health of the host when administered in adequate amounts (FAO/WHO 2001), are thought to play a role in the maintenance of oral health [3]. Enterococci are facultatively anaerobic, Gram-positive cocci that form a part of the normal flora of the gastrointestinal tract of animals and humans.
They are also frequently found in fermented food, such as cheese and meat [4]. Enterococcus faecium and Enterococcus faecalis are the most clinically relevant members of the genus Enterococcus. Traditionally, they are regarded as lowgrade pathogens but have emerged as important causes of nosocomial infections [5]. Clinical use of E. faecium and E. faecalis during food fermentation and as probiotics requires a careful safety evaluation [6].
E. faecium strains have been reported to inhibit biofilm formation by cariogenic bacteria in vitro [7,8]. In addition, a previous double-blind randomized trial in which the subjects cleaned their teeth using a dentifrice containing E. faecium WB2000 or placebo for 4 weeks revealed improvements in salivary flow, salivary buffering capacity, and plaque accumulation [9]. This study aimed to investigate the effect of E. faecium WB2000 on VSC production by P. gingivalis and the mechanism of inhibition of oral malodor by E. faecium WB2000.

Bacterial Strains and Culture
Conditions. The bacterial strains used in the study are listed in Table 1. Enterococcus faecium WB2000, previously classified as Streptococcus faecalis [10], was provided by Wakamoto Pharmaceutical. The selective medium for P. gingivalis consisted of Brucella agar (Becton Dickinson, Le Pont de Claix, France) supplemented with 5% horse blood, hemin (5 g/mL), vitamin K (1 g/mL), and gentamicin (50 g/mL). Lactic acid bacteria were cultivated on BL agar (Nissui, Tokyo, Japan). Bacterial strains were cultivated at 37 ∘ C anaerobically for 40 h and suspended in sterile physiological saline to an optical density at 560 nm (OD 560 ) of 1.0 for P. gingivalis and an OD 560 of 0.02 for lactic acid bacteria.

Cocultivation of P. gingivalis and Lactic Acid Bacteria.
Bacterial cocultivation was carried out using 100 L of P. gingivalis suspension, 100 L of lactic acid bacterial suspension, and 10 mL fresh GAM broth (Nissui, Tokyo, Japan) supplemented with 0.7% glucose, hemin (5 g/mL), and vitamin K (1 g/mL) at 37 ∘ C anaerobically. Viable bacterial counts in the culture medium were determined at 6, 12, 24, and 48 h on the appropriate agar medium at 37 ∘ C anaerobically for 48 h.

Measurement of Volatile Sulfur
Compounds. The VSC concentration in spent medium from P. gingivalis cultured in the presence or absence of E. faecium WB2000 was measured after 24 and 48 h. Aliquots (0.2 mL) of spent culture medium were added to 5 mL conical tubes, which were sealed and incubated at room temperature for 5 min. Then, 0.5 mL of the gas phase was collected and measured by gas chromatography (model GC2014, Shimadzu Works, Kyoto, Japan).

Effect of Lactic Acid
Bacteria on the Growth of P. gingivalis ATCC 33277. The number of viable P. gingivalis ATCC 33277 decreased to less than the detection limit after 6 h in mixed culture with E. faecium WB2000 (Figure 1(a)). In mixed cultures with the other three lactic acid bacteria (S. salivarius JCM 5707, L. salivarius CIP 103140, and L. reuteri JCM 1112), the number of viable P. gingivalis ATCC 33277 decreased to less than the detection limit at 12 h. In mixed culture with P. gingivalis ATCC 33277, E. faecium WB2000 grew more rapidly than it did in mixed cultures with the other three lactic acid bacteria, and its growth plateaued at 6 h ( Figure 1(b)).

Effect of E. faecium WB2000 on the Growth of Various P. gingivalis Strains.
The effect of E. faecium WB2000 on the growth of five P. gingivalis strains (ATCC 33277, ATCC 53978, ATCC BAA-308, 2-1, and 7-1) was evaluated. The numbers of all P. gingivalis strains decreased to less than the detection limit at 24 and 48 h in mixed cultures with E. faecium WB2000 (Figure 2). In contrast, the number of E. faecium WB2000 reached a plateau at 24 h.

Effect of E. faecium WB2000 on VSC Production by P. gingivalis Strains.
The concentrations of VSCs in spent culture medium were measured by gas chromatography ( Table 2). The levels of H 2 S produced by P. gingivalis strains were lower than those of CH 3 SH. The concentrations of H 2 S in mixed culture media were higher than that in medium in which only P. gingivalis was cultured, with the exception of medium from mixed cultures of two P. gingivalis clinical isolates (2-1 and 7-1) and E. faecium WB2000 after 48 h. In contrast, the CH 3 SH concentration in mixed culture medium was lower than that in medium from culture of P. gingivalis alone, with the exception of medium from mixed cultures of two P. gingivalis strains (W50 and 2-1) and E. faecium WB2000 after 24 h. The levels of CH 3 SH in spent medium from P. gingivalis cultured for 48 h in the presence or absence of E. faecium WB2000 are shown in Figure 3. Although CH 3 SH production by P. gingivalis was strain dependent, the CH 3 SH concentration was markedly lower in spent medium from mixed cultures of all P. gingivalis strains and E. faecium WB2000 than in medium from culture of the latter microorganism only.

Discussion
The hypothetical mechanisms of probiotic action in the oral cavity are (1) involvement in binding of oral microorganisms to proteins, (2) effects on plaque formation and its complex ecosystem by competing and interfering with interbacterial attachment, (3) involvement in the metabolism of substrates, and (4) production of compounds that inhibit oral bacteria [11]. E. faecium has been reported to inhibit biofilm formation by cariogenic bacteria [7,8]. Kumada    a protein that inhibited biofilm formation by streptococci. In addition, our previous study suggested that E. faecium inhibits the growth of some mutans streptococci [7]. In this study, E. faecium WB2000 inhibited the growth of, as well as reduced CH 3 SH production by, P. gingivalis. A study on L. salivarius TI 2711 reported that a low pH (≤6.0) and the presence of lactic acid (40-50 mmol/L) induced P. gingivalis death [12]. The pH of E. faecium WB2000 culture medium was 4.4 after 24 h of incubation (data not shown). The growth rate of E. faecium WB2000 was higher than that of the other lactic acid bacteria, suggesting that this organism rapidly inhibited the growth of P. gingivalis. P. gingivalis strains produced CH 3 SH and a low level of H 2 S, as reported previously [13]. E. faecium WB2000 suppressed CH 3 SH production by P. gingivalis but did not inhibit production of H 2 S in the current study. Some probiotic bacteria produce VSCs in the presence of cysteine or methionine [14]. The GAM broth used in the current study contains cysteine, and thus E. faecium WB2000 might have produced H 2 S. Streptococcus thermophilus inhibited the growth and H 2 S, CH 3 SH, and CH 3 SCH 3 production of P. gingivalis [13]. To determine whether E. faecium WB2000 specifically inhibits production of CH 3 SH, different media and culture conditions should be used. Furthermore, future studies are needed to evaluate other P. gingivalis strains producing high levels of H 2 S.
In addition to E. faecium and S. thermophilus, other organisms have been reported to inhibit oral malodor. Streptococcus salivarius K12 suppressed the growth of Solobacterium moorei, which produces H 2 S and is involved in halitosis [15,16]. Lactobacillus salivarius WB21 and L. reuteri have been reported to inhibit oral malodor in clinical trials [17][18][19]. L. salivarius WB21 reduced the number of Fusobacterium nucleatum and ubiquitous bacteria in saliva [19].
E. faecium WB2000 has been used in traditional Japanese medicine (Strong Wakamoto5) to treat gastrointestinal discomfort, and its effect on dry eye was reported recently [20]. Moreover, the effect of dentifrice containing E. faecium WB2000 on plaque control has been investigated [9]. The findings of this study suggest that E. faecium WB2000 may reduce oral malodor by inhibiting the growth of P. gingivalis and neutralizing CH 3 SH. The effect of E. faecium WB2000 on oral malodor will be investigated in a future clinical trial involving the use of dentifrice.