Identification and phylogenetic analysis of oral <i>Veillonella </i>species isolated from the saliva of Japanese children

Ariadna A. Djais; Citra Fragrantia Theodorea; Izumi Mashima; Maiko Otomo; Masato Saitoh; Futoshi Nakazawa

doi:10.12688/f1000research.18506.5

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Research Article

Revised

Identification and phylogenetic analysis of oral Veillonella species isolated from the saliva of Japanese children

[version 5; peer review: 2 approved]

Ariadna A. Djais ¹, Citra Fragrantia Theodorea^1,2, Izumi Mashima³, Maiko Otomo⁴, Masato Saitoh⁴, Futoshi Nakazawa²

Ariadna A. Djais ¹, Citra Fragrantia Theodorea^1,2, [...] Izumi Mashima³, Maiko Otomo⁴, Masato Saitoh⁴, Futoshi Nakazawa²

PUBLISHED 13 Sep 2019

Author details Author details

¹ Department of Oral Biology, Faculty of Dentistry, Faculty of Dentistry, Universitas Indonesia, Jakarta 10430, Indonesia, Jakarta, 10430, Indonesia
² Department of Oral Microbiology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido, 061-0293, Japan
³ Department of Microbiology, School of Pharmacy, Aichi Gakuin University, Kusumoto-cho, Nagoya, 470-0915, Japan
⁴ Department of Pediatric Dentistry, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido, 061-0293, Japan

Ariadna A. Djais
Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Writing – Original Draft Preparation

Citra Fragrantia Theodorea
Roles: Investigation, Methodology, Supervision, Writing – Review & Editing

Izumi Mashima
Roles: Conceptualization, Investigation, Methodology, Project Administration, Supervision, Validation

Maiko Otomo
Roles: Resources, Visualization

Masato Saitoh
Roles: Resources, Visualization

Futoshi Nakazawa
Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Project Administration, Supervision, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background: As the most frequent infectious disease among children worldwide, dental caries have a strong relationship with oral hygiene status, specifically in the development of infection. However, the study regarding the identification and distribution of oral Veillonella are limited. The oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status. Here, we studied the oral hygiene status by examining the composition and proportion of oral Veillonella species in saliva of Japanese children.
Methods: Microbial samples collected from 15 Japanese children divided into three oral hygiene groups were cultured under anaerobic conditions after homogenization and dilution, and inoculated onto brain heart infusion and selective medium Veillonella agar. Genomic DNA was extracted from each isolate. Veillonella species were detected by one-step PCR using rpoB species-specific primers. To analyse the phylogenetic properties of the unknown Veillonella strains, PCR amplification and sequence analysis of rpoB were conducted for 10 representative strains.
Results: Although V. rogosae was found as the predominant species among all groups, its prevalence was significantly lower in the children with poor oral hygiene than in those with good oral hygiene. V. parvula was the prevalent species in the poor oral hygiene group. Approximately 10% of the isolated Veillonella strains were not classified to any established species; the phylogenetic analysis showed that they were most closely related to V. infantium
Conclusions: This study demonstrates that the composition and proportion of oral Veillonella species in the saliva of Japanese children is correlated with different oral hygiene status. Changes in detection ratios of V. parvula and V. rogosae can be useful indicators of oral hygiene status. Furthermore, new strains closely related to V. infantium were isolated from the saliva of Japanese children.

Keywords

Oral Veillonella species, dental caries, oral hygiene status, indicator, phylogenetic, saliva, children, Japan

Corresponding author: Ariadna A. Djais

Competing interests: No competing interests were disclosed.

Grant information: This study was supported in part by grants-in aid from the Japan Society for the promotion of Science Fellows (15J30007), Scientific Research from KAKENHI (26462793), 2016-2017 Research Project of Institute of Personalized Health Sciences, Health Sciences University of Hokkaido, 2016-2017 research grant from the Institute for Fermentation, Osaka, Japan, NKB-0246/UN2.R3.1/HKP.05.00/2019 and NKB-1936/UN2.R3.1/HKP.05.00/2019 Universitas Indonesia, Jakarta, Indonesia.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2019 Djais AA et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Djais AA, Theodorea CF, Mashima I et al. Identification and phylogenetic analysis of oral Veillonella species isolated from the saliva of Japanese children [version 5; peer review: 2 approved]. F1000Research 2019, 8:616 (https://doi.org/10.12688/f1000research.18506.5) First published: 03 May 2019, 8:616 (https://doi.org/10.12688/f1000research.18506.1) Latest published: 13 Sep 2019, 8:616 (https://doi.org/10.12688/f1000research.18506.5)

Revised Amendments from Version 4

Addition Grant number Information. It was necessary to our university.

See the authors' detailed response to the review by Juni Handajani
See the authors' detailed response to the review by Takuichi Sato

Introduction

The oral biofilm comprises multiple bacterial species and develops as a result of adhesion of pioneer bacterial species to adsorption of salivary proteins and glycoproteins on the enamel surface. These biofilms are not formed by random simultaneous colonization, but rather by selective, reproducible, and sequential colonization^1,2. Oral biofilms are a source of bacteria that cause oral infections, for instance dental caries and periodontal disease, and they sometimes lead to or worsen systemic diseases³.

Saliva is an acknowledged pool of biological markers that range from biochemical molecules changes such as DNA, RNA, and proteins, to those in microbiota structural composition⁴. Furthermore, saliva has an important role in oral biofilm development and maintenance. Recently, metagenomic analysis from saliva samples of Thai children demonstrated that Streptococcus and Veillonella were the predominant bacterial genera in the samples, and the proportion of Streptococcus decreased, while that of Veillonella increased in the children with poor oral hygiene status⁵.

The genus Veillonella consist of multiple gram-negative bacterial species, obligate anaerobic, non-motile, non-spore forming, small cocci belonging to the family Veillonellaceae⁶.

No Veillonella species ferment carbohydrates or amino acids, except for V. criceti, V. ratti, and V. seminalis. The metabolic end products of Veillonella species from trypticase-glucose-yeast extract are mainly acetic acid and propionic acid⁶. Veillonella species are present as commensal organisms in the oral cavity, intestinal tract and genitouritary and respiratory systems of humans and animals. Previous studies have reported that Veillonella species are rare causative organisms of meningitis, endocarditis, bacteraemia, discitis, vertebral osteomyelitis, and prosthetic joint infection^7–9. Generally, Veillonella species are known to be resistant to tetracycline and sensitive to penicillin and ampicillin. However, some Veillonella strains resistant to both penicillin and ampicillin have recently emerged¹⁰.

There are 14 species reported to belong to genus Veillonella including V. infantium which was assign as a novel species in 2018¹¹. Of the 14 documented species, V. atypica, V. denticariosi, V. parvula, V. rogosae, V. dispar, V. infantium, and V. tobetsuensis have been found in human saliva or on tongue or dental biofilms^12–17. Periasamy and Kolenbrander reported that oral Veillonella species are an early colonizer during the formation of oral biofilm, along with Streptococcus species, which were reported as initial colonizers in developing multispecies communities of oral biofilm¹⁸. Therefore, it is important to determine the role of oral Veillonella species in formation of oral biofilm to improve the prevention and treatment of oral infectious diseases.

Veillonella strains are relatively easy to identify at the genus level, but remain difficult to identify at the species level, since there are no useful phenotypic or biochemical examinations to distinguish them¹⁹. To resolve this problem, Mashima et al. established a novel one-step PCR method with species-specific primer sets based on the variable region of the rpoB gene sequences of oral Veillonella species¹². Additionally, 1,442 Veillonella strains isolated from the saliva of 107 Thai children were identified by this method as V. dispar, V. parvula, V. rogosae, V. atypica, V. denticariosi, and V. tobetsuensis in our previous study²⁰. In that study, V. parvula was significantly more prevalent in the poor oral hygiene, and the detection rate of oral Veillonella species in the saliva was indicative of the oral hygiene status of Thai children²⁰. Additionally, another study suggested that several novel Veillonella species may inhabit the human oral cavity²¹. However, the study regarding the identification and distribution of oral Veillonella are limited. The oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status.

Therefore, in this study, we examined composition and proportion of oral Veillonella species in saliva of Japanese children with different oral hygiene status.

Furthermore, we determined the phylogenetic position of the unknown Veillonella strains evaluated by the genus-specific PCR primer set as members of the genus Veillonella with a phylogenetic tree.

Methods

Subjects

The 15 children selected to take part in the study were 6 boys and 9 girls, aged 4 to 14 years old. Participants were recruited in-person during appointments at the Dental Hospital, Health Sciences University of Hokkaido. The subjects who had a history of immunosuppression or systemic diseases (e.g. leukemia, hepatitis), or any conditions requiring antibiotic monitoring or treatment procedures (e.g. heart conditions, bone fractures), or those with mucosal lesions, previous chemotherapy, radiation therapy, or medications that can reduce the salivary flow, and those that underwent treatment with antimicrobials within the previous three months were excluded from this study.

Subjects of this study were divided into three groups based on their evaluation by the Simplified Oral Hygiene Index (OHI-s) into good, moderate, and poor oral hygiene groups, according to the criteria of Greene and Vermillion²². Owing to the small number of children with poor hygiene (n=5), it was decided that 5 children would be chosen for each group. The good oral hygiene group (OHI-S score: 0–1.2) was composed of two males and three females. The moderate group (OHI-S score: 1.3–3.0) was composed of 3 males and 2 females. The poor group (OHI-S score: 3.1–6.0) was composed of 1 male and 4 females.

Sample collection

The saliva samples were collected at the Dental Hospital, Health Sciences University of Hokkaido, Japan, over a period between 2016 and 2017. Saliva was stimulated by paraffin chewing for 1 min and was then collected into sterile plastic tubes, and transferred to an anaerobic box (Hirasawa Works, Inc., Osaka, Japan) containing 10% H₂, 85% N₂, 5% CO₂. These samples (1 ml each) were transferred to 1.5-ml Eppendorf tubes, then homogenized for 1 min with a BioMasher ^®II (Nippi, Incoporated Protein Engineering Office, Tokyo, Japan).

Culture conditions

These homogenized saliva samples were serially diluted by 10-fold with sterile phosphate buffer saline (PBS) from 10^-3 to 10^-7. Aliquots (100 µl) of each diluted sample were inoculated into Bacto^TM Brain Heart Infusion (BHI, Difco Laboratories, Detroit, MI, USA) supplemented with 5% (volume/volume) defibrinated sheep blood (BHI agar), hemin (10 μg/mL, Wako, Osaka, Japan), menadione (5 µg/ml, Wako), and the selective medium Veillonella agar²³. After inoculation, all media were incubated under anaerobic conditions with 10% H₂, 85% N₂, and 5% CO₂ at 37°C. Veillonella agar was incubated for 5 days and BHI agar was incubated for 7 days. The bacterial colonies grown on BHI and Veillonella agar were counted as the total number of bacteria and typical Veillonella colonies in the saliva sample, respectively. Bacterial cells of typical Veillonella colonies were confirmed as gram-negative cocci with light microscopy after gram staining. Standard strains consisted of V. atypica ATCC 17744^T, V. denticariosi JCM 15641^T, V. dispar ATCC 17748^T, V. parvula ATCC 10790^T, V. rogosa JCM 15642^T, and V. tobetsuensis ATCCBAA-2400^T.

DNA extraction

The genomic DNA was extracted from the isolated bacterial cells by using Insta Gene Matrix Kit (Bio-Rad Laboratories, Hercules, CA, USA). The DNA concentration determination was based on fluorescence by using a Qubit 3.0 Fluorometer. (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. Additionally, genomic DNA extracted from the standard strains stated above was used as positive control for PCR.

Identification of Veillonella species

For the identification of Veillonella species at the genus level, a genus-specific PCR primer pair, Veill-rpoBF and Veill-rpoBR, were used according to the protocols described by Arif et al. and Mashima et al.^12,13,24. Strains confirmed by PCR as members of genus Veillonella were then subject to the one-step PCR method with the species-specific primers sets ATYR, DENR, DISR, PARR, ROGR, TOBR, and VF, performed according to the method reported by Mashima et al., for identification at species level¹².

The PCR products were applied to a 2.0% agarose gel, and after electrophoresis, the gel was stained with SYBR^® Safe DNA gel stain (Invitrogen).

Phylogenetic analysis of unknown strains

For phylogenetic analysis of unknown strains, genomic DNA was also extracted from bacterial cells of unknown Veillonella strains showing positive PCR reaction with the genus-specific primer, but negative with the species-specific primer sets. In addition, PCR-based amplification and sequence analysis of rpoB were performed using the previously described primers for genus Veillonella rpoB-forward (5’-GTA ACA AAG GTG TCG TTT CTC G-3’) and rpoB-reverse (5’-GCA CCR TCA AAT ACA GGT GTA GC-3’)²⁴.

The PCR product contained DNA fragments were purified by using QIAquick^® Gel Extraction Kit (Qiagen, Hilden, NW, Germany), according to the manufacturer’s instructions. The DNA concentration after purification was determined based on fluorescence using a Qubit^® 3.0 Fluorometer dsDNA HS Assay Kit (Invitrogen life Technologies, Carlsbad, CA, USA. The PCR reaction was performed with 15–20 ng/µl of DNA template for cycle sequence.

Purified DNA from PCR was sequenced with an BigDye^® Terminator v1.1 Cycle Sequencing kit (Thermo Fisher, Waltham, MA, USA), BigDye^® Terminator 5X Sequencing Buffer (Thermo Fisher, Waltham, MA, USA), single primer 1 pM and PCR product in a final volume of 20 µl. Cycle sequencing of the purified DNA was as follows: preheating at 96°C for 1 minutes; followed by 25 cycles of denaturation at 96°C for 10 seconds and annealing with extension at 60°C for 4 minutes¹². Furthermore, the sequencing of PCR products were purified by using Centri-Sep column (Princeton Separations, Adelphia, NJ, USA), according to the manufacture’s instruction and resolved for the sequencing analysis.

DNA sequences were determined using an ABI PRISM 310 Genetic Analyzer (Applied Biosystem) and were aligned and connected using SEQMAN Pro from the LASERGENE program (DNASTAR). The programs MEGALIGN, which includes CLUSTALW and NJPlot were used to compare sequences and to reconstruct an evolutionary tree by the neighbour-joining method²⁵. Confidence intervals were also assessed by CLUSTALW with bootstrap analysis. Furthermore, pairwise similarity values were determined with MEGALIGN in the LASERGENE program. The rpoB sequences of the unknown Veillonella strains were aligned against the sequence of the established Veillonella species retrieved from GenBank. Unipro UGENE could be use as free alternative for both sequencing and pairwise similarity values.

Ethical considerations

All subjects and their parents were made aware of the objectives and procedures of the study and parents of participants provided written informed consent. This study was conducted with the approval of The Ethics Committee of the Health Sciences University of Hokkaido, Japan, under process number of 2016-015

Results

Colony numbers

The average number of colony forming units (CFU)/ml of all bacteria on BHI agar increased with decreased oral hygiene: 1.38E+08, 2.2E+08 and 4.48E+09 in the good, moderate and poor groups, respectively. Raw CFU data are available on Figshare²⁶.

Species identification

The phenotypic characteristics of Veillonella colonies on the selective medium were 2–4 mm in diameter, and slightly domed in shape with an entire edge, opaque, and greyish white. They were composed of small, gram-negative coccal cells, mainly existing as single cells but with short chains visible. In the good oral hygiene group, a mean number of 1.70E+06 CFU/ml (median, 1.20E+06 CFU/ml) were identified as the genus Veillonella, with 12.3% V. atypica, 19.3% V. dispar, 10.5% V. parvula, 49.1% V. rogosae, and 8.8% unknown species (Table 1). In the moderate group, 2.08E+07 CFU/ml with median 2.00E+06 were identified as the genus Veillonella, with 6.2% V. atypica, 29.6% V. dispar, 12.3% V. parvula, 44.4% V. rogosae, and 7.4% unknown species (Table 2). Meanwhile, in the poor oral hygiene group, 4.48E+09 CFU/ml with median 2.20E+06 were identified as the genus Veillonella, with 7.3% V. atypica, 12.2% V. dispar, 31.7% V. parvula, 34.1% V. rogosae, and 14.6% unknown species proportions (Table 3).

Table 1. Ratio of the number of isolates of each species to the total number of Veillonella isolate in saliva from the good oral hygiene group.

The colony-forming units (CFU) of all anaerobic bacteria on brain heart infusion agar and Veillonella strains on Veillonella agar (detection limit <0.1% of the total count). The total of Veillonella isolates identified by the Veillonella genus-specific PCR primer. Individual species as a percentage of the number of isolates identified by one-step PCR with the species-specific primer sets for each subject (n = 5) from saliva of the good oral hygiene group.

Subject			Total number		Isolated Veillonella species
Name	Age	Sex	All bacteria	Veillonella spp.	Total number	V. atypica	V. denticaruisi	V. dispar	V. parvula	V. rogosae	V. tobetsuensis	Unknown
Name	Age	Sex	CFU/mL	CFU/mL	(100%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)
S8	13	F	7.60E+07	3.00E+05	3	0	0	0	1 (33.3)	2 (66.7)	0	0
S9	6	M	2.04E+08	5.00E+06	5	2 (40.0)	0	0	0	1 (20.0)	0	2 (40.0)
S12	6	F	3.52E+08	1.20E+06	12	0	0	4 (33.3)	0	8 (66.7)	0	0
S25	6	F	2.70E+07	2.00E+06	20	2 (10.0)	0	3 (15.0)	4 (20.0)	9 (45.0)	0	2 (10.0)
S28	8	M	3.20E+07	1.70E+02	17	3 (17.6)	0	4 (23.5)	1 (5.9)	8 (47.1)	0	1 (5.9)

Table 2. Ratio of the number of isolates of each species to the total number of Veillonella isolate in saliva from the moderate oral hygiene group.

The colony-forming units (CFU) of all anaerobic bacteria on the brain heart infusion agar and Veillonella strains on Veillonella agar (detection limit <0.1% of the total count). The total of Veillonella isolates identified by the Veillonella genus-specific PCR primer. Individual species as a percentage of the number of isolates identified by one-step PCR with the species-specific primer sets for each subject (n = 5) from saliva of the moderate oral hygiene group.

Subject			Total number		Isolated Veillonella species
Name	Age	Sex	All bacteria	Veillonella spp.	Total number	V. atypica	V. denticaruisi	V. dispar	V. parvula	V. rogosae	V. tobetsuensis	Unknown
Name	Age	Sex	CFU/mL	CFU/mL	(100%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)
S1	9	M	1.04E+08	2.00E+06	20	3 (15.0)	0	11 (55.0)	2 (10.0)	4 (20.0)	0	0
S3	4	M	5.30E+08	1.20E+07	12	0	0	3 (25.0)	1 (8.3)	4 (33.3)	0	4 (33.3)
S10	7	M	4.16E+08	9.00E+07	9	0	0	0	5 (55.6)	3 (33.3)	0	1 (11.1)
S29	6	F	4.46E+07	2.00E+05	20	2 (10.0)	0	5 (25.0)	2 (10.0)	10 (50.0)	0	1 (5.0)
S32	14	F	3.90E+06	2.00E+04	20	0	0	5 (25.0)	0 (0.0)	15 (75.0)	0	0

Table 3. Ratio of the number of isolates of each species to the total number of Veillonella isolated in saliva from the poor oral hygiene group.

Subject			Total number		Isolated Veillonella species
Name	Age	Sex	All bacteria	Veillonella spp.	Total number	V. atypica	V. denticaruisi	V. dispar	V. parvula	V. rogosae	V. tobetsuensis	Unknown
Name	Age	Sex	CFU/mL	CFU/mL	(100%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)
S15	7	M	4.00E+09	1.40E+08	14	0	0	3 (21.4)	1 (7.1)	8 (57.1)	0	2 (14.3)
S16	9	F	4.64E+09	3.00E+08	3	0	0	0	0	3 (100.0)	0	0
S17	10	F	7.84E+09	5.00E+08	5	3 (60.0)	0	0	0	1 (20.0)	0	1 (20.0)
S21	8	F	1.50E+09	1.00E+08	10	0	0	0	8 (80.0)	0	0	2 (20.0)
S30	8	F	4.41E+09	9.00E+08	9	0	0	2 (22.2)	4 (44.4)	2 (22.2)	0	1 (11.1)

As shown in the results, V. rogosae was found as the predominant species in the saliva samples of all oral hygiene groups. However, V. denticariosi and V. tobetsuensis were not found in all oral hygiene groups (Table 1–Table 3). Figure 1 shows the per cent ratio of the total number of strains of each species to the total number of Veillonella isolates from saliva samples of the good, moderate, and poor oral hygiene groups.

Figure 1. Total isolated number of each Veillonella species isolated.

Data expressed as percentage of total the total isolated number as Veillonella in samples from saliva in good, moderate and poor oral hygiene groups.

Strain characteristics

In this study, 179 strains were identified as Veillonella strains, and 162 strains were identified as V. atypica, V. dispar, V. parvula or V. rogosae. However, 17 (9.5%) of 179 strains failed to be classified as any of the known oral Veillonella species, thus, they were defined as unknown species. Of the 17 unknown Veillonella strains 10 (S3-1, S9-1, S10-1, S15-1, S17-1, S21-1, S25-2, S28-1, S29-1 and S30-1) were selected as representative strains from different hygiene groups for phylogenetic analysis. After determination of the rpoB sequences of these 10 strains, these sequences were aligned toward to the sequences of Veillonella type strains retrieved from GenBank. The evolutionary tree produced by analysing the type strains of the 14 Veillonella species and the 10 unknown strains is shown in the Figure 2. According to this data, the most closely related species was V. infantium, although the 10 unknown strains formed three clusters. The DNA sequence similarity of the 10 unknown Veillonella strains to V infantium JCM 31738^T (LC191258) ranged from 97.1 to 99.7%.

Figure 2. Neighbor-joining tree showing the relationship between the 10 unknown Veillonella strains and the type strains of established species of genus Veillonella, with accession numbers.

Bootstrap values were indicated at corresponding nodes. The scale bar indicated the number of nucleotide substitutions per 100 residues.

Discussion

It was previously reported that a higher number of anaerobic bacteria was detected on BHI agar in saliva from Thai children with poor oral hygiene than those with good and moderate oral hygiene²⁰. This prior study demonstrated that oral Veillonella isolates were detected at a twofold higher frequency in the saliva of Thai children with poor rather than good or moderate oral hygiene²⁰. Here, it was demonstrated that the number of anaerobic bacteria on BHI agar and Veillonella species on the selective medium increased in saliva of Japanese children with worsening oral hygiene status. Therefore, the detection level of anaerobic bacterial strains and oral Veillonella strains in saliva from Japanese children with good, moderate and poor oral hygiene status was similar to that from Thai children.

Using the Illumina MiSeq platform, Mashima et al. demonstrated that Streptococcus and Veillonella species were the predominant bacterial species in the saliva microbiome of Thai children, but that the proportion of Streptococcus decreased while that of Veillonella increased with poor oral hygiene status⁵. They also found that Veillonella species were detected predominantly in the tongue microbiome of Thai children with poor oral hygiene status compared to those with good or moderate oral hygiene status⁵. Taken together with the results of the present study, it is possible that Veillonella species could be a biomarker of oral hygiene status for Thai and Japanese children.

This study demonstrated that V. rogosae was the predominant species detected in all groups of Japanese children (Figure 1). Beighton et al. reported V. rogosae as one of the predominant Veillonella species in tongue biofilms of healthy adults in the UK²⁴. A previous study also showed that V. rogosae was the predominant Veillonella species in tongue biofilms of the children in Thailand¹². Recently, Theodorea et al. isolated 1,609 Veillonella strains from saliva samples of Thai children divided into three groups: good, moderate and poor oral hygiene status²⁰. Then, 1,442 of 1,609 strains were detected by the one-step PCR method with the species-specific primer sets for V. atypica, V. denticariosi, V. dispar, V. parvula, V. rogosae, or V. tobetsuensis. They reported that V. rogosae was the predominant species detected in all groups²⁰. These results of the previous and present studies indicate that V. rogosae is the predominant oral Veillonella species in the human saliva and tongue biofilm.

Furthermore, this study showed that the detection rate of V. rogosae decreased as oral hygiene quality decreased: 49.1%, 44.4%, and 34.1% in the good, moderate, and poor oral hygiene groups, respectively (Figure 1). Similar results were obtained from saliva samples of Thai children were also reported by Theodorea et al.²⁰. Based on these results, it was demonstrated that the detection rate of V. rogosae decreased with aggravation of oral hygiene status in Japanese and Thai children. Additionally, Arif et al. detected V. rogosae only in carious-free lesions of dental plaques¹³. All these data suggest that a human oral cavity with good hygiene status is suitable habitat for V. rogosae.

Conversely, the detection rate of V. parvula in the poor (31.7%) oral hygiene was significantly higher than that in the good (10.5%) and moderate (12.3%) oral hygiene groups, in this study with Japanese children. This result is conformed with data from another study, in which V. parvula was most frequently detected in saliva of Thai children with poor oral hygiene status²⁰. Previously, it was also reported that V. parvula was frequently detected in periodontal pockets²⁷ and active carious-lesions²⁸. These data suggest that oral cavities with poor hygiene status are suitable environments for V. parvula.

In this study, 179 strains were isolated members of the genus Veillonella from saliva of 15 Japanese children, V. denticariosi and V. tobetsuensis were not found in any samples. In the case of saliva samples from Thai children, the detection rate of V. denticariosi (0.4%) and V. tobetsuensis (1.7%) were very low. In this study 1,609 Veillonella strains were isolated from 107 Thai children²⁰. Similarly, it was reported that V. denticariosi was not detected in any of the tongue biofilms of Thai children¹², and V. denticariosi was detected in tongue biofilm of only one young Japanese adult¹⁷. Therefore, V. denticariosi may be the least prevalent oral Veillonella species in the saliva and tongue microbiome. On the other hand, V. tobetsuensis, was not detected in saliva from Thai children with good oral hygiene status. However, the detection rate of V. tobetsuensis was 14.3% and 17.8% in the saliva of Thai children with moderate and poor oral hygiene, respectively (20). Similarly, it was demonstrated that the prevalence of V. tobetsuensis ranged from 7.6% to 20.0% in tongue biofilm samples from Japanese adults¹⁶. Therefore, these data suggest that V. tobetsuensis may be potential to co-occur with other Veillonella species in saliva and tongue biofilms.

In the present study with saliva samples of Japanese children, 17 (9.5%) of 179 strains confirmed as member of genus Veillonella were not belong to any established Veillonella species as unknown species. Theodorea et al., also reported that 167 (10.4%) of 1,609 Veillonella isolates from saliva of Thai children could not be assigned to any species of the genus Veillonella²⁰. Furthermore, it was reported that 43 (9.7%) of the 442 Veillonella isolates from periodontal pockets and gingival sulcus could not be identified as any of the known Veillonella species. These data may indicate that other novel Veillonella species inhabit human oral cavities, although only six species were detected as oral Veillonella species up to this point. Further phylogenetic studies are needed to evaluate the possibilities of novel Veillonella species.

In 2018, Mashima et al.¹¹ proposed V. infantium as a novel species isolated from saliva of Thai children, representing a seventh oral Veillonella species. Therefore, for phylogenetic analysis of the unknown Veillonella strains isolated in this study, the rpoB sequences of type strains of the established Veillonella species, including V. infantium JCM 31738^T, were examined. Consequently, 10 unknown Veillonella strains analysed in this study formed three clusters distinct from V. dispar, the most closely related species was V. infantium. Further studies are required to assign these strains most accurately.

In conclusions, this is the first study to identify oral Veillonella at the species level in the saliva of Japanese children divided into three oral hygiene status groups: good, moderate and poor group. Although V. denticariosi and V. tobetsuensis were not found in any groups in this study because of small number of subjects, the distribution and frequency of V. atypica, V. dispar, V. parvula and V. rogosae, were mostly the same as those in the saliva from Thai children divided into the aforementioned oral hygiene status groups. Additionally, the results of this study demonstrate that changes in the ratio of some Veillonella species, such as an increase of V. parvula and decrease of V. rogosae in those with poor oral hygiene, can be a useful indicator of oral hygiene status, as with results obtained in the study of saliva taken from Thai children. The present study also showed that approximately 10% of the isolated Veillonella strains were not classified to any Veillonella species, and that they will be assigned to V. infantium or novel Veillonella species after further studies.

Data availability

16S rRNA sequences of the 10 unknown Veillonella strains are available from GenBank, accession numbers: LC467206 (S9-1), LC467207 (S28-1), LC467208 (S25-1), LC467209 (S17-1), LC467210 (S15-1), LC467211 (S29-1), LC467212 (S10-1), LC467213 (S30-1), LC467214 (S21-1), LC467215 (S3-1).

Figshare: Raw Data Saliva Japanese Children.xlsx. https://doi.org/10.6084/m9.figshare.7856657.v1²⁶.

This project contains the number of colony-forming units and total number of Veillonella strains isolated from each child.

Data on Figshare are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgments

The authors wish to thank all the children and their parents for their valuable participation in this study.

Faculty Opinions recommended

References

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Comments on this article Comments (0)

Version 5

VERSION 5 PUBLISHED 03 May 2019

Author details Author details

Ariadna A. Djais
Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Writing – Original Draft Preparation

Citra Fragrantia Theodorea
Roles: Investigation, Methodology, Supervision, Writing – Review & Editing

Izumi Mashima
Roles: Conceptualization, Investigation, Methodology, Project Administration, Supervision, Validation

Maiko Otomo
Roles: Resources, Visualization

Masato Saitoh
Roles: Resources, Visualization

Futoshi Nakazawa
Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Project Administration, Supervision, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

This study was supported in part by grants-in aid from the Japan Society for the promotion of Science Fellows (15J30007), Scientific Research from KAKENHI (26462793), 2016-2017 Research Project of Institute of Personalized Health Sciences, Health Sciences University of Hokkaido, 2016-2017 research grant from the Institute for Fermentation, Osaka, Japan, NKB-0246/UN2.R3.1/HKP.05.00/2019 and NKB-1936/UN2.R3.1/HKP.05.00/2019 Universitas Indonesia, Jakarta, Indonesia.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (5)

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Djais AA, Theodorea CF, Mashima I et al. Identification and phylogenetic analysis of oral Veillonella species isolated from the saliva of Japanese children [version 5; peer review: 2 approved] F1000Research 2019, 8:616 (https://doi.org/10.12688/f1000research.18506.5)

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Version 5

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Reviewer Report 16 Sep 2019

Takuichi Sato, Division of Clinical Chemistry, Department of Medical Technology, Niigata University Graduate School of Health Sciences, Niigata, Japan

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Takuichi Sato, Division of Clinical Chemistry, Department of Medical Technology, Niigata University Graduate School of Health Sciences, Niigata, Japan

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Reviewer Report 01 Aug 2019

Takuichi Sato, Division of Clinical Chemistry, Department of Medical Technology, Niigata University Graduate School of Health Sciences, Niigata, Japan

Approved with Reservations

https://doi.org/10.5256/f1000research.21912.r51348

[Suggestions]
I recognized the revised parts in the Abstract (Background) as follows;

"However, the study regarding the identification and distribution of oral Veillonella are limited. The oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status."

I think that the above-mentioned parts should be included in the Introduction, but I was unable to find the revised parts. The authors need to revise the Introduction, like as in the Abstract.

[Typographical errors]
Introduction (6th paragraph)
"difficult to idnetify" should read "difficult to identify".

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Oral Microbiology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Author Response 08 Aug 2019

Ariadna Djais, Department of Oral Biology, Faculty of Dentistry, Faculty of Dentistry, Universitas Indonesia, Jakarta 10430, Indonesia, Jakarta, 10430, Indonesia

08 Aug 2019

Author Response
The authors would like to thank the area editor and the reviewers for their precious time and valuable comments. We have carefully addressed all the comments.

Reviewer: Takuichi Sato, Division of Clinical ... Continue reading
The authors would like to thank the area editor and the reviewers for their precious time and valuable comments. We have carefully addressed all the comments.

Reviewer: Takuichi Sato, Division of Clinical Chemistry, Department of Medical Technology, Niigata University Graduate School of Health Sciences, Niigata, Japan [Suggestions]

I recognized the revised parts in the Abstract (Background) as follows;

"However, the study regarding the identification and distribution of oral Veillonella are limited. The oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status."

I think that the above-mentioned parts should be included in the Introduction, but I was unable to find the revised parts. The authors need to revise the Introduction, like as in the Abstract.

[Typographical errors]
Introduction (6th paragraph)
"difficult to idnetify" should read "difficult to identify".

Authors: Thank you very much for this direction.

We have changed the wording of the relevant in the Introduction 6th Paragraph, L 11, like as in the Abstract.

[Typographical errors] Introduction (6th paragraph) "difficult to idnetify" into "difficult to identify".

Introduction 7th Paragraph, L 2 was removed because in this study we are focused on demonstrating that the composition and proportion of oral Veillonella species in the saliva of Japanese children is correlated with different oral hygiene status.

The revised version of this paper has been submitted.

Thank you for taking the time and energy to help us improve this manuscript. We hope that you will find that these revisions rise to your expectation.
The authors would like to thank the area editor and the reviewers for their precious time and valuable comments. We have carefully addressed all the comments.

Reviewer: Takuichi Sato, Division of Clinical Chemistry, Department of Medical Technology, Niigata University Graduate School of Health Sciences, Niigata, Japan [Suggestions]

I recognized the revised parts in the Abstract (Background) as follows;

"However, the study regarding the identification and distribution of oral Veillonella are limited. The oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status."

I think that the above-mentioned parts should be included in the Introduction, but I was unable to find the revised parts. The authors need to revise the Introduction, like as in the Abstract.

[Typographical errors]
Introduction (6th paragraph)
"difficult to idnetify" should read "difficult to identify".

Authors: Thank you very much for this direction.

We have changed the wording of the relevant in the Introduction 6th Paragraph, L 11, like as in the Abstract.

[Typographical errors] Introduction (6th paragraph) "difficult to idnetify" into "difficult to identify".

Introduction 7th Paragraph, L 2 was removed because in this study we are focused on demonstrating that the composition and proportion of oral Veillonella species in the saliva of Japanese children is correlated with different oral hygiene status.

The revised version of this paper has been submitted.

Thank you for taking the time and energy to help us improve this manuscript. We hope that you will find that these revisions rise to your expectation.
Competing Interests: No competing interests were disclosed. Close
Report a concern
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COMMENTS ON THIS REPORT

Author Response 08 Aug 2019

Ariadna Djais, Department of Oral Biology, Faculty of Dentistry, Faculty of Dentistry, Universitas Indonesia, Jakarta 10430, Indonesia, Jakarta, 10430, Indonesia

08 Aug 2019

Author Response
The authors would like to thank the area editor and the reviewers for their precious time and valuable comments. We have carefully addressed all the comments.

Reviewer: Takuichi Sato, Division of Clinical ... Continue reading
The authors would like to thank the area editor and the reviewers for their precious time and valuable comments. We have carefully addressed all the comments.

Reviewer: Takuichi Sato, Division of Clinical Chemistry, Department of Medical Technology, Niigata University Graduate School of Health Sciences, Niigata, Japan [Suggestions]

I recognized the revised parts in the Abstract (Background) as follows;

"However, the study regarding the identification and distribution of oral Veillonella are limited. The oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status."

I think that the above-mentioned parts should be included in the Introduction, but I was unable to find the revised parts. The authors need to revise the Introduction, like as in the Abstract.

[Typographical errors]
Introduction (6th paragraph)
"difficult to idnetify" should read "difficult to identify".

Authors: Thank you very much for this direction.

We have changed the wording of the relevant in the Introduction 6th Paragraph, L 11, like as in the Abstract.

[Typographical errors] Introduction (6th paragraph) "difficult to idnetify" into "difficult to identify".

Introduction 7th Paragraph, L 2 was removed because in this study we are focused on demonstrating that the composition and proportion of oral Veillonella species in the saliva of Japanese children is correlated with different oral hygiene status.

The revised version of this paper has been submitted.

Thank you for taking the time and energy to help us improve this manuscript. We hope that you will find that these revisions rise to your expectation.
The authors would like to thank the area editor and the reviewers for their precious time and valuable comments. We have carefully addressed all the comments.

Reviewer: Takuichi Sato, Division of Clinical Chemistry, Department of Medical Technology, Niigata University Graduate School of Health Sciences, Niigata, Japan [Suggestions]

I recognized the revised parts in the Abstract (Background) as follows;

"However, the study regarding the identification and distribution of oral Veillonella are limited. The oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status."

I think that the above-mentioned parts should be included in the Introduction, but I was unable to find the revised parts. The authors need to revise the Introduction, like as in the Abstract.

[Typographical errors]
Introduction (6th paragraph)
"difficult to idnetify" should read "difficult to identify".

Authors: Thank you very much for this direction.

We have changed the wording of the relevant in the Introduction 6th Paragraph, L 11, like as in the Abstract.

[Typographical errors] Introduction (6th paragraph) "difficult to idnetify" into "difficult to identify".

Introduction 7th Paragraph, L 2 was removed because in this study we are focused on demonstrating that the composition and proportion of oral Veillonella species in the saliva of Japanese children is correlated with different oral hygiene status.

The revised version of this paper has been submitted.

Thank you for taking the time and energy to help us improve this manuscript. We hope that you will find that these revisions rise to your expectation.
Competing Interests: No competing interests were disclosed. Close
Report a concern

Version 2

VERSION 2

PUBLISHED 20 Jun 2019

Revised

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Reviewer Report 28 Jun 2019

Takuichi Sato, Division of Clinical Chemistry, Department of Medical Technology, Niigata University Graduate School of Health Sciences, Niigata, Japan

Approved with Reservations

https://doi.org/10.5256/f1000research.21331.r50157

The authors have described the following in the Response, but I am unable to find the differences in the abstract (and/or introduction).

"We have changed the wording of the relevant in the abstract to express the reason why did we want to know the detection rate and distribution of oral Veillonella species in Japan. The reasons are the study regarding the identification and distribution of oral Veillonella are limited; also the oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status."

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Oral Microbiology

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Reviewer Report 27 Jun 2019

Juni Handajani, Department of Oral Biology, Faculty of Dentistry, Gadjah Mada University, Yogyakarta, Indonesia

Approved

https://doi.org/10.5256/f1000research.21331.r50156

Thank you for the revision. I now understand the analysis statistic ... Continue reading

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Version 1

VERSION 1

PUBLISHED 03 May 2019

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Reviewer Report 23 May 2019

Takuichi Sato, Division of Clinical Chemistry, Department of Medical Technology, Niigata University Graduate School of Health Sciences, Niigata, Japan

Approved with Reservations

https://doi.org/10.5256/f1000research.20250.r48113

Suggestions:

Abstract and Introduction:
I think that the manuscript, in the Abstract and Introduction, needs to express rationales of this study in more detail, such as, why did the authors want to know the detection rate and distribution of oral Veillonella species in saliva of Japanese children? Why did the authors want to compare the results with those of Thai children?

Figure 1 and Tables 1-3:
I think that the data in Figure 1 and Tables 1-3 are overlapping, and Tables 1-3 could be deleted from the manuscript, because the authors mainly stated the total and the mean (proportions) of the isolates of Veillonella species in the Results and Discussion.

Minor:
The authors should check the meaning of the phrase "although":

P. 7, Discussion: in the 6th paragraph
L. 1-2: "although 179 strains were isolated members of the genus Veillonella from saliva of 15 Japanese children, …".

P. 7, Discussion: in the 6th paragraph
L. 6-7: "although in this study 1,609 Veillonella strains were isolated from 107 Thai children".

P. 8, Discussion: in the 8th paragraph
L. 7-8: "although 10 unknown Veillonella strains analysed in this study formed three clusters distinct from V. dispar, …".

Typographical errors;
P. 4, Results: Species identification
L. 6: "numberof" should read "number of". (Put a space in between.)

P. 7, Discussion: in the 5th paragraph
L. 4: "conforms" should read "conformed".

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Oral Microbiology

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Author Response 20 Jun 2019

Ariadna Djais, Department of Oral Biology, Faculty of Dentistry, Faculty of Dentistry, Universitas Indonesia, Jakarta 10430, Indonesia, Jakarta, 10430, Indonesia

20 Jun 2019

Author Response
Abstract and Introduction:
I think that the manuscript, in the Abstract and Introduction, needs to express rationales of this study in more detail, such as, why did the authors want to ... Continue reading
Abstract and Introduction:
I think that the manuscript, in the Abstract and Introduction, needs to express rationales of this study in more detail, such as, why did the authors want to know the detection rate and distribution of oral Veillonella species in saliva of Japanese children? Why did the authors want to compare the results with those of Thai children?
Response: Thank you very much for this direction. We have changed the wording of the relevant in the abstract to express the reason why did we want to know the detection rate and distribution of oral Veillonella species in Japan. The reasons are the study regarding the identification and distribution of oral Veillonella are limited; also the oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status.
Figure 1 and Tables 1-3:
I think that the data in Figure 1 and Tables 1-3 are overlapping, and Tables 1-3 could be deleted from the manuscript, because the authors mainly stated the total and the mean (proportions) of the isolates of Veillonella species in the Results and Discussion.
Response: Thanks for raising this important point. However, we consider to state

the Table 1-3 as Ratio of the number of isolates of each species from each subject.

Figure 1 was indicated to express the Total isolated number of each Veillonella species from each group of oral hygiene.

Minor:
The authors should check the meaning of the phrase "although":

P. 7, Discussion: in the 6th paragraph
L. 1-2: "although 179 strains were isolated members of the genus Veillonella from saliva of 15 Japanese children, …".
Response: the word of “although” has been removed

P. 7, Discussion: in the 6th paragraph
L. 6-7: "although in this study 1,609 Veillonella strains were isolated from 107 Thai children".
Response: the word of “although” has been removed

P. 8, Discussion: in the 8th paragraph
L. 7-8: "although 10 unknown Veillonella strains analysed in this study formed three clusters distinct from V. dispar, …".
Response: the word of “although” has been removed
Typographical errors;
P. 4, Results: Species identification
L. 6: "numberof" should read "number of". (Put a space in between.)

Response: the word of “numberof” has been revised to “number of”

P. 7, Discussion: in the 5th paragraph
L. 4: "conforms" should read "conformed".

Response: the word of “conforms” has been revised to “conformed”
Thank you for taking the time and energy to help us improve this manuscript. We hope that you will find these revision rise to your expectation.
Abstract and Introduction:
I think that the manuscript, in the Abstract and Introduction, needs to express rationales of this study in more detail, such as, why did the authors want to know the detection rate and distribution of oral Veillonella species in saliva of Japanese children? Why did the authors want to compare the results with those of Thai children?
Response: Thank you very much for this direction. We have changed the wording of the relevant in the abstract to express the reason why did we want to know the detection rate and distribution of oral Veillonella species in Japan. The reasons are the study regarding the identification and distribution of oral Veillonella are limited; also the oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status.
Figure 1 and Tables 1-3:
I think that the data in Figure 1 and Tables 1-3 are overlapping, and Tables 1-3 could be deleted from the manuscript, because the authors mainly stated the total and the mean (proportions) of the isolates of Veillonella species in the Results and Discussion.
Response: Thanks for raising this important point. However, we consider to state

the Table 1-3 as Ratio of the number of isolates of each species from each subject.

Figure 1 was indicated to express the Total isolated number of each Veillonella species from each group of oral hygiene.

Minor:
The authors should check the meaning of the phrase "although":

P. 7, Discussion: in the 6th paragraph
L. 1-2: "although 179 strains were isolated members of the genus Veillonella from saliva of 15 Japanese children, …".
Response: the word of “although” has been removed

P. 7, Discussion: in the 6th paragraph
L. 6-7: "although in this study 1,609 Veillonella strains were isolated from 107 Thai children".
Response: the word of “although” has been removed

P. 8, Discussion: in the 8th paragraph
L. 7-8: "although 10 unknown Veillonella strains analysed in this study formed three clusters distinct from V. dispar, …".
Response: the word of “although” has been removed
Typographical errors;
P. 4, Results: Species identification
L. 6: "numberof" should read "number of". (Put a space in between.)

Response: the word of “numberof” has been revised to “number of”

P. 7, Discussion: in the 5th paragraph
L. 4: "conforms" should read "conformed".

Response: the word of “conforms” has been revised to “conformed”
Thank you for taking the time and energy to help us improve this manuscript. We hope that you will find these revision rise to your expectation.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 20 Jun 2019

Ariadna Djais, Department of Oral Biology, Faculty of Dentistry, Faculty of Dentistry, Universitas Indonesia, Jakarta 10430, Indonesia, Jakarta, 10430, Indonesia

20 Jun 2019

Author Response
Abstract and Introduction:
I think that the manuscript, in the Abstract and Introduction, needs to express rationales of this study in more detail, such as, why did the authors want to ... Continue reading
Abstract and Introduction:
I think that the manuscript, in the Abstract and Introduction, needs to express rationales of this study in more detail, such as, why did the authors want to know the detection rate and distribution of oral Veillonella species in saliva of Japanese children? Why did the authors want to compare the results with those of Thai children?
Response: Thank you very much for this direction. We have changed the wording of the relevant in the abstract to express the reason why did we want to know the detection rate and distribution of oral Veillonella species in Japan. The reasons are the study regarding the identification and distribution of oral Veillonella are limited; also the oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status.
Figure 1 and Tables 1-3:
I think that the data in Figure 1 and Tables 1-3 are overlapping, and Tables 1-3 could be deleted from the manuscript, because the authors mainly stated the total and the mean (proportions) of the isolates of Veillonella species in the Results and Discussion.
Response: Thanks for raising this important point. However, we consider to state

the Table 1-3 as Ratio of the number of isolates of each species from each subject.

Figure 1 was indicated to express the Total isolated number of each Veillonella species from each group of oral hygiene.

Minor:
The authors should check the meaning of the phrase "although":

P. 7, Discussion: in the 6th paragraph
L. 1-2: "although 179 strains were isolated members of the genus Veillonella from saliva of 15 Japanese children, …".
Response: the word of “although” has been removed

P. 7, Discussion: in the 6th paragraph
L. 6-7: "although in this study 1,609 Veillonella strains were isolated from 107 Thai children".
Response: the word of “although” has been removed

P. 8, Discussion: in the 8th paragraph
L. 7-8: "although 10 unknown Veillonella strains analysed in this study formed three clusters distinct from V. dispar, …".
Response: the word of “although” has been removed
Typographical errors;
P. 4, Results: Species identification
L. 6: "numberof" should read "number of". (Put a space in between.)

Response: the word of “numberof” has been revised to “number of”

P. 7, Discussion: in the 5th paragraph
L. 4: "conforms" should read "conformed".

Response: the word of “conforms” has been revised to “conformed”
Thank you for taking the time and energy to help us improve this manuscript. We hope that you will find these revision rise to your expectation.
Abstract and Introduction:
I think that the manuscript, in the Abstract and Introduction, needs to express rationales of this study in more detail, such as, why did the authors want to know the detection rate and distribution of oral Veillonella species in saliva of Japanese children? Why did the authors want to compare the results with those of Thai children?
Response: Thank you very much for this direction. We have changed the wording of the relevant in the abstract to express the reason why did we want to know the detection rate and distribution of oral Veillonella species in Japan. The reasons are the study regarding the identification and distribution of oral Veillonella are limited; also the oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status.
Figure 1 and Tables 1-3:
I think that the data in Figure 1 and Tables 1-3 are overlapping, and Tables 1-3 could be deleted from the manuscript, because the authors mainly stated the total and the mean (proportions) of the isolates of Veillonella species in the Results and Discussion.
Response: Thanks for raising this important point. However, we consider to state

the Table 1-3 as Ratio of the number of isolates of each species from each subject.

Figure 1 was indicated to express the Total isolated number of each Veillonella species from each group of oral hygiene.

Minor:
The authors should check the meaning of the phrase "although":

P. 7, Discussion: in the 6th paragraph
L. 1-2: "although 179 strains were isolated members of the genus Veillonella from saliva of 15 Japanese children, …".
Response: the word of “although” has been removed

P. 7, Discussion: in the 6th paragraph
L. 6-7: "although in this study 1,609 Veillonella strains were isolated from 107 Thai children".
Response: the word of “although” has been removed

P. 8, Discussion: in the 8th paragraph
L. 7-8: "although 10 unknown Veillonella strains analysed in this study formed three clusters distinct from V. dispar, …".
Response: the word of “although” has been removed
Typographical errors;
P. 4, Results: Species identification
L. 6: "numberof" should read "number of". (Put a space in between.)

Response: the word of “numberof” has been revised to “number of”

P. 7, Discussion: in the 5th paragraph
L. 4: "conforms" should read "conformed".

Response: the word of “conforms” has been revised to “conformed”
Thank you for taking the time and energy to help us improve this manuscript. We hope that you will find these revision rise to your expectation.
Competing Interests: No competing interests were disclosed. Close
Report a concern

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Reviewer Report 20 May 2019

Juni Handajani, Department of Oral Biology, Faculty of Dentistry, Gadjah Mada University, Yogyakarta, Indonesia

Approved with Reservations

https://doi.org/10.5256/f1000research.20250.r48110

This study is good for identification and phylogenetic analysis of oral Veillonella species isolated from saliva samples of children in Japan. The aim of this study was to analyze the composition and proportion of oral Veillonella species in the saliva of Japanese children compared to previous studies in Thailand.

The manuscript is certainly well written but I have some major concerns on the data analysis:

The number of samples in this study was fewer (15 children) compared to the number of samples in the previous study from Thailand (107 children). It is suggested that the analysis uses proportions so that it can describe the oral Veillonella species according to the number of samples.
To find out the comparison of the results of this study with the results of a previous study from Thailand, a correlation analysis is suggested.
It is also necessary to add the results of a correlation analysis between the results of Veillonella's oral identification and oral health status.

Minor comment:

Some references used are older than 10 years. I suggest to use current references from at least the last 10 years.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Area of my expertise are oral biology and immunology

CITE

Report a concern

Author Response 20 Jun 2019

Ariadna Djais, Department of Oral Biology, Faculty of Dentistry, Faculty of Dentistry, Universitas Indonesia, Jakarta 10430, Indonesia, Jakarta, 10430, Indonesia

20 Jun 2019

Author Response
This study is good for identification and phylogenetic analysis of oral Veillonella species isolated from saliva samples of children in Japan. The aim of this study was to analyze the composition and ... Continue reading
This study is good for identification and phylogenetic analysis of oral Veillonella species isolated from saliva samples of children in Japan. The aim of this study was to analyze the composition and proportion of oral Veillonella species in the saliva of Japanese children compared to previous studies in Thailand.
Response: Thank you for your valuable comment. The aim of this study was focus to analyze the composition and proportion of oral Veillonella species in the saliva of Japanese children. Furthermore, we compared the results with some reports such as in Thailand. The revise version has been submitted.
The manuscript is certainly well written but I have some major concerns on the data analysis:

The number of samples in this study was fewer (15 children) compared to the number of samples in the previous study from Thailand (107 children). It is suggested that the analysis uses proportions so that it can describe the oral Veillonella species according to the number of samples.

Response: These children visited to the Dental Hospital, Health Sciences University of Hokkaido for dental examination, over a period between 2016 and 2017. Based on the evaluation by the Simplified Oral Hygiene Index, they were divided into three groups, good, moderate and poor. But, among many children, only five children had poor oral hygiene status. Therefore, we used these five children as poor group. And we have selected five children from good and moderate groups, without distinction. As the results, the total subject : 15 subjects.

To find out the comparison of the results of this study with the results of a previous study from Thailand, a correlation analysis is suggested.

It is also necessary to add the results of a correlation analysis between the results of Veillonella's oral identification and oral health status.

Response: Thank you for another good point at No. 2 and No.3. The research of oral Veillonella were very limited. The analysis was use in this study : descriptive analysis with mean value to determine the distribution and proportion of oral Veillonella, then we compare the characteristic of oral veillonella distribution with the previous study.
Minor comment:

Some references used are older than 10 years. I suggest to use current references from at least the last 10 years.

Response: As we mentioned before, that the research of oral Veillonella were very limited. Therefore, some of references were used older that 10 years.
Thank you for taking the time and energy to help us improve this manuscript. We hope that you will find these revision rise to your expectation.
This study is good for identification and phylogenetic analysis of oral Veillonella species isolated from saliva samples of children in Japan. The aim of this study was to analyze the composition and proportion of oral Veillonella species in the saliva of Japanese children compared to previous studies in Thailand.
Response: Thank you for your valuable comment. The aim of this study was focus to analyze the composition and proportion of oral Veillonella species in the saliva of Japanese children. Furthermore, we compared the results with some reports such as in Thailand. The revise version has been submitted.
The manuscript is certainly well written but I have some major concerns on the data analysis:

The number of samples in this study was fewer (15 children) compared to the number of samples in the previous study from Thailand (107 children). It is suggested that the analysis uses proportions so that it can describe the oral Veillonella species according to the number of samples.

Response: These children visited to the Dental Hospital, Health Sciences University of Hokkaido for dental examination, over a period between 2016 and 2017. Based on the evaluation by the Simplified Oral Hygiene Index, they were divided into three groups, good, moderate and poor. But, among many children, only five children had poor oral hygiene status. Therefore, we used these five children as poor group. And we have selected five children from good and moderate groups, without distinction. As the results, the total subject : 15 subjects.

To find out the comparison of the results of this study with the results of a previous study from Thailand, a correlation analysis is suggested.

It is also necessary to add the results of a correlation analysis between the results of Veillonella's oral identification and oral health status.

Response: Thank you for another good point at No. 2 and No.3. The research of oral Veillonella were very limited. The analysis was use in this study : descriptive analysis with mean value to determine the distribution and proportion of oral Veillonella, then we compare the characteristic of oral veillonella distribution with the previous study.
Minor comment:

Some references used are older than 10 years. I suggest to use current references from at least the last 10 years.

Response: As we mentioned before, that the research of oral Veillonella were very limited. Therefore, some of references were used older that 10 years.
Thank you for taking the time and energy to help us improve this manuscript. We hope that you will find these revision rise to your expectation.
Competing Interests: No competing interests were disclosed. Close
Report a concern
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COMMENTS ON THIS REPORT

Author Response 20 Jun 2019

Ariadna Djais, Department of Oral Biology, Faculty of Dentistry, Faculty of Dentistry, Universitas Indonesia, Jakarta 10430, Indonesia, Jakarta, 10430, Indonesia

20 Jun 2019

Author Response
This study is good for identification and phylogenetic analysis of oral Veillonella species isolated from saliva samples of children in Japan. The aim of this study was to analyze the composition and ... Continue reading
This study is good for identification and phylogenetic analysis of oral Veillonella species isolated from saliva samples of children in Japan. The aim of this study was to analyze the composition and proportion of oral Veillonella species in the saliva of Japanese children compared to previous studies in Thailand.
Response: Thank you for your valuable comment. The aim of this study was focus to analyze the composition and proportion of oral Veillonella species in the saliva of Japanese children. Furthermore, we compared the results with some reports such as in Thailand. The revise version has been submitted.
The manuscript is certainly well written but I have some major concerns on the data analysis:

The number of samples in this study was fewer (15 children) compared to the number of samples in the previous study from Thailand (107 children). It is suggested that the analysis uses proportions so that it can describe the oral Veillonella species according to the number of samples.

Response: These children visited to the Dental Hospital, Health Sciences University of Hokkaido for dental examination, over a period between 2016 and 2017. Based on the evaluation by the Simplified Oral Hygiene Index, they were divided into three groups, good, moderate and poor. But, among many children, only five children had poor oral hygiene status. Therefore, we used these five children as poor group. And we have selected five children from good and moderate groups, without distinction. As the results, the total subject : 15 subjects.

To find out the comparison of the results of this study with the results of a previous study from Thailand, a correlation analysis is suggested.

It is also necessary to add the results of a correlation analysis between the results of Veillonella's oral identification and oral health status.

Response: Thank you for another good point at No. 2 and No.3. The research of oral Veillonella were very limited. The analysis was use in this study : descriptive analysis with mean value to determine the distribution and proportion of oral Veillonella, then we compare the characteristic of oral veillonella distribution with the previous study.
Minor comment:

Some references used are older than 10 years. I suggest to use current references from at least the last 10 years.

Response: As we mentioned before, that the research of oral Veillonella were very limited. Therefore, some of references were used older that 10 years.
Thank you for taking the time and energy to help us improve this manuscript. We hope that you will find these revision rise to your expectation.
This study is good for identification and phylogenetic analysis of oral Veillonella species isolated from saliva samples of children in Japan. The aim of this study was to analyze the composition and proportion of oral Veillonella species in the saliva of Japanese children compared to previous studies in Thailand.
Response: Thank you for your valuable comment. The aim of this study was focus to analyze the composition and proportion of oral Veillonella species in the saliva of Japanese children. Furthermore, we compared the results with some reports such as in Thailand. The revise version has been submitted.
The manuscript is certainly well written but I have some major concerns on the data analysis:

The number of samples in this study was fewer (15 children) compared to the number of samples in the previous study from Thailand (107 children). It is suggested that the analysis uses proportions so that it can describe the oral Veillonella species according to the number of samples.

Response: These children visited to the Dental Hospital, Health Sciences University of Hokkaido for dental examination, over a period between 2016 and 2017. Based on the evaluation by the Simplified Oral Hygiene Index, they were divided into three groups, good, moderate and poor. But, among many children, only five children had poor oral hygiene status. Therefore, we used these five children as poor group. And we have selected five children from good and moderate groups, without distinction. As the results, the total subject : 15 subjects.

To find out the comparison of the results of this study with the results of a previous study from Thailand, a correlation analysis is suggested.

It is also necessary to add the results of a correlation analysis between the results of Veillonella's oral identification and oral health status.

Response: Thank you for another good point at No. 2 and No.3. The research of oral Veillonella were very limited. The analysis was use in this study : descriptive analysis with mean value to determine the distribution and proportion of oral Veillonella, then we compare the characteristic of oral veillonella distribution with the previous study.
Minor comment:

Some references used are older than 10 years. I suggest to use current references from at least the last 10 years.

Response: As we mentioned before, that the research of oral Veillonella were very limited. Therefore, some of references were used older that 10 years.
Thank you for taking the time and energy to help us improve this manuscript. We hope that you will find these revision rise to your expectation.
Competing Interests: No competing interests were disclosed. Close
Report a concern

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Version 5

VERSION 5 PUBLISHED 03 May 2019

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Version 5 (revision) 13 Sep 19		read
Version 4 (revision) 08 Aug 19		read
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Version 1 03 May 19	read	read

Juni Handajani, Gadjah Mada University, Yogyakarta, Indonesia
Takuichi Sato, Niigata University Graduate School of Health Sciences, Niigata, Japan

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16 Sep 2019 | for Version 5

Takuichi Sato, Division of Clinical Chemistry, Department of Medical Technology, Niigata University Graduate School of Health Sciences, Niigata, Japan

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Approved

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Oral Microbiology

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08 Aug 2019 | for Version 4

Takuichi Sato, Division of Clinical Chemistry, Department of Medical Technology, Niigata University Graduate School of Health Sciences, Niigata, Japan

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Approved

I have recognized and agreed with the revised parts in the Introduction.

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Oral Microbiology

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01 Aug 2019 | for Version 3

Takuichi Sato, Division of Clinical Chemistry, Department of Medical Technology, Niigata University Graduate School of Health Sciences, Niigata, Japan

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Approved With Reservations

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Reviewer Expertise

Oral Microbiology

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Author Response

08 Aug 2019

Ariadna Djais, Department of Oral Biology, Faculty of Dentistry, Faculty of Dentistry, Universitas Indonesia, Jakarta 10430, Indonesia, Jakarta, 10430, Indonesia

The authors would like to thank the area editor and the reviewers for their precious time and valuable comments. We have carefully addressed all the comments.

Reviewer: Takuichi Sato, Division of Clinical Chemistry, Department of Medical Technology, Niigata University Graduate School of Health Sciences, Niigata, Japan [Suggestions]

I recognized the revised parts in the Abstract (Background) as follows;

"However, the study regarding the identification and distribution of oral Veillonella are limited. The oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status."

I think that the above-mentioned parts should be included in the Introduction, but I was unable to find the revised parts. The authors need to revise the Introduction, like as in the Abstract.

[Typographical errors]
Introduction (6th paragraph)
"difficult to idnetify" should read "difficult to identify".

Authors: Thank you very much for this direction.

We have changed the wording of the relevant in the Introduction 6th Paragraph, L 11, like as in the Abstract.
[Typographical errors] Introduction (6th paragraph) "difficult to idnetify" into "difficult to identify".
Introduction 7th Paragraph, L 2 was removed because in this study we are focused on demonstrating that the composition and proportion of oral Veillonella species in the saliva of Japanese children is correlated with different oral hygiene status.

The revised version of this paper has been submitted.

Thank you for taking the time and energy to help us improve this manuscript. We hope that you will find that these revisions rise to your expectation.

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Competing Interests

No competing interests were disclosed.

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28 Jun 2019 | for Version 2

Takuichi Sato, Division of Clinical Chemistry, Department of Medical Technology, Niigata University Graduate School of Health Sciences, Niigata, Japan

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Approved With Reservations

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No competing interests were disclosed.

Reviewer Expertise

Oral Microbiology

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27 Jun 2019 | for Version 2

Juni Handajani, Department of Oral Biology, Faculty of Dentistry, Gadjah Mada University, Yogyakarta, Indonesia

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Approved

Thank you for the revision. I now understand the analysis statistic correctly, no more changes are required and I approved the article.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Area of my expertise are oral biology and immunology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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23 May 2019 | for Version 1

Takuichi Sato, Division of Clinical Chemistry, Department of Medical Technology, Niigata University Graduate School of Health Sciences, Niigata, Japan

15 Views Cite this report Responses(1)

Approved With Reservations

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Oral Microbiology

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Author Response

20 Jun 2019

Ariadna Djais, Department of Oral Biology, Faculty of Dentistry, Faculty of Dentistry, Universitas Indonesia, Jakarta 10430, Indonesia, Jakarta, 10430, Indonesia

Abstract and Introduction:
I think that the manuscript, in the Abstract and Introduction, needs to express rationales of this study in more detail, such as, why did the authors want to know the detection rate and distribution of oral Veillonella species in saliva of Japanese children? Why did the authors want to compare the results with those of Thai children?
Response: Thank you very much for this direction. We have changed the wording of the relevant in the abstract to express the reason why did we want to know the detection rate and distribution of oral Veillonella species in Japan. The reasons are the study regarding the identification and distribution of oral Veillonella are limited; also the oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status.
Figure 1 and Tables 1-3:
I think that the data in Figure 1 and Tables 1-3 are overlapping, and Tables 1-3 could be deleted from the manuscript, because the authors mainly stated the total and the mean (proportions) of the isolates of Veillonella species in the Results and Discussion.
Response: Thanks for raising this important point. However, we consider to state

the Table 1-3 as Ratio of the number of isolates of each species from each subject.
Figure 1 was indicated to express the Total isolated number of each Veillonella species from each group of oral hygiene.

Minor:
The authors should check the meaning of the phrase "although":

P. 7, Discussion: in the 6th paragraph
L. 1-2: "although 179 strains were isolated members of the genus Veillonella from saliva of 15 Japanese children, …".
Response: the word of “although” has been removed

P. 7, Discussion: in the 6th paragraph
L. 6-7: "although in this study 1,609 Veillonella strains were isolated from 107 Thai children".
Response: the word of “although” has been removed

P. 8, Discussion: in the 8th paragraph
L. 7-8: "although 10 unknown Veillonella strains analysed in this study formed three clusters distinct from V. dispar, …".
Response: the word of “although” has been removed
Typographical errors;
P. 4, Results: Species identification
L. 6: "numberof" should read "number of". (Put a space in between.)

Response: the word of “numberof” has been revised to “number of”

P. 7, Discussion: in the 5th paragraph
L. 4: "conforms" should read "conformed".

Response: the word of “conforms” has been revised to “conformed”
Thank you for taking the time and energy to help us improve this manuscript. We hope that you will find these revision rise to your expectation.

View more View less

Competing Interests

No competing interests were disclosed.

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Reviewer Report

31 Views

20 May 2019 | for Version 1

Juni Handajani, Department of Oral Biology, Faculty of Dentistry, Gadjah Mada University, Yogyakarta, Indonesia

31 Views Cite this report Responses(1)

Approved With Reservations

The number of samples in this study was fewer (15 children) compared to the number of samples in the previous study from Thailand (107 children). It is suggested that the analysis uses proportions so that it can describe the oral Veillonella species according to the number of samples.
To find out the comparison of the results of this study with the results of a previous study from Thailand, a correlation analysis is suggested.
It is also necessary to add the results of a correlation analysis between the results of Veillonella's oral identification and oral health status.

Minor comment:

Some references used are older than 10 years. I suggest to use current references from at least the last 10 years.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Area of my expertise are oral biology and immunology

Respond to this report

Responses (1)

Author Response

20 Jun 2019

Ariadna Djais, Department of Oral Biology, Faculty of Dentistry, Faculty of Dentistry, Universitas Indonesia, Jakarta 10430, Indonesia, Jakarta, 10430, Indonesia

This study is good for identification and phylogenetic analysis of oral Veillonella species isolated from saliva samples of children in Japan. The aim of this study was to analyze the composition and proportion of oral Veillonella species in the saliva of Japanese children compared to previous studies in Thailand.
Response: Thank you for your valuable comment. The aim of this study was focus to analyze the composition and proportion of oral Veillonella species in the saliva of Japanese children. Furthermore, we compared the results with some reports such as in Thailand. The revise version has been submitted.
The manuscript is certainly well written but I have some major concerns on the data analysis:

The number of samples in this study was fewer (15 children) compared to the number of samples in the previous study from Thailand (107 children). It is suggested that the analysis uses proportions so that it can describe the oral Veillonella species according to the number of samples.

Response: These children visited to the Dental Hospital, Health Sciences University of Hokkaido for dental examination, over a period between 2016 and 2017. Based on the evaluation by the Simplified Oral Hygiene Index, they were divided into three groups, good, moderate and poor. But, among many children, only five children had poor oral hygiene status. Therefore, we used these five children as poor group. And we have selected five children from good and moderate groups, without distinction. As the results, the total subject : 15 subjects.

To find out the comparison of the results of this study with the results of a previous study from Thailand, a correlation analysis is suggested.
It is also necessary to add the results of a correlation analysis between the results of Veillonella's oral identification and oral health status.

Response: Thank you for another good point at No. 2 and No.3. The research of oral Veillonella were very limited. The analysis was use in this study : descriptive analysis with mean value to determine the distribution and proportion of oral Veillonella, then we compare the characteristic of oral veillonella distribution with the previous study.
Minor comment:

Some references used are older than 10 years. I suggest to use current references from at least the last 10 years.

Response: As we mentioned before, that the research of oral Veillonella were very limited. Therefore, some of references were used older that 10 years.
Thank you for taking the time and energy to help us improve this manuscript. We hope that you will find these revision rise to your expectation.

View more View less

Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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Identification and phylogenetic analysis of oral Veillonella species isolated from the saliva of Japanese children

Abstract

Keywords

Revised Amendments from Version 4

Introduction

Methods

Subjects

Sample collection

Culture conditions

DNA extraction

Identification of Veillonella species

Phylogenetic analysis of unknown strains

Ethical considerations

Results

Colony numbers

Species identification

Table 1. Ratio of the number of isolates of each species to the total number of Veillonella isolate in saliva from the good oral hygiene group.

Table 2. Ratio of the number of isolates of each species to the total number of Veillonella isolate in saliva from the moderate oral hygiene group.

Table 3. Ratio of the number of isolates of each species to the total number of Veillonella isolated in saliva from the poor oral hygiene group.

Figure 1. Total isolated number of each Veillonella species isolated.

Strain characteristics

Figure 2. Neighbor-joining tree showing the relationship between the 10 unknown Veillonella strains and the type strains of established species of genus Veillonella, with accession numbers.

Discussion

Data availability

Acknowledgments

References

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