The aim of this study was to analyze the distribution and phenotypic properties of the indigenous streptococci in chimpanzee (Pan troglodytes) oral cavities. Eleven chimpanzees (aged from 9 to 44 years, mean ± SD, 26.9 ± 12.6 years) in the Primate Research Institute of Kyoto University were enrolled in this research and brushing bacterial samples collected from them. Streptococci were isolated from the oral cavities of all chimpanzees. The isolates (n = 46) were identified as thirteen species by 16S rRNA genes analysis. The predominant species was Streptococcus sanguinis of mitis streptococci from five chimpanzees (45%). Mutans streptococci were isolated from six chimpanzees (55%). The predominant species in the mutans streptococci were Streptococcus troglodytae from four chimpanzees (36%), this species having been proposed as a novel species by us, and Streptococcus dentirousetti from three chimpanzees (27%). Streptococcus mutans was isolated from one chimpanzee (9%). However, Streptococcus sobrinus, Streptococcus macacae and Streptococcus downei, which are indigenous to human and monkey (Macaca fasciclaris) oral habitats, were not isolated. Of the mutans streptococci, S. troglodytae, S. dentirousetti, and S. mutans possessed strong adherence activity to glass surface.
colony forming unit
DNA Data Bank of Japan
decayed, missing, filled
- MS agar
tryptic soy broth
The distribution of oral streptococci is reportedly a means of predicting dental caries activity and oral infections. In particular, mutans streptococci are an important cause of dental caries in human and animals.
The genus Streptococcus includes gram-positive, facultative anaerobic, catalase-negative, and chain-forming or paired cocci . Based on their 16S rRNA gene sequences, Streptococci have been separated into six major clusters, namely anginosus, bovis, mitis, mutans, pyogenic and salivarius groups [2, 3]. The pyogenic group is composed of strongly β-hemolytic streptococci that are associated with infections in humans and animals. The other groups of oral streptococci have α- or no hemolytic reactions on blood agar. Oral streptococci often cause opportunistic infections in oral or non-oral sites, particularly in immunocompromised patients .
Mutans streptococci are known to form characteristic colonies on MS agar plates and to demonstrate extracellular polysaccharide synthesis. Previously, the mutans group has included seven species, two of which are Streptococcus mutans (serotypes c, e, f, and k) and Streptococcus sobrinus (serotypes d and g). Because they possess acidogenicity, glucan productivity and aciduric activities, these species are accepted as the main bacteria causing human dental caries . They can reportedly be transmitted from mothers to children . The other mutans streptococci are Streptococcus criceti (serotype a), Streptococcus ratti (serotype b), Streptococcus macacae (serotype c), Streptococcus downei (serotype h) and Streptococcus ferus (serotype c) . Additional novel species, such as Streptococcus devriesei from horses, Streptococcus orisuis (serotype d and p) from pigs, Streptococcus dentirousetti (serotype d) from bats, Streptococcus dentapri (serotype c) from wild boars and Streptococcus ursoris from bears have been reported [5-10].
Because chimpanzees are highly intelligent primates and have many human physical and behavioral characteristics, they are attractive animals on which to conduct oral health research. We have previously reported a novel species, Streptococcus troglodytae (serotype c), isolated from chimpanzee oral cavities during investigation of their oral flora . The similarity of the 16S rRNA gene of this species to the type strain of S. mutans is 96.4%, which makes it the most closely related species to S. mutans of all mutans streptococci. S. macacae and S. downei have been isolated from monkeys (Macacu fascicularis) [12, 13] that are categorized as non-human primates and are evolutionarily more ancient apes than chimpanzees. To our knowledge, there is no published data on chimpanzee oral flora, particularly not with a focus on streptococci. Therefore, we investigated the distribution and phenotypic properties of the indigenous streptococci, including a novel species of S. troglodytae, in chimpanzee oral cavities.
MATERIALS AND METHODS
Subjects and collection of bacterial samples
This study was conducted according to the guidelines of the Primate Research Institute of Kyoto University. After obtaining the approval of the Institutional Animal Welfare and Care Committee, oral specimens were collected. Eleven chimpanzees (four male and seven female) ranging in age from 9 to 44 years (mean ± SD, 26.9 ± 12.6 years) in the primate research institutes, Kyoto University (http://gcoe.biol.sci.kyoto-u.ac.jp/pgdb/index.en.html) were enrolled in this study. They were living in a group of 13 chimpanzees in an enriched outdoor compound  and have been used mainly in comparative-cognitive studies [15-18]. The chimpanzees were fed with the usual primate diet of AS pellets (Oriental Yeast, Tokyo, Japan) together with fresh items to balance their diets such as bananas, apples, sweet potatoes, carrots, oranges and cabbage. Sugarcane was also occasionally provided. The bacterial samples were collected by a toothbrush method under general anesthesia, as previously described . Briefly, the bacterial samples were collected by brushing erupted teeth with a sterile toothbrush for 1 min. Plaque adhering to the toothbrush was removed by washing in 35 mL of PBS and this was used as the original sample solution.
Using spiral plating equipment (Eddy Jet, IUL SA, Barcelona, Spain), ten-fold serial dilutions of the brushing bacterial samples with PBS were inoculated on MS agar (Difco MS agar; BD Biosciences, Franklin Lakes, NJ, USA) for isolation of streptococci and on mMSB agar (MS agar plus 150 g sucrose, 20 g yeast extract, 200 units bacitracin, 10 mg colistin, 2 mg gramicidin D, 10 mg nalidixic acid, per 1 L)  for isolation of mutans streptococci. The samples were then incubated at 37°C for 1–2 days under anaerobic conditions in AnaeroPack-Anaero jars (Mitsubishi Gas Chemical, Tokyo, Japan). Appropriate dilutions for the plates (20–200 colonies) were chosen and the number of streptococci and mutans streptococci estimated according to the formula in the manufacturers' instructions for the spiral plating equipment. Colony counts were transformed logarithmically to log10 cfu/mL of the original sample solution. Detection limits for streptococci and mutans streptococci were 1.31 log10 cfu/mL. Morphological typical colonies in grown on MS agar plates were picked up randomly, and purified by several streaking on the MS agars. The purified isolates were incubated with Bacto tryptic soy broth (TSB; BD Biosciences) at 37°C for 1 day for the following identification and characterization studies. The resulting bacterial cells were suspended in TSB in 50% glycerol and stored at − 80°C.
Identification of bacterial species
Bacterial DNA was extracted using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Amplifications of partial 16S rRNA genes and temporary identification of S. mutans and S. sobrinus species were carried out by PCR. Amplification reactions were prepared using 1 µL of templates, 1 µL of 5 µM primers and 98 µL of diluted SapphireAmp Fast PCR Master Mix (Takara Bio, Kyoto, Japan). PCR reaction mixtures were run in a thermal cycler (GeneAmp PCR system 9600; PE-ABI, Carlsbad, CA, USA) with pre-heating at 94°C for 2 mins, followed by 35 cycles of denaturing at 98°C for 5 s, annealing at 55°C for 5 s and elongation at 72°C for 1.5 mins. The PCR products were subjected to electrophoresis on a 1.5% agarose gel containing ethidium bromide and then visualized by ultraviolet trans-illumination. The PCR primer pairs for the temporary identification primers were designed for the species specific glucosyltransferase (gtf) gene sequences . Those for S. mutans were 5′-AGCCATGCGCAATCAACAGGTT-3′, and 5′-CGCAACGCGAACATCTTGATCAG-3′ and for S. sobrinus were 5′-GAAACCAACCCAACTTTAGCTTGGAT-3′ and 5′-ATGGAGTGATTTTCCATCGGTACTTG-3′. The PCR primer pairs for the 16S rRNA sequencing were (27F) 5′-AGAGTTTGATCCTGGCTCAG-3′ and (520R) 5′-ACCGCGGCTGCTGGC-3′, as described by Sakamoto et al. . The PCR products were purified using a MinElute PCR Purification Kit (Qiagen Sciences, Gaithersburg, MD, USA). Cycle-sequencing reactions were performed using an ABI BigDye terminator v1.1 Cycle Sequencing kit (Applied Biosystems, Foster, CA, USA) and ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Approximately 500 bp of the 16S rRNA gene sequences were compared with those that are available at the DNA Data Bank of Japan (DDBJ) using BLAST algorithms software, or the EZ-taxon server (http://eztaxon-e.ezbiocloud.net/) . The isolates, with similarity values for the partial 16S rRNA gene sequences of < 98% were further sequenced using (350F) 5′-TACGGAGGCAGCAG-3′, (780F) 5′-GATTAGATACCCTGGTAG-3′, (1100F) 5′-GCAACGAGCGCAACCC-3′, (920R) 5′-GTCAATTCCTTTGAGTTT-3′, (1240R) 5′-CCATTGATAGCACGTGT-3′ and (1492R) 5′-GGTTACCTTGTTACGACTT-3′ primers. Species with pair-wise similarity of < 97%, which is representatives of a single species according the EZ-taxon server, were designated as “sp.” of the nearest related species.
Adherence to glass surface and bacitracin tolerance test
Adherence to glass surface was determined as previously described . Briefly, glass tubes (13 × 100 mm) containing 4 mL of TSB without glucose + 1% sucrose were inoculated with each isolate and incubated at an angle of 45° for 24 hrs at 37°C. The glass tubes were rotated three times and the culture fluid poured off. Each culture was washed with 4 mL of PBS and gently mixed with a Vortex blender for 10 s to remove any cells that had loosely adhered to the glass surface; The tubes were scored as −, ± or + for adherence as follows: −, no visible adherence; ± , small amounts of cells and WIG on the bottom and sides of the tube; +, confluent coating of cells and WIG on the bottom and sides of the tube. Bacitracin tolerance tests were performed by streaking overnight cultures on Mitis-Salivarius-Bacitracin (MSB) agar (bacitracin 200 units/L) .
The number of colonies grown on mitis–salivarius and modified mitis–salivarius–bacitracin agars for each chimpanzee
The number of cfus grown on MS and mMSB agars and information about each chimpanzee are shown in Table 1. Streptococci were isolated from the oral cavities of all chimpanzees. The number of colonies grown on MS agar ranged from 5.64 ± 0.08 to 7.68 ± 1.25 log10 cfu/mL (mean ± SD, 6.88 ± 0.64 log10 cfu/mL). The number of colonies grown on mMSB agar ranged from zero to 5.60 ± 0.39 (mean ± SD, 2.45 ± 2.51 log10 cfu/mL).
|Samples from:||Age||Sex||MS (log10 cfu/mL)||mMSB (log10 cfu/mL)|
|Pal||9||♀||7.68 ± 1.25||5.60 ± 0.39|
|Cleo||9||♀||6.87 ± 0.14||4.42 ± 0.10|
|Ayumu||10||♂||7.54 ± 0.44||2.67 ± 2.67|
|Pan||25||♀||6.48 ± 1.72||Not detected|
|Popo||28||♀||6.86 ± 0.74||Not detected|
|Reo||29||♂||5.64 ± 0.08||Not detected|
|Pendesa||32||♀||7.44 ± 0.64||5.57 ± 0.55|
|Mari||34||♀||7.59 ± 1.89||Not detected|
|Akira||34||♂||6.74 ± 0.13||Not detected|
|Gon||42||♂||6.65 ± 0.17||5.40 ± 0.25|
|Reiko||44||♀||6.23 ± 0.83||3.30 ± 0.01|
|Mean ± SD||26.9 ± 12.6||6.88 ± 0.64||2.45 ± 2.51|
Identification of streptococcal isolates
Streptococci were isolated from the oral cavities of all chimpanzees. The isolates (n = 46) were identified as 13 different species by 16S rRNA genes analysis (Table 2). The pair-wise similarity of all isolates with their type strains was > 99% with the exception of two isolates whose similarity value were < 97%: their most closely related species were Streptococcus infantis and Streptococcus peroris. The most dominant species, Streptococcus sanguinis of the mitis streptococci (nine isolates, 18% of all isolates) was isolated from five chimpanzees (45%). Mutans streptococci were isolated from six chimpanzees (55%). The predominant species of mutans streptococci were S. troglodytae, which has been proposed as a novel species by us  (seven isolates from four chimpanzees), and S. dentirousetti (six isolates from three chimpanzees). Both S. troglodytae and S. dentirousetti were simultaneously isolated from one chimpanzee (Cleo). These two species comprised 26% of all streptococcal isolates. Two isolates of S. mutans were recovered from one chimpanzee (Pal, 9%). However, S. sobrinus, S. macacae and S. downei, which are indigenous to humans and monkeys, were not isolated.
|Popo||1||S. dysgalactiae subsp. equisimilis|
|1||S. infantis sp.|
|Akira||1||S. gallolyticus subsp. pasteurianus|
|1||S. peroris sp.|
Adherence, temporal identification by species specific gtf genes and bacitracin tolerance testing
Phenotypic characters of the isolates, such as adherence to glass surfaces, temporal identification of isolates with gtf genes and bacitracin tolerance, are shown in Table 3. The isolates that possessed sucrose-dependent adherence to glass surfaces were identified as S. troglodytae, S. dentirousetti, and S. mutans of the mutans streptococci. S. salivarius of the salivarius streptococci, S. infantis sp. and S. perosis sp. also possessed adherence activity. The other species, including S. sanguinis, S. gordonii, S. mitis, S. oralis, S. cristatus and S. dysgalactiae, possessed no or a variation of adherence activity. S. mutans-specific gtf gene positive isolates were obtained from three chimpanzees and S. sobrinus-specific gtf gene positive isolates from three chimpanzees by PCR. S. troglodytae, S. mutans, and S. dysgalactiae showed bacitracin resistance.
|Isolates||No. of chimpanzees (n)||No. of isolates (n)||Adherence (n)||PCR for S. mutans||PCR for S. sobrinus||Bacitracin tolerance|
|S. infantis sp.||+||1|
|S. peroris sp.||+||1|
Indigenous species of periodontopathic pathogens isolated from monkey oral cavities have been reported previously . In the present study, we used 16S rRNA gene sequencing to investigate the distribution of streptococci in chimpanzee oral cavities and found that the predominant mutans streptococcal species were S. troglodytae and S. dentirousetti (55%). We isolated none or a few S. sobrinus or S. mutans, which harbor in human oral cavities. We isolated neither S. downei nor S. macacae, species which harbor in monkeys' oral cavities. These results suggest that specific indigenous bacterial species harbor in the oral cavities of different animals species, depending on the evolution of the host animals.
S. dentirousetti, which resembles S. sobrinus and S. downei phylogenetically, has been isolated from bat oral cavities . We are interested in determining why S. dentirousetti is distributed in both bat and chimpanzee oral cavities.
Mutans streptococci reportedly can transmit from mothers to children . An indigenous species could be conserved in a species' oral cavities over generations by repeated transmission from mother to child. On the other side, because chimpanzees are closely related to humans, zoonotic pathogens could be transmitted to both chimpanzees and humans . Yamasaki et al. performed detailed analyses of gene sequences in isolates from humans and animals and reported that several periodontopathic species can be transmitted between humans and their companion dogs . In the present study, S. mutans was isolated from one chimpanzee (Pal); we suggest that this organism could have been transmitted from humans.
Recently, researchers have developed a pyrosequencing method for determining oral microbiota and concluded that these microbiota may include many uncultivable species . Microbiota should be cultured for identification and detailed analysis of their characteristics. In the present study, two of the isolates (S. infantis sp. and S. peroris sp.) had similarity values in 16S rRNA gene sequences of < 97%, which suggests there is a novel species in chimpanzee oral cavities.
Initially, species specific PCR designed according to gtf gene sequences was performed for temporal identification . Subsequently, the S. mutans-specific and S. sobrinus-specific PCR positive isolates were identified as S. troglodytae and S. dentirousetti, respectively, by 16S rRNA gene sequencing. According to analysis of the sequences of PCR products, the gtf sequence of S. troglodytae is similar to that of S. mutans gtf primers (Sm F5 and Sm R4) (Supplementary Fig. A). Similarly, the gtfI sequence of S. dentirousetti (accession number AB355819) is similar to the primer pairs of S. sobrinus (Ss F3 and Ss R1) (Supplementary Fig. B). S. troglodytae could be cross-acted with S. mutans gtf primers and S. dentirousetti with the primer pairs of S. sobrinus gtfI genes. Based on phylogenetic correlations, Sinozaki-Kuwahara et al. have reported that, among mutans streptococci, as well as the 16S rRNA genes and the house keeping genes, the gtf gene could be used for taxonomic determinations. The water-insoluble glucan-producing gtf gene sequences of S. sobrinus is in the cluster of S. downei, S. cricetii and S. orisuis, which is a different cluster of the S. mutans gtf gene .
Adherence to tooth surfaces, aggregation, and subsequent biofilm formation are important in the onset and progression of dental caries. In the present study, we examined the isolates for adherence activity to glass surfaces in the presence of sucrose. The mutans streptococci, such as S. troglodytae and S. dentirousetti showed strong adherence activities, which was not surprising because their gtf genes are similar to those of S. mutans and S. sobrinus. The isolate of S. salivarius also possessed adherence activity. S. salivarius reportedly possess several water insoluble gtf genes [31, 32].
Gross et al. have reported caries-associated microbial communities in young children that they identified by 16S rRNA community analysis . Large numbers of S. salivarius, S. sobrinus and S. parasanguinis are associated with caries, especially in subjects with no or small numbers of S. mutans, suggesting these species are alternative pathogens. The S. mitis/S. infantis/S. oralis group and S. sanguinis are found at more advanced stages of caries. On the other hand, S. gordonii, S. cristatus and others may have a beneficial role in the caries process. In the present study, the predominant species was S. sanguinis of the mitis streptococci (nine isolates) from five chimpanzees (45%). S. gordonii (seven isolates), S. mitis (three isolates) and S. cristatus (three isolates) were also recovered.
Momoi et al. performed simultaneous dental examinations when we obtained the brush samples for microbiological study and reported that all chimpanzee subjects were in good oral health . They counted 279 teeth in all and recorded that decay was present only in one tooth in each of three chimpanzees: Gon, Cleo and Ayumu. Breakage was the reason for the other missing teeth; there were 10 DMF teeth and the DMF index was 1.11.
Previous studies have reported that numbers of S. mutans in saliva can be used to predict caries risk. S. mutans > 6 log10 cfu/mL in saliva indicates a high caries risk and 4–5 log10 cfu/mL indicates a low to moderate risk . In the present study, mutans streptococci (S. troglodytae and/or S. dentirousetti) were isolated from three chimpanzees (Cleo, Ayumu and Gon; 4.42 ± 0.10, 2.67 ± 2.67 and 5.40 ± 0.25 log10 cfu/mL, respectively). These numbers are small and indicate moderate caries risks if the caries risk in chimpanzees is the same as in humans. However, these species were also isolated from three other chimpanzees, Pal, Pendesa and Reiko, who were caries free.
Because of the discrepancies between good oral health and the results of this microbiological study in chimpanzees, the significance of the presence of specific species of mutans streptococci should be further assessed from various viewpoints.
We are grateful to Dr. Takako Miyabe-Nishiwaki, Dr. Akihisa Kaneko, Dr. Akino Watanabe, Dr. Shohei Watanabe and Dr. Misato Hayashi of the Primate Research Institute, Kyoto University, for collaboration on this study. This study was supported in part by Grants-in-Aid from the Japan Society for the Promotion of Science (20002001, 22592350, 23220006, and 24000001) and by the Cooperative Research Program of the Primate Research Institute, Kyoto University (2009-C-20 and 2010-B-54).
The authors declare that they have no conflicts of interest.