Novel Isolation and Identification Methods for Veillonella Species Involved in Halitosis

Purpose: The purpose of the present study was to develop a novel selective medium (OVSM) and useful identification method for oral Veillonella species, to assess the prevalence of this species using the selective medium, and to investigate whether the monitoring of oral Veillonella species levels is useful as a clinical indicator for the diagnosis of halitosis. Methods: To examine bacterial populations in the oral cavity, a novel selective medium (OVSM) and multiplex PCR method were developed for isolating and identifying oral Veillonella species. OVSM consists of tryptic soy agar, sheep blood, sodium lactate, vancomycin, and oxacillin. Results: Oral Veillonella species grew well on OVSM. Oral Veillonella species accounted for 5.4% and 27.2%, respectively, of all bacteria in the tongue biofilm samples of healthy and halitosis subjects. Oral Veillonella species produced larger amounts of volatile sulfur compounds than other representative oral bacteria. Conclusion: These results indicate that monitoring the levels of these organisms is useful as a clinical indicator for the diagnosis of halitosis.

early colonizers during the formation of oral biofilms, along with Streptococcus species, which were identified as the initial colonizers in multispecies communities developing on oral biofilms [9]. Oral Veillonella species are associated with severe early childhood caries [10] and intraradicular infections, including abscesses [11], apical root canals [12], and dental tubules [13]. Oral Veillonella species were also predominant in the subgingival biofilm samples of patients with chronic periodontitis [14]. Previous findings showed that their distribution and frequency at the species level varied depending on the oral site and oral hygiene status [15] [16] [17] [18] [19]. Therefore, it is important to elucidate the role of oral Veillonella species in the formation of oral biofilms in order to improve the prevention and treatment of oral infectious diseases.
Halitosis is caused due by the metabolic products of bacteria in the oral cavity, particularly those on the dorsum of the tongue [20]. Some cases of halitosis have been associated with periodontitis [21] [22], and, thus, various periodontitis-related bacteria species have been detected in tongue coatings. These findings also suggest that the tongue coating plays a role as a reservoir of these bacteria [23]. Most of these bacteria have the ability to produce hydrogen sulfide (H 2 S), one of the major components of halitosis [24]. Washio et al. focused on halitosis in patients without oral diseases, such as periodontitis and caries, and found that the predominant H 2 S-producing bacteria were not periodontitis-related bacteria, but were mainly indigenous bacteria of the oral cavity, such as Veillonella and Actinomyces [25]. Moreover, Veillonella species, including V. atypica, V. dispar, and V. parvula, were predominant among these microorganisms.
Previous studies [5] [6] [26] [27] [28] that examined the prevalence of oral Veillonella species used the well-known selective medium for Veillonella species, Veillonella agar (VA) [29]. In a pilot study, we attempted to detect oral Veillonella species in oral samples using VA. However, it was impossible to detect some bacterial species among these organisms because VA inhibited their growth. There-fore, previous findings may not have precisely reflected the prevalence of oral Veillonella species. A novel suitable selective medium is needed to accurately assess the prevalence of oral Veillonella species involved in dental caries, intraradicular infections, and halitosis in the oral cavity.
Veillonella strains are relatively easy to identify at the genus level, but remain difficult to identify at the species level because there are no useful phenotypic or biochemical examinations to distinguish them [30]. A sequence analysis of several target genes is the most reliable method for identifying bacterial species; however, it is expensive, laborious, and time-consuming. Therefore, a simple and more reliable assay for identifying oral Veillonella species at the species level, such as a polymerase chain reaction (PCR) amplification method, is needed.
The purpose of the present study was to develop a novel selective medium for the isolation of oral Veillonella species, to assess the prevalence of these species in the oral cavity, and to investigate whether the monitoring of Veillonella species levels is useful as a clinical indicator for the diagnosis of halitosis. Furthermore, the ability of each oral Veillonella species to produce volatile sulfur compounds (VSCs) was investigated in vitro and quantitatively compared to other representative oral bacteria using sensor gas chromatography.

Bacterial Strains and Culture Conditions
All bacterial strains used in the present study are listed in Tables 1-4. Strains of anaerobic bacteria except for Veillonella species were maintained by cultivating them on Anaerobic Blood Agar (CDC) that consists of a Tryptic soy agar (Becton, Dickinson and Co., Sparks, MD, USA) supplemented with vitamin K 1 , hemin, L-cysteine, yeast extract, and sheep blood. Veillonella strains were maintained by cultivating them on tryptic soy agar blood (TS-LAC-Blood) composed of tryptic soy agar (TS) (Becton, Dickinson and Co., Sparks, MD, USA) supplemented with 2.1% sodium lactate and 5% sheep blood. These organisms were cultured at 37˚C for 48 h in an anaerobic jar with a gas pack system (Anaero-Pack  , Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan).

Evaluation of the Base Medium
TS, TS with 2.1% sodium lactate (TS-LAC), TS with 5% sheep blood (TS-blood), and TS-LAC-Blood were examined as the base medium in the selective medium. Ten-fold dilutions of cultures were made in 0.9 ml of Tris-HCl buffer (0.05 M, pH 7.2) and aliquots of 0.1 ml were spread onto the test media. The plates were cultured at 37˚C for 4 days under anaerobic conditions. After cultivation, the number of colony-forming units (CFU)/ml was counted. Open Journal of Stomatology

Susceptibility Tests
Preliminary studies on antibiotic selection were performed using disk susceptibility tests (Sensi-Disk, Becton Dickinson Co., MD, USA). The microbroth dilution method was employed for susceptibility testing [31].

Recovery of Oral Veillonella Species
The recoveries of the Veillonella reference strains and Veillonella isolates were calculated as CFU/ml on selective medium and compared with those on TS-LAC-Blood for total cultivable bacteria and on conventional selective medium (VA) for Veillonella species. All Veillonella strains used in the present study are listed in Table 1. VA contained 5 g of tryptone, 5 g of yeast extract, 0.75 g of sodium thioglycolate, 0.002 g of basic fuchsin, 2.1% sodium lactate, and 15 g of agar.
All bacterial strains were pre-incubated in TS (Becton, Dickinson and Co., Sparks, MD, USA) supplemented with 2.1% sodium lactate at 37˚C overnight under anaerobic conditions. Ten-fold dilutions of cultures were made in 0.9 ml of Tris-HCl buffer (0.05 M, pH 7.2) and aliquots of 0.1 ml were spread onto the test media. The plates were cultured at 37˚C for 4 days under anaerobic conditions. After cultivation, the number of CFU/ml was counted.

Clinical Samples
One hundred volunteers (43 men, 57 women; mean age 34 years, range 19 -61 years) participated in the present study. They had no systemic disease and

Identification of Oral Veillonella Species Isolated from Clinical Samples
Twenty-four out of the approximately 50 colonies that grew on a novel selective medium for oral Veillonella species per subject were randomly isolated and subcultured, and their identity was then confirmed by a PCR analysis.

Identification of Bacteria Species Isolated from Clinical Samples
Bacterial species, i.e., Streptococcus, Fusobacterium, oral Veillonella, Porphyromonas, and Prevotella species, except for oral Veillonella species on each selective medium, were identified by colony morphology and Gram staining.

Development of a Multiplex PCR Method for Identifying Oral Veillonella Species Using Designed Primers
Bacterial cells were cultured in TS supplemented with 2.1% sodium lactate for 24 h, and 1 ml of samples was then collected in microcentrifuge tubes and resuspended at a density of 1.0 McFarland standard (approximately 10 7 CFU/ml) in 1 ml of sterile distilled water. A total of 3.6 μl of the suspension was then used as the PCR template. The detection limit of PCR was assessed by serially diluting known numbers of bacterial cells in sterile distilled water and then subjecting each suspension to PCR. The multiplex PCR mixture contained 0.2 μM of each primer, 10 μl of 2 × MightyAmp Buffer Ver.3 (Takara Bio Inc., Shiga, Japan), 0.4 μl of MightyAmp DNA Polymerase (Takara), and 5 μl of the template in a final volume of 20 μl. PCR reactions were performed in a DNA thermal cycler (Applied Biosystems 2720 Thermal Cycler; Applied Biosystems, CA, USA). PCR conditions included an initial denaturation step at 98˚C for 2 min, followed by 30 cycles consisting of 98˚C for 30 s, 63˚C for 30 s, and 72˚C for 30 s. PCR products were analyzed by 2.0% agarose gel electrophoresis before being visualized by electrophoresis in 1 × Tris-borate-EDTA on a 2% agarose gel stained with ethidium bromide. A 100-bp DNA ladder (Takara Biomed, Shiga, Japan) was used as a molecular size marker. All experiments were performed in triplicate.

Assessment of VSC-Producing Abilities
The VSC-producing abilities of oral Veillonella species and other representative oral bacteria in vitro were assessed as previously reported [32]. In the present study, VSC levels were measured using a sensor gas chromatograph (ODSA-P3-A, Yamato Scientific Co., Ltd., Tokyo, Japan). All bacterial strains were pre-incubated as described above. Each bacteria culture solution was inoculated into 19 × 55-mm sample vials (1880 SV10; AGC Techno Glass Co., Ltd., Shizuoka, Japan) containing TS supplemented with vitamin K 1 (10 μg/ml), hemin (5 μg/ml), 0.5% yeast extract, L-cysteine (25 mM), and L-methionine (25 mM). All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). After the inoculation, sample vials were loosely sealed with silicon caps and cultured at 37˚C under anaerobic conditions until a density of 3.0 McFarland Standard. After the incubation, sample vials were immediately sealed tightly with silicon caps. One milliliter of each sample vial headspace air was aspirated by puncturing a silicon cap with the needle of the gastight syringe (Termosyringe  SS-02SZ2232), and immediately injected into the sensor gas chromatograph. The VSC levels of the sample vial headspace were measured using the sensor gas chromatograph (ODSA-P3-A).

Statistical Analysis
The number of total bacteria and oral Veillonella species and the proportions of the anaerobes, Streptococcus species, Fusobacterium species, oral Veillonella species, and Porphyromonas and Prevotella species in samples of tongue biofilms from healthy subjects without halitosis and halitosis subjects were compared using the Mann-Whitney U test. P values < 0.05 were considered to be significant.

Selection of the Base Medium
A base medium was selected for the growth of oral Veillonella species. All of the oral Veillonella species grew well on TS-LAC-Blood. The growth of V. atypica, V. denticariosi, V. parvula, and V. dispar was enhanced by supplementing with 2.1% sodium lactate, whereas that of V. tobetsuensis and V. rogosae was markedly inhibited on TS and TS-LAC free from sheep blood (data not shown). Therefore, TS-LAC-Blood was ultimately selected as the base medium.

Susceptibility to Antibiotics
Oral Veillonella species were more resistant to vancomycin than oral Gram-positive bacteria, such as Streptococcus, Actinomyces, and Corynebacterium species. The minimal inhibitory concentration (MIC) of vancomycin for oral Veillonella species was 60 μg/ml. Oral Gram-positive bacteria were sensitive to 16 μg/ml of vancomycin. Oral Veillonella species were more resistant to oxacillin than oral Gram-negative rods, such as Fusobacterium, Porphyromonas, and Prevotella species. The MIC of oxacillin for oral Veillonella species was 8 μg/ml. Oral Gram-negative bacteria were sensitive to 1 μg/ml of oxacillin. Neisseria species, which are Gram-negative cocci similar to Veillonella species, were also resistant to vancomycin and oxacillin, but were inhibited in an aerobic culture.

Composition of the New Selective Medium
The new selective medium, designated Oral Veillonella selective medium (OVSM), comprised the following (per liter): 40 g of tryptic soy agar, 21 ml of Open Journal of Stomatology sodium lactate, 50 ml of sheep blood, 16 mg of vancomycin, and 1 mg of oxacillin. Sheep blood and antibiotics, i.e., vancomycin and oxacillin, were added after the base medium had been sterilized and cooled to 50˚C.

Primer Design
Twelve specific primers covering the upstream region of the rpoB, gltA, and dnaK gene sequences of six oral Veillonella species were designed in the present study (Figures 1-3

Detection Limit
Our multiplex PCR method for identifying and detecting six oral Veillonella species, i.e., V. atypica, V. tobetsuensis, V. rogosae, V. parvula, V. dispar, and V. denticariosi successfully amplified DNA fragments of the expected size for each species (Figure 4). The detection limit was assessed in the presence of titrated bacterial cells, and the sensitivity of the PCR assay was between 5 × 1 and 5 × 10     CFU per PCR template (5.0 μl) for the V. atypica-specific primer set with strain DSM 20739, the V. tobetsuensis-specific primer set with strain JCM 17876, the V. tobetsuensis-specific primer set with strain JCM 17876, the V. rogosae-specific primer set with strain JCM 15642, the V. parvula-specific primer set with strain JCM 12972, the V. dispar-specific primer set with strain DSM 20735, and the V. denticariosi-specific primer set with strain JCM 15641 (data not shown).

Assay of Representative Oral Bacteria
Some Streptococci, Actinomyces, Neisseria, and Corynebacterium species were used as representative oral bacteria in PCR using the designed primer set. No amplicons were produced from any of the representative oral bacteria ( Figure   4). Table 1 shows the recovery of oral Veillonella species on VA and OVSM relative to TS-LAC-Blood. The average growth recoveries of the oral Veillonella species reference strains and isolates on VA and OVSM were 14.5 (range: 0 -29.7) and

Recovery of Oral Veillonella Species and Inhibition of Other Representative Oral Bacteria on Selective Medium
98.4% (range: 93.3 -99.9), respectively. The growths of V. tobetsuensis and V. denticariosi were markedly inhibited on VA.

Clinical Examination
The detection frequencies of oral Veillonella species in tongue biofilm samples from healthy subjects without halitosis (n = 50) and halitosis subjects (n = 50) are shown in Table 2. The total VSC levels of healthy and halitosis subjects were 78.2 and 812.6 ppb, respectively. The mean numbers of total bacteria in healthy Open Journal of Stomatology and halitosis subjects were 1.43 × 10 7 CFU/ml and 5.21 × 10 7 CFU/ml, respectively. Oral Veillonella species were detected in all samples from healthy and halitosis subjects. The mean numbers of oral Veillonella species in healthy and halitosis subjects were 3.97 × 10 6 CFU/ml (range: 7.0 × 10 5 -1.3 × 10 7 ) and 2.58 × 10 7 CFU/ml (range: 4.7 × 10 6 -5.4 × 10 7 ), respectively. The mean numbers of total bacteria and oral Veillonella species in tongue biofilm samples were significantly higher in halitosis subjects than in healthy subjects (P < 0.01). Figure 5 shows a comparison of the bacteria flora in tongue biofilms between the 2 groups. The proportions of anaerobe and Veillonella species were significantly higher in halitosis subjects than in healthy subjects (P < 0.05). Increases in anaerobe species in the tongue biofilm samples of halitosis subjects were attributed to elevations in oral Veillonella species, which are anaerobic Gram-negative cocci. No significant differences were observed in the proportions of Fusobacterium, Porphyromonas, and Prevotella species between the 2 groups. Comparisons of oral Veillonella species in tongue biofilms between the 2 groups are shown in Table 3. The average CFU of each oral Veillonella species was higher in halitosis subjects than in healthy subjects. V. rogosae was predominant and detected in all subjects, followed by V. tobetsuensis, V. atypica, and V. dispar. V. denticariosi and V. parvula were rarely detected in both groups. The threshold level for genuine halitosis was defined as >200 ppb total VSCs in mouth air. *Mann-Whitney U test; p < 0.01.   In the first isolation, colonies of oral Veillonella species on OVSM commonly had a smooth and circular appearance. The colony color and average colony size of oral Veillonella species on OVSM were translucent light white and 1.0 mm in diameter, respectively ( Figure 6).

VSC-Producing Abilities of Oral Veillonella Species and Other Representative Oral Bacteria in Vitro
Comparisons of VSC-producing abilities among six oral Veillonella species and other representative oral bacteria are shown in Table 4. F. nucleatum produced the highest VSC level, followed by F. nucleatum, P. gingivalis, P. nigrescens, P. intermedia, and six oral Veillonella species.

Discussion
Halitosis is a relatively common condition in which an individual either has bad breath or perceives oneself to have an offensive mouth odor. Although it has multifactorial origins, bad breath originates from the oral cavity in most cases [33]. The causes of bad breath, except for an oral origin, include nasal and pharyngeal infections, respiratory conditions, gastrointestinal issues, metabolic conditions, such as diabetes, and liver diseases [33]. Halitosis originating from the oral cavity is caused by products generated through bacterial metabolic degradation. In individuals with a healthy oral cavity, an important site for the accumulation of bacteria associated with halitosis is the dorsum of the tongue, particu-larly its posterior portion, which provides a suitable environment for the growth of anaerobes because a low oxygen concentration is present in the deep crypts of the tongue [33]. Poor oral hygiene that results in the accumulation of bacterial biofilms in subgingival areas and that may lead to periodontal disease has also been associated with halitosis [34]. VSCs, which include H 2 S, CH 3 SH, and (CH 3 ) 2 S, are considered to be the primary compounds responsible for halitosis originating from the oral cavity, although amines (cadaverine, putrescine, and indole) and short-chain fatty acids (isobutyric and isovaleric acids) also contribute to oral malodor. VSCs are generated through enzymatic modifications to sulfur-containing amino acids (cysteine and methionine), which are made available following the proteolytic degradation of proteins or glycoproteins [33]. Bacteria that have been classically associated with halitosis include oral Veillonella species, F. nucleatum, P. intermedia, P. gingivalis, and Treponema denticola [25] [33]. Most of these bacteria have the ability to produce H 2 S, one of the major components of halitosis. Washio et al. [25] focused on oral malodor in patients without oral diseases, such as periodontitis and dental caries, and reported that the predominant H 2 S-producing bacteria were not periodontitis-related bacteria, but were mainly indigenous bacteria of the oral cavity, such as the genus Veillonella.
VA is used worldwide as the selective medium for Veillonella species. In our pilot study, VA inhibited the growth of some Veillonella species. The findings obtained also indicated that VA was not appropriate for investigating the prevalence of oral Veillonella species. Therefore, a novel suitable selective medium is needed to accurately assess the prevalence of oral Veillonella species involved in severe early childhood caries and intraradicular infections as well as halitosis in the oral cavity. In the present study, oral Veillonella species grew better on TS-LAC-Blood than on media that did not contain lactic acid and sheep blood.
Veillonella species utilize lactic acid as carbon and energy sources instead of carbohydrates [3] [4]. Lactic acid and animal blood have been identified as growth factors that promote the growth of these organisms.
Oral Veillonella species were more resistant to vancomycin and oxacillin than other representative oral bacteria. The growth of the representative oral bacterial strains used in the present study was inhibited by the addition of 16 mg/L vancomycin and 1 mg/L oxacillin to TS-LAC-Blood. All of the oral Veillonella reference strains and isolates tested grew well on the new selective medium, designated as OVSM, while the growth of other bacteria was markedly inhibited (data not shown). Moreover, OVSM allowed the identification of oral Veillonella species based on their characteristic colony morphologies.
Moreover, characteristic colony morphologies on OVSM allowed oral Veillonella species to be easily distinguished.
Veillonella strains are relatively easy to identify at the genus level, but remain difficult to identify at the species level because there are currently no useful phenotypic or biochemical examinations to distinguish them [19]. To overcome this issue, Mashima et al. established a novel one-step PCR method with spe-cies-specific primer sets based on the variable region of the rpoB gene sequences of oral Veillonella species [27]. However, the findings of our pilot study showed that the size of each PCR fragment by this method was similar; therefore, it was difficult to accurately identify each oral Veillonella species. Moreover, including DNA extraction, it took more than 5 hours to finish the identification. Therefore, a reliable identification method is needed to accurately assess the prevalence of oral Veillonella species.
In the present study, we designed species-specific primers with the already mentioned methods for the identification and detection of six Veillonella species using a PCR-based method. These primers were able to distinguish each Veillonella species and did not display cross-reactivity with representative oral bacte-  [28] investigated the prevalence of oral Veillonella species using VA. In the present study using OVSM, oral Veillonella species were detected in all samples from healthy and halitosis subjects. This result demonstrated that oral Veillonella species are one of the main members of the human oral microbiome, which is consistent with previous findings [5] [6]. In the present study, V. rogosae was predominant and was detected in the samples of all subjects, followed by V. tobetsuensis, V. atypica, and V. dispar. Beighton et al. [26], Mashima et al. [27], and Djais et al. [28] also reported that V. rogosae, V. atypica, and V. dispar were the predominant Veillonella species in the human oral cavity, which is consistent with the present results. However, previous studies using VA reported that V. tobetsuensis was rarely detected in the human oral cavity [27] [28]. On the other hand, V. tobetsuensis was the second predominant Veillonella species in the present study using the novel selective medium, OVSM. Since VA inhibits the growth of V. tobetsuensis, previous findings may not have accurately reflected the prevalence of oral Veillonella species. In the present study, V. denticariosi and V. parvula were rarely detected in both groups, as previously reported [26] [27] [28]. Therefore, V. denticariosi and V. parvula do not appear to be a part of the normal microbiome in human tongue biofilms.
The mean numbers of oral Veillonella species in tongue biofilm samples were significantly higher in halitosis subjects than in healthy subjects (P < 0.01). The proportions of Veillonella species were significantly higher in halitosis subjects than in healthy subjects (P < 0.05), compared with those of Fusobacterium, Porphyromonas, and Prevotella species. In addition, six oral Veillonella species produced high VSC levels, particularly H 2 S, in vitro. Higher production levels of VSCs were observed in the presence of cysteine, which is transformed into H 2 S, ammonia, and pyruvate by cysteine desulfhydrase [35]. Accordingly, the monitoring of oral Veillonella species levels may be useful as a clinical indicator for the diagnosis of genuine halitosis.

Conclusion
We developed a selective medium, designated OVSM, to isolate oral Veillonella species in the oral cavity of humans. Since OVSM is highly selective for oral Veillonella species, it will be useful for assessing the distribution and role of these organisms at various locations in humans. A novel selective medium (OVSM) and our multiplex PCR method as isolation and identification methods, respectively, for oral Veillonella species may contribute to the diagnosis of halitosis as well as severe early childhood caries and intraradicular infections.

Authors' Contribution
Ono Y., Fukatsu A. and Tsuzukibashi O. corrected the data. Ono Y. and Tsuzukibashi O. drafted and wrote the manuscript. The concept of this manuscript was devised by Ono Y. All authors read and approved the final manuscript.