Chapter 7 - Taxonomy of Oral Bacteria

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Abstract

The oral cavity is a collection of diverse microenvironments, each inhabited by a community of microorganisms, the majority of which are bacteria and their phages. Given the appropriate conditions, some of these bacteria can cause destruction of the teeth or their supporting hard and soft tissues. For over 300 years microbiologists have been characterising these microbial communities, in both oral health and disease. In this chapter, we take the reader on a journey through time as we discuss the various methods that have been utilised in the characterisation of the bacteria calling the oral cavity home, and how the use of these methods has informed our understanding of oral bacterial communities and the diversity of their members.

Introduction

The various structures that make up the oral cavity: the tongue, cheeks, palate, gingiva and teeth allow us to talk, chew, taste, whistle and smile. These structures provide the oral cavity with a diverse range of microenvironments, each inhabited by a community of microorganisms, the majority of which are bacteria and their phages. Community membership is influenced by myriad factors including available nutrients, pH, redox potential, substratum for adherence, receptors for adhesion and microbial interactions both agonistic and antagonistic.

The Human Oral Microbiome Database currently lists 688 prokaryotic taxa as having been found in the oral cavity (Chen et al., 2010), a figure that is still thought to underestimate the true microbial diversity in this part of the human gastrointestinal tract (Siqueira & Rocas, 2017) and its multiple unique environments (Simon-Soro et al., 2013). The oral cavity of a healthy individual is likely to provide a home to about 30–70 predominant prokaryotic species (Aas, Paster, Stokes, Olsen, & Dewhirst, 2005); friends, foes and some we do not yet know well enough to determine.

Since 1683, when Antony Van Leeuwenhoek described ‘living animalcules’ upon viewing dental plaque using a hand-crafted microscope of his own design (Porter, 1976), the quest to characterise the microbiota inhabiting the oral cavity has continued unabated. From morphologic descriptions of dental plaque in the classic microscopy studies of Listgarten and colleagues (Listgarten, 1976; Listgarten, Mayo, & Tremblay, 1975) to the comprehensive culture studies of W.E. Moore and L.V. Moore and into the genomics era of the 21st century our appreciation of the true diversity of prokaryotes in the oral cavity continues to evolve.

Studying the diversity of and relationships between prokaryotes in the oral cavity requires taxonomy: a circinate process that involves the classification of organisms based on similarities and differences, naming them according to a defined code (Parker, Tindall, & Garrity, 2015) and characterisation which allows an organism to be classified and named, or reclassified and renamed (Brenner, Staley, & Krieg, 2005). As such, prokaryotic taxonomy is not fact (Garrity, 2016) but is in a constant state of flux and is influenced in large part by the evidence provided by different laboratory techniques and methods of characterisation used at any one time. Which raises the question of why bother? In the oral cavity, classification of microorganisms allows the microbial communities at different sites, and associated with states of health or disease, to be defined. This may then enable the monitoring of health and disease with predictive models to inform targeted prevention. The aim of this chapter is to review the methods used to characterise the bacteria calling the oral cavity home, and point to the clinical significance of classifying oral prokaryotes not only at a species level, but at the whole community level.

With its combination of mucosal surfaces, and nonshedding hard tooth surfaces, the oral cavity is not one single environment, but a collection of unique microenvironments (Fig. 1). Different sites in the oral cavity such as the tongue, buccal mucosa, teeth and gingival crevice have been found to exhibit differences in the bacterial composition of their associated microbial communities (Eren, Borisy, Huse, & Mark Welch, 2014; Hall et al., 2017; Mager, Ximenez-Fyvie, Haffajee, & Socransky, 2003; Simon-Soro et al., 2013; Xu et al., 2015; Zhou et al., 2013). Further variability exists within these sites. For example, the location of the tooth surface in the oral cavity may affect the bacterial composition of the complex polymicrobial biofilm (plaque) forming at a site (Haffajee et al., 2009), and even different surfaces of the same tooth (Simon-Soro et al., 2013). This variation between microbial communities at different oral sites is likely to be the combined result of multiple factors including the surface available for colonisation (Mager et al., 2003), salivary flow and composition (Proctor et al., 2018) and the presence of other species (Eren et al., 2014). Oral microbial community profiles can also be affected by myriad environmental perturbations including dietary sugar intake, pH, oral hygiene practices, plaque mass and gingival inflammation (Haffajee et al., 2009; Haffajee, Teles, & Socransky, 2006; Kianoush et al., 2014; Uzel et al., 2011).

The tissues of the oral cavity are at risk of several diseases resulting from bacteria residing in dental plaque biofilms. The two most common of these diseases, dental caries and periodontal diseases, are considered polymicrobial, opportunistic diseases which can result in damage to the hard tooth structure, or supporting bone and soft tissues, respectively. Unlike diseases caused by exogenous pathogens, dental caries and periodontal diseases are caused by bacteria (pathobionts) that are part of our normal microbiota, the associated microorganisms being able to reside in the human mouth at low levels without causing disease. Under certain conditions, however, these bacteria can proliferate and cause destruction of soft and/or hard tissue either directly or indirectly via the host immune response (Marsh, Head, & Devine, 2015).

While evidence of both dental caries and periodontal diseases exists in ancient populations (Eshed, Gopher, & Hershkovitz, 2006; Lopez-Valverde, Lopez-Cristia, & de Diego, 2012), there has been a marked increase in the prevalence of these diseases in modern populations (Muller & Hussein, 2017; Raitapuro-Murray, Molleson, & Hughes, 2014). The oral cavity has been home to pathobionts associated with oral disease since at least prehistoric times (Warinner et al., 2014; Weyrich et al., 2017), signifying an enduring association between these microorganisms and the oral cavity. However, not only has there been an increase in the prevalence of pathobionts such as Streptococcus mutans and Porphyromonas gingivalis in modern times (Adler et al., 2013) but also an increase in the pathogenic potential of the oral microbiome. This is illustrated by the rapid evolution of sugar transport, metabolism and acid tolerance in S. mutans in response to dietary changes associated with the move from hunter-gatherer to agricultural lifestyles and the accompanying increased consumption of grains and fermentable carbohydrates (Cornejo et al., 2013). Cooccurrence of additional risk factors for oral disease such as smoking may also be related to the increased prevalence of periodontal diseases in modern populations (Hujoel, del Aguila, DeRouen, & Bergstrom, 2003). While it is possible to coexist with our oral microbiome and remain in a state of oral health, the ubiquitous nature of oral diseases in modern times raises the question of which is the atypical environment? The modern diseased oral cavity? Or are oral diseases now so prevalent that the modern disease-free oral cavity should be considered the atypical environment? Both dental caries and periodontal diseases are largely preventable with sufficient control of oral microbial biofilms and appropriate diet, and it can be argued that while our modern lifestyle leaves us more susceptible to some oral diseases than our ancestors, the disease-free oral cavity is an appropriate and achievable aim of modern dental care (Axelsson, 2014; Axelsson, Nystrom, & Lindhe, 2004; Moynihan & Kelly, 2014).

A single 10 s kiss can transfer 80 million bacterial cells from one mouth to another (Kort et al., 2014), but what is the vast and diverse collection of bacteria humans carry in their oral cavity composed of? Dewhirst and colleagues characterised the most prevalent and abundant bacterial taxa in the human oral cavity as being in the phyla Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Spirochaetes, Fusobacteria and Saccharibacteria (TM7) using an impressively comprehensive analysis of oral clones and isolates (Table 1) (Chen et al., 2010; Dewhirst et al., 2010). The Human Microbiome Project determined the bacterial community composition of 18 sites in and on the human body of over 200 healthy individuals, including 9 sites in the oral cavity (Huttenhower et al., 2012). Across all oral samples, taxa from between 185 and 322 genera were found to inhabit various oral sites which included buccal mucosa (cheek), keratinised gingiva (gums), hard palate, saliva, tongue dorsum and both supragingival (above the gingival margin) and subgingival (below the gingival margin) plaque. Individuals were found to harbour between 41 and 72 genera at a site and saliva and subgingival dental plaque exhibited the greatest diversity of microbiota (Zhou et al., 2013). At a genus level, similarity exists across sites, with Streptococcus and Pasteurellaceae unclassified identified as core genera, having been found at an abundance of > 1%, in > 80% of samples from all nine oral sites and Fusobacteria, Prevotella and Veillonella present in > 80% of samples from at least five of the nine oral sites sampled (Li, Bihan, & Methe, 2013).

While some species such as Streptococcus mitis and Gemella haemolysans appear ubiquitous across all oral sites (Aas et al., 2005; Hall et al., 2017), others show a site preference, such as Simonsiella muelleri with its specificity to the hard palate (Aas et al., 2005; Eren et al., 2014). Comparison of communities across sites indicates similarity in community membership, with site preference demonstrated by an increased abundance at a site or sites. For example, while Rothia aeria, Lautropia mirabilis, Streptococcus sanguinis and Streptococcus salivarius are found across all oral sites, their abundance indicates site preference, where R. aeria, L. mirabilis and S. sanguinis show a preference for supragingival and subgingival plaque, S. salivarius shows a preference for soft tissues being present in low abundance in supragingival and subgingival plaque (Eren et al., 2014).

Dental caries is a disease of the hard tissues of the teeth whereby acids produced by the bacterial metabolism of dietary sugars results in a reduced pH in the plaque fluid surrounding the tooth which disrupts the chemical equilibrium with the tooth and results in dissolution of calcium and phosphate ions from the tooth (Pitts et al., 2017). S. mutans has long been associated with dental caries due to its abundance in supragingival dental plaque over carious lesions (Becker et al., 2002; Loesche, Rowan, Straffon, & Loos, 1975); and its ability to rapidly convert a wide range of dietary sugars to acid (Ajdic et al., 2002; de Soet, Nyvad, & Kilian, 2000), its ability to adapt to and survive in low pH environments (Dashper & Reynolds, 1992) and the production of extracellular polysaccharides (Bowen & Koo, 2011). The importance of S. mutans as the major microbial aetiologic factor associated with dental caries is considered doubtful with findings from both bacterial culture and molecular studies unveiling a far more complex bacterial community associated with dental caries, including Lactobacillus gasseri (Aas et al., 2008; Schulze-Schweifing, Banerjee, & Wade, 2014), Lactobacillus fermentum, S. salivarius, Streptococcus parasanguinis and Veillonella sp. (Aas et al., 2008; Becker et al., 2002) and Actinomyces gerencseriae (Becker et al., 2002; Tanner et al., 2011). While most of these species can produce lactic acid from the fermentation of dietary sugars and directly contribute to the dissolution of tooth mineral, Veillonella sp. do not produce, but consume lactate. Veillonella sp. have been found to be predictive of future dental caries and have been proposed as an indicator of total acid production in the biofilm, regardless of the microbial source (Gross et al., 2012). Not only is dental caries a polymicrobial disease, the microbial aetiology may be tissue dependent with different microbial communities associated with disease in dentin and enamel (Simon-Soro, Guillen-Navarro, & Mira, 2014). These observations serve to highlight the importance of what the bacteria in these communities are doing, rather than who is present as will be discussed in the sections on metagenomics and metatranscriptomics.

Periodontitis is a multifactorial, polymicrobial infection characterised by irreversible destruction of the supporting tissues of the teeth. If left untreated, it can progress to the point at which teeth exfoliate (Kinane, Stathopoulou, & Papapanou, 2017). Based on considerable human and animal studies it has been proposed that in a susceptible host under particular environmental conditions, P. gingivalis is able to dysregulate protective host responses to shape and stabilise a dysbiotic, disease-provoking microbial community, and subsequently work with a variety of other pathobionts to sustain an uncontrolled inflammatory state resulting in irreversible periodontal bone loss (Hajishengallis, 2014). Treponema denticola and Tannerella forsythia are frequently found with the aforementioned P. gingivalis, with all three species associated with periodontal pocket depth (Gatto, Montevecchi, Paolucci, Landini, & Checchi, 2014; Pradhan-Palikhe et al., 2013; Socransky, Haffajee, Cugini, Smith, & Kent, 1998), and P. gingivalis and T. denticola also associated with disease progression (Byrne et al., 2009). However, 16S rRNA gene sequencing of subgingival dental plaque samples in cross-sectional studies has implicated a wider range of species to be associated with disease including Filifactor alocis (Camelo-Castillo, Novoa, et al., 2015; Chen et al., 2015; Griffen et al., 2012), Peptostreptococcus stomatis (Griffen et al., 2012), Treponema maltophilum (Hong et al., 2015), Desulfobulbus propionicus (Camelo-Castillo, Mira, et al., 2015; Camelo-Castillo, Novoa, et al., 2015), Fretibacterium sp. (Park et al., 2015) and Rothia sp. (Park et al., 2015). However, the role of these species in disease initiation and progression, if any, has yet to be elucidated.

Section snippets

Methods for Characterising Oral Bacteria

In 1876, Alexander Graham Bell invented the telephone and around the same time, German physician Robert Koch was attempting to culture bacteria on pieces of boiled potato (Simonson, Robinson, Pranger, Cohen, & Morton, 1992). Just as technology has advanced to allow video pictures to be sent around the world in seconds to and from wireless telephones that fit in the palm of a hand, laboratory techniques have advanced such that tiny fragments of DNA can be used to detect and identify

Conclusions

So where does this leave the characterisation of the oral microbiome? We have hope that the continued development and combination of novel laboratory methods coupled with comprehensive prospective longitudinal studies of oral health and diseases has the potential to expand our understanding of our microbial partners and how we might harness their power in an attempt to maintain oral health in a time of increased susceptibility to oral microbial biofilm-associated diseases.

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