Oral Flora Composition and Its Connection to Oral Health

J. Oral Biosci. 53(3):206-212, 2011

REVIEW(A New Aspect for Systemic Disease Induced by Oral Disease)

Oral Flora Composition and Its Connection to Oral Health Yoshihisa Yamashita§ and Toru Takeshita

Section of Preventive and Public Health Dentistry, Division of Oral Health, Development and Growth, Kyushu University Faculty of Dental Science

Fukuoka 812-8582, Japan

〔Received on April 13, 2011;Accepted on June 9, 2011〕

Key words:commensal bacteria/oral health/etiology of oral disease/comprehensive analysis

Abstract:More than 700 species of commensal bacteria inhabit the human oral cavity, of which many have been of keen interest due to their pathogenicity in oral diseases(e.g., dental caries and periodontal diseases);however, the interactions between the pathogens and the remaining commensal bacteria are not well known, thus preventing us from understanding the genuine etiologies of oral diseases. To overcome this challenge, it is essential to comprehensively identify the species compositions of individual oral flora in order to associate them with various conditions of oral health and understand the virulence derived from the oral flora community. In this review, we refer to modern molecular genetic technologies, such as termi- nal restriction fragment length polymorphism, DNA microarray, and pyrosequencing analyses using bioinformatics. We also discuss their potential to further our comprehension of the complexities of floral composition.

Introduction

The etiologies of two major oral disease, dental car- ies and periodontitis, have been extensively sought for more than a century. Several specific oral bacterial species have been implicated as pathogens in these oral conditions, although both ailments show signs of being multifactorial diseases. W. D. Miller sought the specific pathogens in oral diseases in Koch’s labora- tory, but failed to identify specific bacterial species responsible for these maladies. Finally, he concluded in his publication, “The Micro-organisms of the Human Mouth”(1890), that the dental caries process was mediated by microorganisms capable of producing acid and digesting protein. His conclusion on the etiology of dental caries somewhat conforms to the mod- ern concept based on the mutual interaction of multi- bacterial species.

On the other hand, authentic culprits among the aciduric bacteria were chased by many of his succes- sors in accordance with Koch’s postulate and Miller’s chemo-parasitic theory. The group o“f mutans Streptococci”1,2),mainlycomposedofStreptococcusmutans, was the favored candidate for cariogenic bacteria, based on animal experiments that clearly demon- strated the virulence of the mutans streptococci groupindentalcaries3).Inaddition,epidemiological studies supported the etiology of the mutans;how- ever, the subjects in these studies were biased to those with deciduous or permanent teeth immedi- ately after tooth eruption and few epidemiological studies proved the mutans hypothesis in adults. Inter- estingly, a recent study with adult subjects clearly rejectedthemutanstheory4).Theetiologyofperiodontal diseases is further complicated due to modifi- cations made by multiple host immune-inflammatory responses. Porphyromonas gingivalis, Tannerella for- sythia, and Treponema denticola are most likely periodontopathogens, because they are frequently detected in subgingival sites with periodontitis. These bacterial species release proteolytic enzymes causing host tissue damage in addition to various puta- tive virulence factors;however, it is still unclear whether these particular bacterial species act as causative species or are only resultant inhabitants adapted to the anaerobic conditions of the deep pocket of periodontal lesions.

Does it seem reasonable to seek the pathogen responsible for oral diseases without considering the more than 700 bacterial species that inhabit the human oral cavity? We must take into account the interactions among or between pathogens and the sur- rounding non- or low-virulent bacterial species in order to confirm the etiology of oral diseases. Name- ly, it is better to consider the virulence of the oral microflora as a whole rather than that of particular species that constitute the flora. In this review, we delineate modern molecular genetic technologies that can be used to identify oral commensal bacterial flora and discuss the potentials of such techniques, includ- ing our own recent investigations.

Evaluation of Microfloral Composition Using Molecular Genetic Technology

Limitations of conventional cultivation techniques have been overcome by the molecular genetic approach using DNA extracted from microbial communities. The use of the 16S ribosomal RNA (rRNA)gene has become the most common method for community analysis, especially in combination with polymerase chain reactions (PCRs). The 16S rRNA gene(approximately 1,500 bp in length)is pres- ent in all bacterial species and contains nine variable regions useful for the grouping of bacteria down to the species level5), although its nucleic acid sequence remains relatively conserved among bacterial species. PCRs, with a primer set designed from the conserva- tive regions, amplify the gene from nearly all members of the bacterial community. This technique allows for the characterization of variable regions in hundreds of amplicons, thus facilitating the classifica- tion of the bacterial community. Alternatively, variable regions can be used to discriminate among the varia- tions based on the primer’s ability to hybridize to the t a r g e t c a p t u r e p r o b e.

1 .Clone library analysis

The PCR method cannot equivalently quantify the 16S rRNA gene DNA sequences from different spe- cies;however, the cloning and sequencing approach is currently regarded as the gold standard of methods analyzing amplicons to identify the composition of microbial consortia. Some researchers have sug- gested that it is much better to clone intact DNA extracted from a sample without PCR amplification and to only collect the gene to classify the actual flo- ral composition;however, such an attempt is time- consuming and not cost-effective, making it unrealis- tic when the number of samples increases, despite the use of pyrosequencing(which will be described in the latter part of this section). In general, PCR ampli- cons are cloned into a plasmid vector followed by transformation of Escherichia coli with the resultant plasmids. The inserts of the clone library are sequenced individually and are then compared to a large number of sequences deposited in a public data- base to identify their origins or the species that is most similar.

More than 1,400,000 16S rRNA gene sequences are presently registered in the Ribosomal Database Project(http://rdp.cme.msu.edu), which is frequently updated. The compositions of different oral bacterial communities from subjects with various symptoms have been investigated using clone library analysis. More than 700 bacterial species have since been revealed in oral microflora and, of these, more than 300 were not cultivated6―8). Despite the fact that clone library analysis resulted in a paradigm shift in studies associated with flora communities, several dis- advantages to using the technique are that it is labor intensive, time-consuming, and expensive. It also pre- vents us from comparing a large number of bacte- rial communities and monitoring shifts in their

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2 .Denaturing (DGGE) gradient gel electrophoresis

DGGE, the so-called fingerprinting analysis of 16S rRNA gene amplicons, provides a pattern of commu- nity diversity based on the physical separation of unique nucleic acid species9). Internal regions(around 200―700 bp)of the gene are amplified by PCR and are separated by electrophoresis with a denaturing gel. Because amplicons with different nucleic acid compositions(i.e., GC content)denature under differ- ent conditions, they separate into distinct bands under a particular denaturing gradient. The diversity of bacterial community composition can be seen in the pattern of bands. In addition, specific bands of interest can be excised from the gels and identified via direct sequencing.

The similarity in the bacterial composition of saliva between mother and child10)and the temporal stabil- ity of these bacteria in saliva11)were demonstrated by this method. Although its phylogenetic resolution limi- tations are well known, this method has confirmed dif- ferences in community structure associated with oral diseases. In combination with sequencing, DGGE is currently one of the most useful tools applicable in the comparative analysis of microbial communities.

3 .Terminal restriction fragment length poly- morphism(T-RFLP)

T-RFLP is another technique that utilizes PCR

reports of Socransky et al.14,15), whole genome DNA was used as probes;however, whole genomic probes occasionally cannot distinguish among closely related species and are limited to identifying cultivable species. Paster et al.16) introduced PCR-based re- verse-capture checkerboard hybridization to over- come these limitations. The partial region of the 16S rRNA gene, the sequence of which is unique to the target bacterial species, is designed to be a reverse- capture probe. Artificially synthesized oligonucleotide probes are deposited on a nylon membrane and the digoxigenin-labeled 16S rRNA gene amplicons, ampli- fied from the microbial community, are hybridized. Although a disadvantage of this method is the lim- ited number of species that can be selected, micro- array hybridization on glass slides, known as the Human Oral Microbe Identification Microarray17), enabled us to simultaneously analyze more than 300 species. This technique may be the most powerful approach available for characterizing the total bacte- rial community in the oral cavity, but comparing quan- titative differences in signal intensities among differ- ent bacterial species remains a challenge.

4 .Checkerboard hybridization and microar- ray analysis

Socransky et al.(1994)first described the checker- board hybridization assay of oral microbial communi- ties14), and five distinct bacterial complexes related to periodontal health were subsequently discovered when 13,261 subgingival plaque samples from 185 subjects were studied using this method15).

5 .Pyrosequencing analysis

Pyrosequencing, which became available with the development of next-generation sequencers, is a promising technology for the analysis of oral bacterial communities. Next-generation sequencing offers vast amounts of sequence data at a much lower cost than conventional sequencing techniques. This technology can handle large numbers of sequences from a single sample, but not from multiple samples;however, the addition of a tag sequence to a primer enables the simultaneous analysis of multiple distinct microbial community samples. Another disadvantage of this highly processed method is the short length gener- ated for each individual read. Indeed, in early phases of development of this technique, target sequences were restricted to about 100 bases and only the hypervariable region of the 16S rRNA gene(e.g., V6 region)18);however,readablelengthhasincreasedup to 400 bases in the current GS FLX Titanium series and more accurate bacterial taxonomic grouping is expected.

Pyrosequencing produces tremendous amounts of sequence data, and it is difficult to manually develop procedures that involve classifying based on tag sequences, trimming sequences by removing an adap- tor sequence, eliminating low-quality sequences, and searching genetic databases for corresponding bacte- rial species. Bioinformatic support for this method is expected to develop in the near future.

Relationship between Oral Indigenous Microbiota Composition and Health Conditions

Among the various approaches for analyzing 16S rRNA genes, T-RFLP is an effective method that can be used to compare large numbers of complex bacte- rial communities. In addition, accurate bacterial pre- dictions have been made possible by our efforts to improve fragment sizing accuracy19) and its applica- tionintheanalysisoforalmicrobiota20).Usingthis method and multivariate statistical analysis, we have reported the relationship between microbiota com- position patterns in the oral cavity and health conditions21―23).

1 .Oral malodor

Oral malodor primarily originates from the meta- bolic activities of indigenous bacterial populations within the oral cavity. Whether characteristic patterns of healthy and oral malodor-related microbiota or non- specific bacterial overgrowth result in oral malodor remains unclear. We examined the bacterial composi- tions in the saliva of 240 patients complaining of oral malodor that visited an oral malodor clinic23). Figure 2 shows the schema of the analytic strategy for explor- ing health conditions based on T-RFLP patterns. Using T-RFLP analysis, microbial community compo- sitions showing a similar pattern were clustered based on T-RFLP profiles, and microbial patterns of those patients exhibiting higher and lower malodor were explored. When the bacterial compositions were divided into four groups(clusters I, II, III, and IV), striking differences in malodor productivity were observed. Both the concentration of volatile sulfur compounds(VSCs)in the mouth air and organoleptic scores were much lower in cluster I than in clusters

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II and IV. Cluster grouping was highly influenced by proportional areas of the dominant peaks detected in most subjects. The T-RFLP profiles of cluster I were associated with bacterial populations with higher pro- portions of Streptococcus, Granulicatella, Rothia, and Treponema species. On the other hand, cluster II was associated with the more predominant microbiota Prevotella and Veillonella, whereas cluster IV was dominated by Neisseria, Parvimonas, Fusobacterium, and Porphyromonas. Using logistic regression analy- sis, significant differences in the levels of oral mal- odor were detected after adjustment for potential con- founding factors, such as total bacterial count, mean periodontal pocket depth, and tongue-coating score(p <0.001). These results suggested that oral malodor is a symptom based on the characteristic occupation of indigenous oral bacterial populations.

2 .Gingival condition

The relationship between gingival conditions and oral microbiota composition was investigated using thesameapproachasdescribedfororalmalodor21). The bacterial composition of the saliva of 200 sub- jects aged 15 to 40 years was depicted as peak pat- terns by T-RFLP analysis and compared using clus- ter analysis. While previous studies have aimed to identify some specific bacterial species that exhibit a

positive correlation with disease as putative patho- gens, this study focused on the relationship between the whole colonization pattern of dominant indige- nous bacteria in the microbiota and periodontal health. The bacterial composition susceptible to peri- odontitis was characterized by a predominance of Prevotella and Veillonella species, whereas microbiota with higher proportions of Neisseria, Haemophilus, and Aggregatibacter species and Porphyromonas spe- cies were implicated as a healthy pattern of oral microbiota. It is likely that this study characterized the bacterial environment surrounding a specific potential pathogen and might affect the periodontal condition through synergistic and antagonistic interac- tions in the microbial community.

3 .Incidence of pneumonia and fever in insti- tutionalized elderly adults

Pneumonia is a leading cause of death in elderly adults inhabiting nursing homes, and has been associ- ated with the aspiration of oral contents. To examine the relationship between the community structure of oral indigenous microbiota and pneumonia, tongue microbiota of 343 long-term hospitalized people over the age of 65 years were analyzed using T-RFLP22). Among the four groups classified by cluster analysis, two groups dominated by Prevotella, Veillonella, and

Treponema species showed significantly greater risks of pneumonia and fever than the cluster with a pre- dominance of Streptococcus and Rothia species. Signifi- cant differences in the incidences of pneumonia and fever were also observed after multivariate analysis to adjust for the effects of confounding factors. Another cluster was correlated with dysphagia and dif- ficulty with oral food consumption. This cluster could not be characterized using the sequences in the oral bacterial database, suggesting that the bacteria associ- ated with dysphagia are rarely detected in normal oral cavities and mostly dominate under the particular oral conditions associated with dysphagia.

Concluding Remarks

In this review, we introduced modern techniques of microfloral analysis and evaluated their abilities to effectively characterize oral microbial flora. There is no doubt that a close relationship exists between the composition of microbiota in the human oral cavity and oral health. On the other hand, a decisive method for identifying oral microflora communities as a whole has yet to be established due to the extraordinary complexity of these systems;however, even with such limitations, we can approach new infectious eti- ologies with the wealth of information gained by previ- ous analyses of indigenous bacterial species, as shown in this review. We hope that this review pro- vides readers will new insights into the field of dental research.

Acknowledgements

This study was supported in part by The Uehara Memorial Foundation (Y. Y.), Grants-in Aid for Young Scientists 23792517(T. T.)from the Ministry of Education, Culture, Sports, Science and Technology

(MEXT)of Japan and a grant of“Strategic Research Base Development”Program for Private Universities from MEXT of Japan, 2010―2014(S1001024).

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