RESEARCH ARTICLE

Screening of Probiotic Candidates in Human

Oral Bacteria for the Prevention of Dental
Disease
Tomohiko Terai1*, Takekazu Okumura1, Susumu Imai2, Masumi Nakao1, Kazuaki Yamaji1,
Masahiko Ito1, Tsuyoshi Nagata1, Kimiyuki Kaneko1, Kouji Miyazaki1, Ayako Okada2,
Yoshiaki Nomura2, Nobuhiro Hanada2
1 Yakult Central Institute, Kunitachi, Tokyo, Japan, 2 Department of Translational Research, Tsurumi
University School of Dental Medicine, Yokohama, Kanagawa, Japan
* tomohiko-terai@yakult.co.jp

Abstract
OPEN ACCESS
Citation: Terai T, Okumura T, Imai S, Nakao M,
Yamaji K, Ito M, et al. (2015) Screening of Probiotic
Candidates in Human Oral Bacteria for the
Prevention of Dental Disease. PLoS ONE 10(6):
e0128657. doi:10.1371/journal.pone.0128657
Academic Editor: Markus M. Heimesaat, Charit,
Campus Benjamin Franklin, GERMANY
Received: November 18, 2014
Accepted: April 29, 2015
Published: June 8, 2015
Copyright: 2015 Terai 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 author and source are
credited.

The oral cavity in healthy subjects has a well-balanced microbiota that consists of more
than 700 species. However, a disturbance of this balance, with an increase of harmful microbes and a decrease of beneficial microbes, causes oral disorders such as periodontal
disease or dental caries. Nowadays, probiotics are expected to confer oral health benefits
by modulating the oral microbiota. This study screened new probiotic candidates with potential oral health benefits and no harmful effects on the oral cavity. We screened 14 lactobacillus strains and 36 streptococcus strains out of 896 oral isolates derived from healthy
subjects. These bacteria did not produce volatile sulfur compounds or water-insoluble glucan, had higher antibacterial activity against periodontal bacteria, and had higher adherence activity to oral epithelial cells or salivary-coated hydroxyapatite in vitro. We then
evaluated the risk of primary cariogenicity and infective endocarditis of the selected oral isolates. As a result, Lactobacillus crispatus YIT 12319, Lactobacillus fermentum YIT 12320,
Lactobacillus gasseri YIT 12321, and Streptococcus mitis YIT 12322 were selected because they showed no cariogenic potential in an artificial mouth system and a lower risk of
experimental infective endocarditis in a rat model. These candidates are expected as new
probiotics with potential oral health benefits and no adverse effects on general health.

Data Availability Statement: All relevant data are

within the paper.
Funding: This work was funded by the Yakult Central
Institute (http://institute.yakult.co.jp/). The funder
provided support in the form of salaries for authors
TT, TO, MN, KY, MI, TN, KK, and KM, but did not
have any additional role in the study design, data
collection and analysis, decision to publish, or
preparation of the manuscript. SI and NH received
research funds from the Yakult Central Institute. All
remaining authors declare no potential conflicts of
interest with respect to the authorship and publication

Introduction
The oral cavity in healthy subjects has a well-balanced microbiota that consists of approximately 1.0 1011 microbes/g of dental plaque, and more than 700 species reside on the tongue dorsum, buccal epithelium, hard and soft palates, and other surfaces [1]. Many studies have
demonstrated that a disturbance of this balance, with an increase of harmful microbes and a
decrease of beneficial microbes, causes oral disorders, such as periodontal disease and dental
caries [2]. One of the purposes of oral health is to retain as many teeth as possible, even in the
elderly. In fact, the proportion of young adults with dental caries is decreasing and the

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Screening of Probiotic Candidates for the Prevention of Dental Disease

of this article. The specific roles of the authors are

detailed in the Author Contributions section.
Competing Interests: TT, TO, MN, KY, MI, TN, KK,
and KM are paid employees of Yakult Central
Institute. This does not alter the authors adherence
to the PLOS ONE policies on sharing data
and materials.

proportion of the elderly population with many adult teeth is increasing in developed countries
because of the establishment of daily dental care for improving oral health [3]. However, the
prevalence of periodontal disease has been estimated at approximately 75% and 74% in the
USA and Japan, respectively [4,5]. In particular, it has been revealed that periodontal disease
not only deteriorates the quality of life but also increases the risk of systemic disorders such as
coronary heart disease, cardiovascular disease, cerebrovascular disease, diabetes, pre-term low
birth weight babies, preeclampsia, and respiratory infections [6,7]. Therefore, the importance
of the prevention of periodontal disease has been recognized in countries where lifestyle-related
diseases are increasing in prevalence.
Antibiotics have been used in the treatment of infectious disease including oral disease,
however, their use should be restricted because the spread of antibiotic-resistant bacteria has
become a serious problem. Another tool that can be used to modulate the microbiota is probiotics, which are defined as living microorganisms that confer a health benefit on the host when
administered in a sufficient amount [8]. Lactobacilli and bifidobacteria are typical probiotics
that can induce many health benefits such as anti-constipation, anti-diarrhea, anti-infection,
anti-carcinogenesis, anti-inflammation, and immune modulation, based on the action of their
metabolites and cellular components [9,10]. There is accumulating evidence showing the
health benefits of oral probiotics. For example, one or more strains of lactobacilli, streptococci,
and/or bifidobacteria isolated from human oral specimens reduce the oral malodor caused by
volatile sulfur compounds (VSCs) [11,12] and prevent dental caries [1316], periodontal disease [17], and other infections such as candidiasis in the human oral cavity [18].
Conversely, the presence of some oral streptococci and lactobacilli is associated with an increased risk of cariogenicity and infectious endocarditis in humans. Mutans streptococci
(Streptococcus sobrinus and S. mutans) have the ability to convert sucrose into sticky water-insoluble glucan (WIG), one of the main factors in cariogenicity [19]. Furthermore, they are well
known and potent pathogens of dental caries caused by the accumulation of lactic acid in dental plaques after their adherence to the tooth surface. Hence, potent acid producers are considered to have the potential to cause dental caries. However, although oral streptococci such as S.
sanguinis, S. oralis, and S. salivarius are reported to produce organic acid, their acid tolerance is
weaker than that of mutans streptococci, restricting the contribution of these non-mutans oral
streptococci to cariogenicity. Several species of oral streptococci, including these three strains,
are isolated frequently from patients with endocarditis [20]; in contrast, lactobacilli are isolated
very rarely in such patients [21]. As oral bacteria always invade the blood vessels of the gum in
the oral cavity, they are potential causes of bacteremia [22]. Therefore, it is important that the
association between oral bacteria and infectious endocarditis is evaluated. The Food and Agriculture Organization/World Health Organization guidelines state that probiotics must have no
toxicity in vitro, in animal models, and in clinical trials. Oral probiotics have been screened so
far by checking their in vitro efficacy and attributes, including reduced acid production, no risk
of cariogenicity, no potential of propagating antibiotic resistance, and no general toxic parameters [23,24]. However, an evaluation of the risk for infectious endocarditis by oral probiotics
has never been reported.
This study aimed to screen new probiotic candidates from oral isolates derived from healthy
subjects with potential oral health benefits and no harmful effects by evaluating the lack of production of VSCs and WIG, antibacterial activity against pathogenic bacteria of periodontal disease and dental caries, higher adherence to salivary-coated hydroxylapatite (S-HA) or oral
epithelial cells in vitro, no cariogenicity in an artificial mouth system (AMS), and a lower risk
of experimental infectious endocarditis in a rat model. Four such probiotic strains were
successfully selected.

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Screening of Probiotic Candidates for the Prevention of Dental Disease

Materials and Methods

Subjects
We recruited 56 volunteers (34 men, 22 women; mean age 40.6 11.7 years, range 2566
years). Volunteers with a smoking habit, dental caries of more than C2, probing pocket depth
of more than 4 mm with bleeding on probing, or oral malodor evaluated by the OralChroma
(Abimedical Corporation, Kanagawa, Japan) method [25] were excluded. Finally, 32 healthy
volunteers (21 men, 11 women; mean age 39.4 10.3 years, range 2666 years) were enrolled
as subjects for the isolation of oral bacteria. The purpose and content of this study were explained fully to all subjects, whose signed informed consent was obtained prior to enrollment.
The protocol of this study was specifically approved by the Human Studies Committee of the
Yakult Central Institute, Tokyo, Japan, in accordance with the guidelines of the Helsinki Declaration and the Human Studies Committee (approval number: 017).

Collection of oral specimens and isolation of oral bacteria

Dental plaque and tongue coatings were collected from each subject and were mixed and diluted in an anaerobic transport medium [26]. Immediately after sampling, they were spread on
deMan, Rogosa, and Sharpe (MRS) agar plates (Difco; Becton Dickinson, Detroit, MI), Lactobacillus selection (LBS) agar plates (Nikken Biomedical Laboratory, Kyoto, Japan), and Mitissalivarius (MS) agar plates (Difco; Becton Dickinson). These plates were incubated at 37C for
3 days under anaerobic conditions in a globe box (Anaerobic work station Concept mini;
GSI Creos, Tokyo, Japan) or an anaerobic jar containing an AnaeroPack (Mitsubishi Gas
Chemical, Tokyo, Japan). For colonies with different morphology, 2 or 3 colonies with the
same morphology were picked up and cultured in MRS broth or brain heart infusion (BHI)
broth (Difco; Becton Dickinson) at 37C for 2448 h under the above-mentioned anaerobic
conditions. The cultured bacterial cells were collected by centrifugation (1,912 g, 10 min,
4C), suspended in the stock medium (2-fold concentrated BHI broth and glycerol, 1:1), and
frozen at -80C.

Production of volatile sulfur compounds

Each oral isolate was pre-cultured in MRS broth at 37C for 24 h under the above-mentioned
anaerobic conditions. Forty microliters of the culture were inoculated into 4 mL Gifu anaerobic
medium broth (Nissui, Tokyo, Japan) containing 1% D-glucose and 0.5 mM DL-methionine
and cultured at 37C for 24 h under anaerobic conditions. After the addition of 0.16 mL of 6 M
HCl to the culture to reduce the pH below 1, VSCs (hydrogen sulfide, methyl mercaptan, and
dimethyl sulfide) in the headspace of each culture were analyzed with an OralChroma portable
gas chromatograph according to a previous report [27]. The optical density at 550 nm (OD550)
of each culture was also measured to calculate the amount of VSCs/mL culture/1 OD550. Fusobacterium nucleatum YIT 6069T, a potent VSC producer [28], was used as a positive control.
Streptococcus mitis YIT 2035T, a low VSC producer, was used as a negative control because S.
mitis is an indigenous oral bacterium in humans and dominant in subjects without oral malodor [29]. Bacteria that produced less VSCs than S. mitis YIT 2035T were selected.

Production of water-insoluble glucan

According to a method in a previous report [30], the ability of bacteria to produce WIG was
evaluated. Briefly, each oral isolate was pre-cultured in MRS broth or BHI broth at 37C for 24
h under anaerobic conditions. Forty microliters of the culture were inoculated into 4 mL heart
infusion (HI) broth (Difco; Becton Dickinson) or a mixed broth of MRS and HI (7:3, v/v)

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Screening of Probiotic Candidates for the Prevention of Dental Disease

containing 1% sucrose and cultured at 37C at a 45 angle under anaerobic conditions. After
24 h, the culture supernatant was removed from the glass tube and 4 mL sterilized phosphate
buffered saline (sPBS) were poured slowly along the inner surface of the glass tube. The tube
was rotated gently 3 times by hand to wash its inner surface. After removing the sPBS, WIG on
the inner surface of the glass tube was evaluated visually, and its presence was used to determine whether the bacterium was a WIG producer. S. mutans ATCC 25175 and S. sobrinus
ATCC 33478 were employed as positive controls with high WIG producing potential.

Antibacterial activity against oral pathogenic bacteria

A radial diffusion assay [31] was employed to evaluate antibacterial activity against periodontal
pathogens, namely Porphyromonas gingivalis ATCC 33277, Prevotella intermedia ATCC
25611, and Aggregatibacter actinomycetemcomitans Y4, also known as ATCC 43718, and cariogenic pathogens, namely S. mutans ATCC 25175 and S. sobrinus ATCC 33478. These bacteria
were maintained at Tsurumi University. P. gingivalis ATCC 33277 and P. intermedia ATCC
25611 were pre-cultured in tryptic soy (TS) broth (Difco; Becton Dickinson) supplemented
with 5 g/mL hemin (Wako Pure Chemical Industries Ltd.) and 0.5 g/mL menadione (Wako
Pure Chemical Industries Ltd.), while the other pathogenic bacteria were pre-cultured in BHI
broth at 37C for 24 h under anaerobic conditions. The culture supernatant of the pre-culture
for 24 h of each oral isolate obtained by centrifugation under the above conditions was filtered
through a 0.22-m membrane filter (Merck Millipore, Billerica, MA). In this screening, both
the isolates with lower antibacterial activity and the isolates with lower growth potential were
excluded by adopting the same incubation time of 24 h for each oral isolate. TS agar medium,
composed of 6 mg TS, 2 L Tween 20 (Wako Pure Chemical Industries Ltd., Osaka, Japan),
and 100 mg agarose (Wako Pure Chemical Industries Ltd.) in 10 mL deionized water, and
0.1 mL of the culture of each pathogenic bacterium were mixed sufficiently to prepare an agar
plate. The agar plate was punched to make a 2.5 mm diameter well, into which 5 L of the filtrate of each oral isolate culture were added and pre-incubated at 37C for 60 min. Ten milliliters of TS agar medium without Tween 20 were poured on the agar plate to prepare the
overlaid agar plate, which was incubated at 37C for 1848 h. When P. gingivalis ATCC 33277
or P. intermedia ATCC 25611 was used as a target, 5 g/mL hemin and 0.5 g/mL menadione
were added to the TS medium. As a positive control, 82.5 U/mL bacitracin (Wako Pure Chemical Industries Ltd.) and 0.050.2 mg/mL tetracycline-HCl (Wako Pure Chemical Industries
Ltd.) were employed for cariogenic pathogens of streptococci and other periodontal disease
pathogens, respectively. Antibacterial activity was determined by the diameter of the inhibited
zone before and after adjustment of the filtrate of each oral isolate culture with a 2 N NaOH solution to pH 7 (neutralization).

Adherence activity to salivary-coated hydroxyapatite

To evaluate retention ability in the oral cavity, the ability of the bacteria to adhere to S-HA was
measured by a previously reported method [32]. Briefly, human saliva was filtered through a
0.22-m filter (Merck Millipore) after being heated at 60C for 30 min and centrifuged (10,000
g, 10 min, 4C), and S-HA beads were prepared by incubating HA beads (ceramic hydroxyapatite; Bio-Rad Laboratories, Inc., Hercules, CA) in sterilized human saliva at 37C for 30 min
with shaking. The bacterial cells of each oral isolate, pre-cultured in MRS broth under the
above conditions, were collected and washed twice in sPBS by centrifugation, and they were
then re-suspended in sPBS to adjust the OD550 to 1. Five milligrams of S-HA beads and 2 mL
of the bacterial cell suspension were incubated at 37C for 60 min with shaking. After the test
tube was left for 10 min for the S-HA beads to settle, 1 mL of the collected supernatant was

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mixed vigorously with 0.1 mL of a 0.1 M EDTA solution to dissolve the remaining HA particles. Both the OD550 of the mixture and the control containing the bacterial cell suspension
alone were measured. The adherence rate (%) to the S-HA beads was calculated using the following formula:
Adherence rate%OD550 of control  OD550 of sample=OD550 of control  100
Its adherence rate was used to determine whether the bacterium was adherent to S-HA.

Adherence to oral epithelial cells

To evaluate the retention ability of the bacteria in the oral cavity, bacterial adherence to oral epithelial cells was measured. Oral epithelial cells, JCRB0831 HO-1-N-1 (HO) cells and
JCRB0623 HSC-3 (HSC) cells, which originated from human buccal mucosa carcinoma and
human tongue carcinoma, respectively, were obtained from the JCRB cell bank (National Institute of Biomedical Innovation, Osaka, Japan). HO and HSC cells were pre-cultured in Dulbeccos modified Eagles medium (D-MEM; Gibco Life Technologies, Carlsbad, CA) and
D-MEM/F12 medium (Gibco Life Technologies), respectively, containing 10% calf serum at
37C under 5% CO2 + 95% humidified air. The cells were seeded in an 8-well Lab-Tek II chamber slide (Nunc Thermo Fisher Scientific, Rockford, IL) at 2.0 104 cells/well in 0.2 mL medium, cultured for 4896 h under the above conditions, and washed 3 times in Roswell Park
Memorial Institute (RPMI) 1640 medium (Gibco Life Technologies) to remove non-adhering
cells. The bacterial cells of each oral isolate, pre-cultured in MRS broth, collected, and washed
in sPBS under the above conditions, were suspended in sPBS and the OD660 was adjusted to
0.25. The suspension was added at 0.2 mL/well and incubated at 37C for 10 min. After washing the chamber slide in RPMI 1640 medium 3 times to remove non-adhering bacteria, the adhering bacteria were visualized after Gram staining. Thereafter, the number of adhering
bacteria per 0.16 mm2 of the sheet of oral epithelial cells was counted and averaged for 6 different fields per well under a microscope (BX50; Olympus Corporation, Tokyo, Japan). The obtained value was converted into cell count per mm2.

Experimental infective endocarditis

To assess the safety of the oral isolates when they invade blood vessels, the risk of infective endocarditis from the oral isolates was evaluated in a pre-clinical experiment using rats, according to previous reports [33,34]. Isolated bacterial cells cultured in MRS broth, collected, and
washed in sterilized saline under the above conditions, were suspended in sterilized saline to
make 4.09.0 106 colony forming units (CFU)/mL. L. rhamnosus YIT 0227 (PHLS A103/70),
a clinical isolate and an inducer of experimental infective endocarditis [35], was employed as a
positive control. The protocol of this study was specifically approved by the Ethical Committee
for Animal Experiments of Yakult Central Institute (approval numbers: 080229 and 08
0280). Male SD (Crl:CD) rats at 8 weeks of age were obtained from Charles River Laboratories
(Kanagawa, Japan) and maintained and treated in accordance with the guidelines of the Ethical
Committee for Animal Experiments of Yakult Central Institute. All efforts were made to minimize suffering. Under anesthesia with a ketamine-xylazine drug mixture (18:5), the external
jugular artery on the right side of the neck was exposed. A polyethylene catheter with an external diameter of 0.61 mm and a length of approximately 20 cm (SP10; Natsume Seisakusho,
Tokyo, Japan) was passed down the artery to the left ventricle of the heart and placed to induce
experimental infective endocarditis. The next day, each suspension of bacterial cells was injected via the trail vein at 2.05.0 106 CFU/0.5 mL/rat for 67 rats per group. After 4 days, venous blood was collected under anesthesia with Nembutal (Abbott Laboratories, Chicago, IL)

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and diluted in sterilized saline and then spread on an MRS agar plate. After the rats were sacrificed by bleeding, vegetation at the heart valve was removed, weighed, homogenized, diluted in
sterilized saline, and spread on an MRS agar plate. After incubation at 37C for 3 days, CFU/
mL of the blood and vegetation in each sample were determined. Inducing potential was judged
as positive when the bacteria administered were detected from either the blood or vegetation in
more than 2 rats per group.

Potential of primary cariogenicity

To evaluate the potential of primary cariogenicity, demineralization of bovine tooth enamel
and the production of WIG on the enamel were examined in an AMS according to a previous
report [36]. The bacterial cells of each oral isolate, pre-cultured in MRS broth, collected, and
washed in sPBS under the above conditions, were suspended in sPBS to adjust the OD540 to 1.
S. sobrinus 6715, maintained at Tsurumi University and available as ATCC 27351, was pre-cultured in TS broth at 37C for 16 h under anaerobic conditions. The AMS consisted of 3 chambers, and 4 bovine enamel slabs were placed on a Teflon holder around the bulb of a flat pH
electrode in each chamber. Each bacterial suspension and nutrient medium (MRS/TS) containing 2.5% sucrose (final concentration 1% on the flat pH electrode) were dropped continuously
on the bovine enamel slabs and incubated at 37C for 20 h to form a biofilm, while the pH underneath the artificial biofilm that formed on the flat bulb of the pH electrode was monitored
with a multiple pH recorder. After 20 h, enamel slabs and flat bulb were sufficiently washed
with sPBS to remove cell-free glucan, water-soluble glucan and the added sucrose. The biofilm
that formed on enamel slabs and flat bulb was suspended in a 0.5 N NaOH solution to dissolve
the WIG produced in the biofilm. The suspension was centrifuged (1,912 g, 20 min, 4C) to
separate WIG from the bacterial cells. The amount of WIG in the NaOH solution was quantified colorimetrically by the phenol-sulfuric acid method. Moreover, the Vickers hardness values of the enamel slabs were determined and compared before and after incubation. The
differences in hardness (gf) were used to infer the degree of demineralization.

Identification of isolates based on 16S rDNA partial sequencing

DNA was extracted using the benzyl-chloride method [37]. Cell pellets of each isolate were collected from each culture by centrifugation (1,912 g, 10 min, 4C). The pellets were re-suspended in 200 L extraction buffer (100 mM Tris-HCl, 40 mM EDTA; pH 9.0). Glass beads
(diameter, 0.1 mm; 300 mg), in 400 L benzyl chloride, were added to each resuspension and
mixed vigorously for 30 s using a FastPrep-24 (M.P. Biomedicals, Irvine, CA) at a power level
of 6.5. After mixing, 50 L of 10% sodium dodecyl sulfate were added and incubated at 50C
for 20 min, and 150 L of 3 M sodium acetate were added to each sample, which was then
cooled on ice for 10 min. After centrifugation (20,630 g, 5 min, 4C), the supernatant was collected. DNA was precipitated with isopropanol and washed with 70% ethanol. Finally, each
DNA pellet was diluted in 60 L TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH 8).
Isolates were identified at the species level through polymerase chain reaction (PCR) sequencing of the 16S rRNA gene using the universal primers 63F (50 -GCYTAAYACATGC
AAGTMG-30 ) and 15R (50 -AAGGAGGTGATCCARCCGCA-30 ) [38]. PCR was carried out in
a 35 L reaction volume containing 3.2 L of 10 PCR buffer (1 PCR buffer: 10 mM Tris-HCl
[pH 8.3], 50 mM KCl, 1.5 mM MgCl2,), 200 M each dNTP, 0.5 U Ex Taq HS DNA polymerase (TAKARA, Shiga, Japan), 0.4 M of each respective primer, and 10 ng DNA template. The
PCR amplification program consisted of an initial heating step at 94C for 20 s, 30 cycles at
94C for 20 s, 55C for 20 s, and 72C for 20 s, and a final extension step at 72C for 3 min. All
amplifications were performed on a DNA Engine Thermal Cycler (Bio-Rad Laboratories, Inc.).

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Amplicons were purified using a Montage PCR Centrifugal Filter Device (Merck Millipore)
and sequenced using the primers 63F (50 -GCYTAAYACATGCAAGTMG-30 ) and 520R (50 ACCGCGGCTGCTGGC-30 ) [39], and BigDye Terminator v1.1 Chemistry (Life Technologies)
on a 3130xl Genetic Analyzer (Life Technologies). The resulting sequences were used to search
the DDBJ database using the BLAST algorithm (http://blast.ddbj.nig.ac.jp/blast/blastn?lang =
jp), and the isolates were identified on the basis of the highest scores.

Results
Number of isolated bacteria
A total of 896 oral isolates were obtained from 32 healthy subjects. They consisted of 350 isolates from MS agar plates, 376 isolates from MRS agar plates, and 170 isolates from LBS agar
plates, and deposited as an oral bacterial library to apply the following screening steps (Fig 1).

Production of volatile sulfur compounds and water-insoluble glucan

F. nucleatum YIT 6069T, as a positive control, produced a high amount of VSCs (hydrogen sulfide, 929 g/10 mL culture/1 OD550; methyl mercaptan, 7.02 mg/10 mL culture/1 OD550). In
contrast, S. mitis YIT 2035T produced much less VSCs (hydrogen sulfide, 0.7 g/10 mL culture/1 OD550; methyl mercaptan, 1.2 g/10 mL culture/1 OD550). Dimethyl sulfide was below
the detection limit for the controls and all isolates. As shown in Table 1, 391 oral isolates in the
library had higher VSC production than S. mitis YIT 2035T. In addition, 146 oral isolates were
non-producers and 359 oral isolates were lower producers.
S. mutans ATCC 25175 and S. sobrinus ATCC 33478, recognized as cariogenic pathogens,
produced a remarkable amount of WIG. As shown in Table 1, 448 oral isolates were producers
of WIG (+). In addition, 172 oral isolates (-) and 272 oral isolates (+/-) were non-producers
and pseudo-producers of WIG, respectively. By using this screening approach, 241 (22 (WIG
(-):VSC(-)), 47 (WIG(-):VSC(+/-)), 72 (WIG(+/-):VSC(-)), and 100 (WIG(+/-):VSC(+/-)))
oral isolates, which were neither VSC nor WIG producers, were screened initially from
the library.

Identification based on 16S rDNA partial sequencing

Table 2 shows the classification of the screened 241 oral isolates identified using 16S rDNA
partial sequencing. The isolates consisted of 6 genera and 29 species. Several species that are
known to be associated with disease (e.g., S. pneumoniae causes pneumonia and S. anginosus
causes aspiration pneumonitis) were excluded. We then applied 206 isolates (62 lactobacillus
isolates and 144 streptococcus isolates) of oral lactic acid-producing bacteria (LAB) to the following screening step.

Antibacterial activity against oral pathogenic bacteria

Fig 2 shows the antibacterial activity of the culture supernatants of 206 LAB oral isolates
against 5 oral pathogenic bacteria. Antibacterial activity against P. gingivalis ATCC 33277 was
observed in all lactobacillus isolates before neutralization and most lactobacillus isolates also
showed activity after neutralization. Fifty-six lactobacillus isolates showed antibacterial activity
against P. intermedia ATCC 25611, but no strain showed antibacterial activity after neutralization. Similarly, 61 lactobacillus isolates showed antibacterial activity against A. actinomycetemcomitans Y4; however, only L. crispatus YIT 12319 still showed antibacterial activity against A.
actinomycetemcomitans Y4 after neutralization. Moreover, antibacterial activity against S.

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Fig 1. Screening of human oral probiotic candidates with potential oral health benefits and no harmful effects.
doi:10.1371/journal.pone.0128657.g001

sobrinus ATCC 33478 and S. mutans ATCC 25175 was detected in 23 and 21 lactobacillus isolates, respectively, before neutralization, but not after neutralization.
Antibacterial activity against P. gingivalis ATCC 33277 was found in 80 and 70 streptococcus isolates before and after neutralization, respectively. Forty-seven and 69 streptococcus

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Table 1. Ability of oral isolates to produce VSCs and WIG.

WIG productionb

VSC productiona

Total

+/

Total

22

47

77

146

+/

72

100

187

359

82

125

184

391

176

272

448

896

VSC (volatile sulfur compound) production in vitro was evaluated with an OralChroma. , < detection limit;

+/, produced H2S < 0.7 g/10 mL/1 OD550 or CH3SH < 1.17 g/10 mL/1 OD550; +, produced H2S > 0.7 g/
10 mL/1 OD550 or CH3SH > 1.17 g/10 mL/1 OD550; (CH3)2SH was below the detection limit in all strains.
WIG (water-insoluble glucan) production in vitro: , non-producer; +/, attachment of bacterial cells;

+, producer.
doi:10.1371/journal.pone.0128657.t001

Table 2. Identification of oral isolates based on 16S-rDNA partial sequencing.

Genus and species

Number of strains

Streptococcus salivarius

38

Streptococcus oralis

26

Streptococcus sanguinis

24

Streptococcus mitis

22

Streptococcus parasanguinis

20

Streptococcus anginosus

Streptococcus sp.

Streptococcus cristatus

Streptococcus infantis

Streptococcus pneumoniae

Streptococcus constellatus

Streptococcus gordonii

Streptococcus intermedius

Streptococcus vestibularis

Lactobacillus gasseri

31

Lactobacillus fermentum

16

Lactobacillus casei

Lactobacillus crispatus

Lactobacillus salivarius

Lactobacillus vaginalis

Lactobacillus mucosae

Lactobacillus oris

Lactobacillus ultunensis

Actinomyces sp.

Actinomyces odontolyticus

Alloscardovia omnicolens

Veillonella dispar

Veillonella atypica

Bidobacterium dentium

doi:10.1371/journal.pone.0128657.t002

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Fig 2. Antibacterial activity of oral LAB isolates against five oral pathogenic bacteria. Antibacterial activity of 206 LAB ([A] 62 lactobacilli and [B] 144
streptococci) isolates was evaluated by the radial diffusion assay. The diameter of the inhibited zone was determined before and after neutralization of the
filtrate of each isolate culture.
doi:10.1371/journal.pone.0128657.g002

isolates showed antibacterial activity against P. intermedia ATCC 25611 and A. actinomycetemcomitans Y4 before neutralization, while zero and 36 streptococcus isolates showed activity
after neutralization, respectively. No streptococcus isolate showed activity against S. mutans
and S. sobrinus before and after neutralization. Meanwhile, the culture supernatants of oral lactobacilli and streptococci had a pH of 3.85.6 and 4.26.5, respectively.

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Table 3. Adherence of oral LAB isolates to salivary-coated hydroxyapatite.

Genus and species

Number of test strains

Adherence activity to
S-HAa
+b

-c

S. salivarius

38

31

S. oralis

26

23

S. parasanguinis

20

16

S. sanguinis

24

19

S. mitis

22

13

Streptococcus sp.

S. cristatus

S. infantis

L. fermentum

16

L. gasseri

31

23

L. casei

L. salivarius

L. crispatus

L. mucosae

L. oris

L. ultunensis

The adherence of 206 LAB (62 lactobacilli and 144 streptococci) isolates was evaluated in vitro.
a

The number of strains adhesion to salivary-coated hydroxyapatite (S-HA).

+; Adherence rate >0%.

-; Includes unmeasurable samples.

b
c

doi:10.1371/journal.pone.0128657.t003

Adherence to salivary-coated hydroxylapatite

Table 3 shows the adherence of 206 oral LAB isolates to S-HA. One hundred and nine streptococcus isolates and 19 lactobacillus isolates adhered to S-HA (+). Most isolates of S. salivarius
and S. oralis demonstrated adherence to S-HA. The number of lactobacilli which adhered to
S-HA was less than that of streptococci. Due to cellular aggregation in the reaction mixture, 4
isolates of L. crispatus and 1 isolate of L. fermentum could not be evaluated.

Adherence to oral epithelial cells

Fig 3 shows the adherence of 206 oral LAB isolates to human oral squamous cell carcinoma,
that is, HO and HSC cells. As a result, 17 streptococcus isolates and 7 lactobacillus isolates
demonstrated adherence, showing more than 60 bacterial cells/mm2 for HO and/or HSC cells.
There were 3 types of isolates showing similar levels of adherence to both HSC and HO cells,
specific adherence to HSC cells by lactobacilli, and specific adherence to HO cells by streptococci. Especially, higher adherence activity with more than 600 bacterial cells/mm2 was observed for L. crispatus YIT 12319, L. crispatus LBS 1711, S. mitis MS 0632, S. mitis MS 09
51, S. salivarius MRS 0971, S. salivarius MS 0612, and S. salivarius MS 0722. Consequently,
4 lactobacillus isolates, showing both higher antibacterial activity against oral pathogenic bacteria and higher adherence to oral epithelial cells, and 10 isolates of streptococci, showing higher
antibacterial activity against oral pathogenic bacteria and higher adherence to S-HA, were selected and applied to the next screening step.

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Screening of Probiotic Candidates for the Prevention of Dental Disease

Fig 3. Adherence of oral LAB isolates to two different human oral epithelial cells. The adherence of 206 LAB (62 lactobacilli and 144 streptococci)
isolates was evaluated on cell sheets of HO and HSC cells derived from human buccal and tongue tissue, respectively, in vitro. The number of adhering cells
was averaged for 6 different fields per well. L. crispatus YIT 12319 (*1) and L. fermentum LBS 1731 (*2) show higher adherence activity to HSC cells. S.
mitis MS 2111 (*3) and S. salivarius MS 0722 show higher adherence activity to HO cells.
doi:10.1371/journal.pone.0128657.g003

Experimental infective endocarditis

Table 4 shows the results of the intravenous administration of 14 oral LAB isolates and a positive control regarding their potential to induce experimental infective endocarditis in a rat
model. L. rhamnosus YIT 0227, adopted as a positive control, was detected in the blood of 5
rats and vegetation of 100% (6/6) rats, showing an average of 8.4 CFU/mL blood and 4.7 104
CFU/g vegetation. Therefore, this strain was judged as a potent inducer of infective endocarditis in rats. Four lactobacillus isolates were not detected in the blood of any rat and their levels
were below the detection limit in the vegetation, suggesting that they had a low risk for infective
endocarditis. In contrast, 9 streptococcus isolates were detected in the blood or vegetation of
more than 2 rats of the 27 rats examined, suggesting they had a risk for infective endocarditis.
However, S. mitis YIT 12322 was not detected in the blood and was only detected in the vegetation of 1 of the 5 rats examined, suggesting a lower risk. As a result, 5 oral isolates, L. crispatus

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Screening of Probiotic Candidates for the Prevention of Dental Disease

Table 4. Potential to induce experimental infective endocarditis of oral LAB isolates.

Strain No.

L. rhamnosus YIT 0227b

Bacterial number (CFU/g/mLa)

Detection number (detection rate)

Judgment

Blood

Vegetation

Blood

Vegetation

5/6 (83%)

6/6 (100%)

8.4

4.7 104

Positive
Negative

L. crispatus YIT 12319

0/4 (0%)

0/4 (0%)

< 3.5

L. crispatus LBS 1711

0/5 (0%)

0/5 (0%)

<3.5c

Negative

L. fermentum YIT 12320

0/6 (0%)

0/6 (0%)

<3.5c

Negative

L. gasseri YIT 12321

0/5 (0%)

0/5 (0%)

<3.5c

Negative

S. infantis MRS 2031

1/3 (33%)

6/7 (86%)

33

4.7 105

Positive

S. mitis YIT 12322

0/5 (0%)

1/5 (20%)

8.5 102

Negative

S. mitis MRS 0821

3/6 (50%)

6/6 (100%)

9.3

2.7 106

Positive

S. mitis MRS 0941

5/5 (100%)

5/5 (100%)

70

1.2 106

Positive

Positive

S. oralis MRS 1981

0/5 (0%)

5/5 (100%)

2.9 10

S. salivarius MRS 0971

5/5 (100%)

5/5 (100%)

7.0 102

1.2 106

Positive

S. salivarius MRS 4932

2/2 (100%)

2/2 (100%)

9.2 104

Positive

S. salivarius MS 0722

1/1 (100%)

7/7 (100%)

1.5 103

8.4 106

Positive

S. salivarius MS 1011

3/3 (100%)

3/3 (100%)

7.9 102

1.8 106

Positive

S. salivarius MRS 1831

5/5 (100%)

5/5 (100%)

1.5 102

6.2 104

Positive

The potential to induce experimental infective endocarditis of 14 LAB (4 lactobacilli and 10 streptococci) isolates was evaluated in a rat model. The
inducing potential was judged as positive when the bacteria administered were detected in either the blood or vegetation in more than 2 rats per group (6
7 rats). Rows in which there are less than 6 rats in a group show death due to either a technical mishap or infection during the experiment.
Averaged value of the number of detected bacteria.

a
b

Positive control.

Below the detection limit.

doi:10.1371/journal.pone.0128657.t004

YIT 12319, L. crispatus LBS 1711, L. fermentum YIT 12320, L. gasseri YIT 12321, and S. mitis
YIT 12322, were screened further.

Potential of primary cariogenicity

Fig 4 shows the effects of 4 oral LAB isolates, which were selected from the 5 oral isolates
screened, and a positive control on the demineralization of bovine tooth enamel slabs, pH
change, and the production of WIG on the enamel in an AMS. After incubation in nutrient
medium (MRS/TS) containing 2.5% sucrose (final concentration 1% on the flat pH electrode),
S. sobrinus 6715 decreased the pH to 3.5, reduced the Vickers hardness value of the enamel
slabs by approximately 80%, and produced WIG, suggesting that the positive control had the
potential to demineralize the enamel as the initial step of cariogenicity. All isolates, L. crispatus
YIT 12319, L. fermentum YIT 12320, L. gasseri YIT 12321, and S. mitis YIT 12322, maintained
the pH above 6 and showed neither a reduction of the Vickers hardness value of the enamel
slabs nor WIG production. The utilization of sucrose was evaluated for the 4 isolates in ILS medium containing 2% sucrose as the sole source of sugar. L. gasseri YIT 12321 did not grow in
the medium, showing no utilization of sucrose (Fig 5), but the others utilized sucrose and grew.

Discussion
This study screened new probiotic candidates with potential oral health benefits and no harmful effects, including cardiovascular disease. We evaluated their production of VSCs, antibacterial activity against pathogenic bacteria causing periodontal disease and dental caries,

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Screening of Probiotic Candidates for the Prevention of Dental Disease

Fig 4. Effects on demineralization of bovine tooth enamel slabs in an AMS. (A) Demineralization was evaluated using the Vickers hardness values of
the enamel slabs before and after incubation with 4 LAB isolates (3 lactobacilli and 1 streptococcus) and a positive control (S. sobrinus 6715). gf (%) shows
the ratio of hardness after/before incubation. Values are mean SD (n = 4). (B) Values indicate the pH values underneath the artificial biofilm that formed on
the flat bulb of the pH electrode after incubation.
doi:10.1371/journal.pone.0128657.g004

adherence activity to S-HA or oral epithelial cells in vitro, cariogenic potential in an AMS, and
risk of experimental infectious endocarditis in a rat model.
The transit time of foods is shorter in the oral cavity than in the other areas of the digestive
tract, and oral bacteria are transferred to the stomach together with saliva, which is secreted in
large volumes. Therefore, oral probiotics must have the potential to adhere to oral tissues. S.
sanguinis, S. gordonii, S. oralis, and S. mitis are dominant oral species that reportedly adhere to
the pellicle of the tooth surface and contribute to the formation of an oral biofilm at the early
stage of its development [40]. S. sanguinis and S. salivarius also reportedly have adherent pili
that contribute to their adherence to mucin [41], a major component of saliva, and to tooth
surfaces [2]. In contrast, lactobacilli are generally known to have lower adherence ability to
teeth than streptococci. In our screening (Table 3), most of S. salivarius, S. oralis, S. parasanguinis, and S. mitis strains demonstrated adherence to S-HA, which was used as an alternative to
human teeth. Adherence activity was lower in lactobacilli than in streptococci, except for L. fermentum and L. gasseri. Our observations are consistent with findings reported previously
[41,42].
In the intestine, bacteria do not adhere to the mucosal layer directly because the thick
mucin layer acts as a reservoir of microbes. In contrast, oral epithelial tissue is coated with a
thin mucosal layer, which consists of mucin from the saliva in the oral cavity. Oral microbes
can adhere directly to oral epithelial tissue or teeth via this mucosal layer. Therefore, saliva can

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Screening of Probiotic Candidates for the Prevention of Dental Disease

Fig 5. Utilization of sucrose for growth of L. gasseri YIT 12321. Three strains of L. gasseri (2 clinical
isolates and 1 type strain) grown in 3 different media: ILS broth without lactose, ILS containing lactose, and
ILS containing sucrose instead of lactose. The degree of growth of each strain was evaluated by the O.D. 660
nm value.
doi:10.1371/journal.pone.0128657.g005

function as a reservoir of harmful and beneficial oral microbes via their adherence to tissue
[43]. In fact, tongue coating, which contains food residue and many microbes, is one of the
sites at which VSCs are produced as a source of oral malodor [44]. Therefore, it is considered
important that oral probiotics have a higher adherence potential to oral epithelial tissue, such
as the tongue, rather than to teeth. In our screening (Fig 3), L. crispatus YIT 12319 and LBS
1711 had higher adherence activity to HSC cells, which are derived from the human tongue.
In addition, we classified the bacteria into 3 types on the basis of their adherence activity: isolates showing similar adherence to both HSC and HO cells, and isolates adhering specifically to
either HSC cells or HO cells. These results suggest that common and specific receptor-ligand
systems may exist between epithelial cells and bacteria. As an intestinal-dwelling species of L.
crispatus reportedly possesses S-layer proteins that facilitate its adherence to epithelial HT-29
cells and Caco-2 cells from the colon [45], oral L. crispatus is expected to adhere to the epithelial cells of the tongue via S-layer-like proteins.
LAB reportedly show antibacterial activity against periodontal disease pathogens such as P.
gingivalis and P. intermedia [46]. As P. gingivalis and P. intermedia are highly sensitive to acid
[47], most of their activity is considered to be due to the lactic acid and other organic acids they
produce. Meanwhile, S. sobrinus and S. mutans, as cariogenic pathogens, are acid tolerant

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Screening of Probiotic Candidates for the Prevention of Dental Disease

because they express a proton-ATPase [48]. Recently, it was reported that lactic acid and other
organic acids contribute to less than 50% of the antibacterial activity of several LAB [49], and
bacteriocins have been isolated from some lactobacilli and streptococci, such as salivaricin
from a strain of L. salivarius [50] and a strain of S. salivarius K12 [51], reuterin from a strain of
L. reuteri [52], and plantaricin from a strain of L. plantarum [53]. In our screening (Fig 2),
many oral LAB isolates showed antibacterial activity against P. gingivalis and A. actinomycetemcomitans after neutralization, suggesting that the LAB may have the potential to produce
bacteriocins or other antibacterial substances in the culture supernatant.
VSCs (hydrogen sulfide, methyl mercaptan, and dimethyl sulfide) are produced by periodontal pathogenic bacteria from sulfur compounds such as L-cysteine and L-methionine
under anaerobic conditions. A recent study showed that the production of VSCs is associated
with the progression of periodontal disease [7]. Therefore, oral probiotics must have no potential to produce VSCs. Six potent VSC producers were found among 46 isolates of oral lactobacilli [23]. In our study (Table 1), 391 isolates from 896 oral isolates with higher VSC producing
ability than S. mitis YIT 2035T were excluded from the screening. For the lactobacilli, L. oris
YIT 0277 (hydrogen sulfide, 5.73 g/10 mL culture/1 OD550; methyl mercaptan, 1.2 g/10 mL
culture/1 OD550) and L. paracasei LBS 2613 (hydrogen sulfide, 9.65 g/10 mL culture/1
OD550; methyl mercaptan, 1.36 g/10 mL culture/1 OD550) showed a high level of VSC production. L. paracasei LBS 2613 was isolated from a healthy subject with no oral malodor. Lactobacilli were detected at approximately 105 CFU/g dental plaque in this healthy subject and
estimated at 0.004% of the total number of oral anaerobes (data not shown). It is suggested that
oral lactobacilli are a very minor habitant in this subject, whose oral concentration of VSCs
was lower than the detection limit. It is also indicated that a potent producer of VSCs can exist
endogenously even in healthy subjects, and the control of endogenous harmful bacteria is important to maintain oral health.
Infective endocarditis is inflammation of the inner tissue of the heart valves caused by infectious bacteria, whose attachment to the surface of an abnormal valve triggers the formation of
vegetation containing microcolonies of bacteria, immune cells, fibrin, and blood platelets [54].
It has been reported that several species of oral streptococci, such as S. sanguinis, S. salivarius,
and S. oralis, are isolated frequently from the vegetation of patients with endocarditis [20], but
lactobacilli are isolated very rarely from such patients [21]. In addition, a clinical isolate of L.
rhamnosus reportedly induces experimental infective endocarditis at a dose of 1.0 106 CFU
in a rat model [55] and a rabbit model [35]. In our screening (Table 4), 4 lactobacillus isolates
showed no induction of experimental infective endocarditis at the same dose in a rat model;
however, 9 streptococcus isolates did induce infective endocarditis, except for S. mitis YIT
12322. In further research, we will examine the differences between S. mitis YIT 12322 and the
other streptococci. There are possible factors associated with the induction of infective endocarditis, such as adherence activity to fibrin, laminin, fibronectin, collagen, and blood platelets
as component of vegetation and the expression of adherence genes.
Tooth decay remains one of the most common oral diseases worldwide, although the proportion of the elderly population with many teeth is increasing in developed countries due to
the development of daily dental care for the improvement of oral health [3]. Tooth decay is initiated by the adherence of early colonizers such as S. oralis, followed by pathogenic bacteria
such as S. sobrinus and S. mutans to tooth surfaces to form a dental plaque, which is a biofilm
of colonized bacteria with WIG-producing abilities. Thereafter, the demineralization and destruction of enamel are caused by lowering the pH below 5.5 due to the acid produced by the
bacteria from sugars in the plaque [56]. Therefore, oral probiotics must have no cariogenic potential, such as enamel demineralization associated with no or low adherence to S-HA, no production of WIG, and maintaining the pH above 5.5. In our screening (Fig 4), L. crispatus YIT

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Screening of Probiotic Candidates for the Prevention of Dental Disease

Table 5. List of probiotic candidates selected from oral LAB isolates.

Strain No.

Utilization of
sucrose

Antibacterial activity against oral pathogenic

bacteriaa,b
Pg

Pi

Aa

Sm

Ss

Adherence activity to
S-HAc

Total
(mm)

HO
cellsd

HSC
cellse

(cells/1.0 mm2)

()

(+)

()

(+)

()

(+)

()

(+)

()

(+)

()

(+)

L. crispatus YIT 12319

4.7

6.1

5.2

0.0

2.0

3.0

0.3

0.0

0.8

0.0

13.0

9.1

N.D.

66.7

864.6

L. fermentum YIT
12320

5.4

2.6

4.5

0.0

4.5

0.0

0.0

0.0

0.0

0.0

14.3

2.6

93.75

265.63

L. gasseri YIT12321

3.5

3.6

2.2

0.0

0.0

0.0

0.0

0.0

0.0

0.0

5.7

3.6

41.7

169.8

S. mitis YIT 12322

3.2

4.6

4.5

0.0

0.0

0.0

0.0

0.0

0.0

7.7

4.6

128.1

72.9

Pg, P. gingivalis ATCC 33277; Pi, P. intermedia ATCC 25611; Sm, S. mutans ATCC 25175; Ss, S. sobrinus ATCC 33478; Aa, A.

actinomycetemcomitans Y4.
b
c

(); Without neutralization, (+); with neutralization.

S-HA; salivary-coated hydroxylapatite.

Cells originating from human buccal mucosa carcinoma.

Cells originating from human tongue carcinoma.

doi:10.1371/journal.pone.0128657.t005

12319, L. fermentum YIT 12320, L. gasseri YIT 12321, and S. mitis YIT 12322 showed neither
enamel demineralization, decrease of pH below 5.5, nor the production of WIG on the enamel
following incubation in a 1.0% sucrose solution in an AMS, despite potent acid production
from glucose. It is indicated that the lack of adherence to tooth surfaces or S-HA is associated
with the absence of primary cariogenic potential in potent acid producers such as L. fermentum
YIT 12320 and probably L. crispatus YIT 12319. It is also suggested that the no or low acid production from sucrose by L. gasseri YIT 12321 and S. mitis YIT 12322 with adherence activity to
S-HA could be a reason for their lack of primary cariogenic potential.
In conclusion, this study demonstrates that 4 oral isolates from healthy subjects, L. crispatus
YIT 12319, L. fermentum YIT 12320, L. gasseri YIT 12321, and S. mitis YIT 12322 (Table 5),
are considered as new probiotic candidates because of their lack of VSC production, higher antibacterial activity against some oral pathogenic bacteria, higher adherence activity to oral epithelial cells in vitro, no potential of primitive cariogenicity, such as no demineralization of
tooth enamel or production of WIG on the enamel in the AMS, and a lower risk of experimental infectious endocarditis in a rat model. These candidates are expected as new probiotics with
potential oral health benefits and no harmful effects.
Further studies are necessary to establish a probiotic strain showing neither the potential to
propagate antibiotic resistance, general and genetic toxicity in animal models, nor side-effects
during human studies. After the safety of candidate probiotics for humans is examined in animal
and human studies (Phase 1), it is necessary to examine whether the selected oral probiotic candidates can remain in the human oral cavity to decrease the levels of oral pathogenic bacteria, and
whether these candidates have an effect and are safe as probiotics in human clinical trials (Phase
2). At the same time, it is also necessary to examine what product form, such as tablets or dairy
products, and what amount of intake is sufficient to enhance their retention in the oral cavity.

Acknowledgments
We thank the volunteers and dentists for their cooperation and sampling of oral specimens.
We thank Drs. Fumiyasu Ishikawa, Masanobu Nanno, Haruji Sawada, and Masashi Sakai at

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Screening of Probiotic Candidates for the Prevention of Dental Disease

the Yakult Central Institute for their helpful advice and good coaching. We also thank Dr.
Kenji Oishi and Ms. Wakae Yokoi for their good suggestions. We also thank Ms. Yukiko Yamamoto in our laboratory and Mr. Isamu Oosuga, Ms. Kayoko Komatsu, and Mr. Junta Murakami at the Yakult Central Institute for their technical support. We are also grateful to the staff
of the Department of Translational Research, Tsurumi University School of Dental Medicine,
for their great assistance in completing this study.

Author Contributions
Conceived and designed the experiments: TT TO MI KK KM SI YN NH. Performed the experiments: TT TO MN KY TN AO. Analyzed the data: TT TO MN MI KK KM SI YN NH. Contributed reagents/materials/analysis tools: TT TO MN KY TN AO. Wrote the paper: TT TO
KM SI YN NH.

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