A practical guide to the oral microbiome and its relation to health and disease

“With pure bacterial cultures, PCR amplicons (approximately 1,500 bp) of 16S rRNA genes were simply sequenced using Sanger sequencing (Sanger et al., 1977).”

“org, is the new HOMIM, which utilizes standard NGS methodologies (Caporaso et al., 2010), and is capable of species level identification for most of the prevalent oral bacterial taxa.”

“The length of sequencing reads varies depending upon the primers used, but many studies utilize about 500 base-reads for a typical sequencing run, which allows for microbial community identification (Liu et al., 2007, Liu et al., 2008, Wang et al., 2007, Mougeot et al., 2016).”

“The 16S rRNA approach was refined further using NGS methodologies to rapidly sequence hypervariable regions of the 16S rRNA genes (Caporaso et al., 2011).”

“DNA microarrays used signals from hybridization of DNA fragments to hundreds or thousands of complementary probes arrayed on a glass slide for expression profiling (Schena et al., 1995).”

“This method clonally amplified fragmented DNA on beads within an emulsion (Margulies et al., 2005).”

“This complex contained P. gingivalis, T. forsythia, and Treponema denticola and depended on earlier colonization of the pocket by the orange complex (Socransky & Haffajee, 2005, Socransky et al., 1998).”

“…anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript 16S rRNA gene sequencing The 16S rRNA has been used as an evolutionary clock (Woese, 1987) for the identification and classification of pure cultures of bacteria as well as for estimation of bacterial diversity in environmental…”

“For example, species of Rothia typically colonize the tongue or tooth surfaces, Simonsiella colonizes only the hard palate, Streptococcus salivarius mainly colonizes the tongue and treponemes are typically restricted to the subgingival crevice (Aas et al., 2005, Kazor et al., 2003, Mager et al., 2003, Segata et al., 2…”

“…of bacteria from cultural and phenotypic characterization studies, but mostly from identification of bacteria from culture-independent molecular studies using 16S rRNA gene comparative analyses (Paster et al., 2001, Aas et al., 2005, Dewhirst et al., 2010, Paster et al., 2006, Aas et al., 2008).”

“The “keystone pathogen” hypothesis suggests that specific low-abundance pathogens can influence periodontal disease by altering the “healthy” microflora to a disease state (Hajishengallis et al., 2012).”

“This instrument is sensitive enough to detect a single fluorescently labeled nucleotide (Korlach et al., 2008, Levene et al., 2003, Lundquist et al., 2008) and is purportedly able to generate ~10,000 bp reads.”

“This is achieved by an in silico search for specific ‘probe’ sequences, called ProbeSeq for HOMINGS, that targets approximately 600 species.”

“…typically colonize the tongue or tooth surfaces, Simonsiella colonizes only the hard palate, Streptococcus salivarius mainly colonizes the tongue and treponemes are typically restricted to the subgingival crevice (Aas et al., 2005, Kazor et al., 2003, Mager et al., 2003, Segata et al., 2012).”

“An array platform with broad range, higher taxa probes can help to identify/estimate a community population composition at a family or phylum level even if species level specificity is absent (Hamady & Knight, 2009).”

“(Mark Welch et al., 2016, Valm et al., 2011).”

“Molecular methods such as the 16S rRNA approach or microarrays (Aas et al., 2008) have demonstrated that in carious lesions with S. mutans, additional species belonging to the genera Atopobium, Propionibacterium, and Lactobacillus, were present at significantly higher levels.”

“Bacterial-bacterial and host-bacterial interactions It has long been known that oral bacteria exhibit specificity for their respective colonization sites and to each other, directed by adhesin-receptor binding (Kolenbrander, 2000).”

“…first pioneering microorganisms to colonize the oral cavity with Streptococcus salivarius found mostly on the tongue dorsum and in saliva, Streptococcus mitis on the buccal mucosa and Streptococcus sanguinis on the teeth (Gibbons & Houte, 1975, Socransky & Manganiello, 1971, Smith et al., 1993).”

“…of bacteria from cultural and phenotypic characterization studies, but mostly from identification of bacteria from culture-independent molecular studies using 16S rRNA gene comparative analyses (Paster et al., 2001, Aas et al., 2005, Dewhirst et al., 2010, Paster et al., 2006, Aas et al., 2008).”

“Long read lengths enable a more accurate assembly of the genomes present in the population, however the huge volume of data generated still poses a bioinformatic challenge (Grice & Segre, 2012).”

“In the primary dentition (Becker et al., 2002), S. mutans was typically detected at high levels.”

“The length of sequencing reads varies depending upon the primers used, but many studies utilize about 500 base-reads for a typical sequencing run, which allows for microbial community identification (Liu et al., 2007, Liu et al., 2008, Wang et al., 2007, Mougeot et al., 2016).”

“Furthermore, oral bacteria may be linked or serve as biomarkers for certain systemic diseases, such as pancreatic cancer (Farrell et al., 2012), diabetes type II (Demmer et al, 2015), pediatric Crohn’s Disease (Docktor et al., 2012), heart disease (Leishman et al., 2010), and low weight, preterm…”

“, 2002) and GeoChip for functional analysis (He et al., 2007).”

“The length of sequencing reads varies depending upon the primers used, but many studies utilize about 500 base-reads for a typical sequencing run, which allows for microbial community identification (Liu et al., 2007, Liu et al., 2008, Wang et al., 2007, Mougeot et al., 2016).”

“Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS), http:// homings.forsyth.org, is the new HOMIM, which utilizes standard NGS methodologies (Caporaso et al., 2010), and is capable of species level identification for most of the prevalent oral bacterial taxa.”

“This technique does rely on PCR amplification and care should be taken on which region of the 16S rRNA gene is used in analysis for accurate classification of the population (Yang et al., 2016), but nevertheless is a valuable tool to identify species in a population.”

“…techniques such as denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) separated DNA of the same size, which in turn could be sequenced for identification purposes (Anderson & Cairney, 2004, Deng et al., 2008, Nishigaki et al., 2000).”

“Several studies suggested that there may be additional red complex species (Kumar et al., 2003, Paster et al., 2001) that are associated with chronic periodontitis.”

“…typically colonize the tongue or tooth surfaces, Simonsiella colonizes only the hard palate, Streptococcus salivarius mainly colonizes the tongue and treponemes are typically restricted to the subgingival crevice (Aas et al., 2005, Kazor et al., 2003, Mager et al., 2003, Segata et al., 2012).”

“One of the most recent technologies, released in May 2015, enables sequencing of single DNA molecules (Mikheyev & Tin, 2014, Quick et al., 2014).”

“…typically colonize the tongue or tooth surfaces, Simonsiella colonizes only the hard palate, Streptococcus salivarius mainly colonizes the tongue and treponemes are typically restricted to the subgingival crevice (Aas et al., 2005, Kazor et al., 2003, Mager et al., 2003, Segata et al., 2012).”

“…P. micra, Filifactor alocis, P. alactolytcus, species of Dialister, Fusobacterium nucleatum, Treponema denticola, Porphyromonas endodontalis, P. gingivalis, T. forsythia, Prevotella baroniae, Prevotella intermedia, Prevotella nigrescens and Bacteroidaceae [G-1] HOT272 (Siqueira & Rocas, 2009).”

“…of systemic diseases including bacterial endocarditis (Berbari et al., 1997), ischemic stroke (Joshipura et al., 2003), cardiovascular disease (Beck & Offenbacher, 2005; Teles & Wang, 2011); pancreatic cancer (Farrell et al 2012), pediatric Crohn’s Disease (Docktor et al., 2012), and…”

“These advances have led to a plethora of genomic and metagenomic studies investigating the role of microbes in several different ecosystems (Gilbert & Dupont, 2011).”

“, 1997), ischemic stroke (Joshipura et al., 2003), cardiovascular disease (Beck & Offenbacher, 2005; Teles & Wang, 2011); pancreatic cancer (Farrell et al 2012), pediatric Crohn’s Disease (Docktor et al.”

“…first pioneering microorganisms to colonize the oral cavity with Streptococcus salivarius found mostly on the tongue dorsum and in saliva, Streptococcus mitis on the buccal mucosa and Streptococcus sanguinis on the teeth (Gibbons & Houte, 1975, Socransky & Manganiello, 1971, Smith et al., 1993).”

“The resultant amplicons were cloned into Escherichia coli, and the 16S rDNA inserts were sequenced to determine species identity (Hugenholtz & Pace, 1996, Paster et al., 2001).”

“The literature is quite extensive in regards to those species that are associated with periodontal disease (Socransky & Haffajee, 2005, Teles et al., 2013).”

“…uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript Table 1 Newly identified putative periodontal pathogens (from Perez-Chaparro et al 2014) Bacterial taxa Anaeroglobus geminatus HOT 121 cultivable Archaea spp. cultivable Bacteroidales [G-2] sp. oral taxon 274…”

“This was modified to identify microbial populations (Bodrossy & Sessitsch, 2004, Zhou, 2003), such as the PhyloChip to screen for 16S rDNA (Wilson et al., 2002) and GeoChip for functional analysis (He et al., 2007).”

“…including bacterial endocarditis (Berbari et al., 1997), ischemic stroke (Joshipura et al., 2003), cardiovascular disease (Beck & Offenbacher, 2005; Teles & Wang, 2011); pancreatic cancer (Farrell et al 2012), pediatric Crohn’s Disease (Docktor et al., 2012), and pneumonia (Awano et al., 2008).”

“…sequenced to identify changes in gene expression between disease and normal states to identify key pathways upregulated in disease and expression patterns of potential pathogenic factors and microbial diversity (Yost et al., 2015, Duran-Pinedo et al., 2014, Benitez-Paez et al., 2014) (Wade, 2011).”

“(Mark Welch et al., 2016, Valm et al., 2011).”

“…sequenced to identify changes in gene expression between disease and normal states to identify key pathways upregulated in disease and expression patterns of potential pathogenic factors and microbial diversity (Yost et al., 2015, Duran-Pinedo et al., 2014, Benitez-Paez et al., 2014) (Wade, 2011).”

“This was modified to identify microbial populations (Bodrossy & Sessitsch, 2004, Zhou, 2003), such as the PhyloChip to screen for 16S rDNA (Wilson et al., 2002) and GeoChip for functional analysis (He et al., 2007).”

“…anuscript A uthor M anuscript 16S rRNA gene sequencing The 16S rRNA has been used as an evolutionary clock (Woese, 1987) for the identification and classification of pure cultures of bacteria as well as for estimation of bacterial diversity in environmental samples (Rajendhran & Gunasekaran, 2011).”

“This was modified to identify microbial populations (Bodrossy & Sessitsch, 2004, Zhou, 2003), such as the PhyloChip to screen for 16S rDNA (Wilson et al., 2002) and GeoChip for functional analysis (He et al., 2007).”

“Oral bacteria as biomarkers for non-oral diseases Oral bacteria have been linked to a number of systemic diseases including bacterial endocarditis (Berbari et al., 1997), ischemic stroke (Joshipura et al., 2003), cardiovascular disease (Beck & Offenbacher, 2005; Teles & Wang, 2011); pancreatic…”

“This instrument is sensitive enough to detect a single fluorescently labeled nucleotide (Korlach et al., 2008, Levene et al., 2003, Lundquist et al., 2008) and is purportedly able to generate ~10,000 bp reads.”

“Preza et al (Preza et al., 2008) demonstrated that S. mutans was also associated with root surface caries, but that the predominant taxa included Actinomyces spp., Lactobacillus, Enterococcus faecalis, Mitsuokella sp. HOT131, Atopobium and Olsenella spp., Prevotella multisaccharivorax, P.…”

“In contrast, those adolescents in Sweden, who had very good care, were colonized only infrequently with S. mutans and S. sobrinus., but were colonized more with species of Actinomyces, Selenomonas, Prevotella, and Capnocytophaga (Johansson et al. 2016).”

“One of the most recent technologies, released in May 2015, enables sequencing of single DNA molecules (Mikheyev & Tin, 2014, Quick et al., 2014).”

“Newer technology with a similar emulsion PCR for amplification technique but with an underlying semiconductor technology (Nakano et al., 2003).”

“…to identify ~290 oral bacterial species and has been used in several disease related and oral microbiome characterization studies (Colombo et al., 2009, Colombo et al., 2012, Olson et al., 2011, Lif Holgerson et al., 2011, Duran-Pinedo et al., 2011, Torlakovic et al., 2012, Belstrom et al., 2015).”

“An intriguing suggestion has been that oral bacteria may play a role in nitric oxide (NO) homeostasis, which is important in renal and cardiovascular health (Hezel & Weitzberg, 2015).”

“The Human Oral Microbe Identification Microarray(HOMIM), another reverse-capture protocol, was developed using 379 specieslevel probes to identify ~290 oral bacterial species and has been used in several disease related and oral microbiome characterization studies (Colombo et al., 2009, Colombo et al., 2012, Olson et…”

“HOMINGS has been used in several recent studies demonstrating bacterial associations with endodontic lesions (Gomes et al., 2015), salivary microbiomes in caries and periodontitis (Belstrom et al., 2016b), temporal differences in salivary microbiomes (Belstrom et al., 2016a), and in biofilm models…”

“…sequenced to identify changes in gene expression between disease and normal states to identify key pathways upregulated in disease and expression patterns of potential pathogenic factors and microbial diversity (Yost et al., 2015, Duran-Pinedo et al., 2014, Benitez-Paez et al., 2014) (Wade, 2011).”

“In Alzheimer’s disease, inflammation, a key feature of the disease (Olsen & Singhrao, 2015), could be caused in part by peripheral infections, such as periodontal disease.”

“Oral Health The oral microbiome changes during the life of an individual from bacterial acquisition at birth (Berkowitz & Jones, 1985, Asikainen & Chen, 1999) to bacterial colonization of the elderly (Preza et al., 2008).”

“At great depth of short read sequencing, metagenomic analysis also allows quantification of copy number and allelic variants of genes within the microbial population (Vincent et al., 2016).”

“…sequenced to identify changes in gene expression between disease and normal states to identify key pathways upregulated in disease and expression patterns of potential pathogenic factors and microbial diversity (Yost et al., 2015, Duran-Pinedo et al., 2014, Benitez-Paez et al., 2014) (Wade, 2011).”

“Furthermore, oral bacteria may be linked or serve as biomarkers for certain systemic diseases, such as pancreatic cancer (Farrell et al., 2012), diabetes type II (Demmer et al, 2015), pediatric Crohn’s Disease (Docktor et al., 2012), heart disease (Leishman et al., 2010), and low weight, preterm birth (Shira Davenport…”

“Similarly, the presence of several anaerobic oral bacterial species has been shown to predispose to bacterial pneumonia including Actinobacillus actinomycetemcomitans and Streptococcus constellatus (Venkataramani et al., 1994, Shinzato & Saito, 1994).”

“A recent study showed that a consortium of A. actinomycetemcomitans, Streptococcus parasanguinis, and Filifactor alocis may serve as a biomarker of disease, i.e., predict bone loss before it occurs (Fine et al., 2013).”

“Shaddox et al (2012) showed that A. actinomycetemcomitans, Filifactor alocis, Tannerella spp. Solobacterium moorei, Parvimonas micra, and Capnocytophaga spp were most abundant in aggressive periodontitis.”

“Periodontal disease has been shown to predispose individuals to cardiovascular disease through its ability to induce chronic inflammation (Valtonen, 1991, Syrjanen, 1990).”

“Although this method requires more computing power, it is able to maximize the percentage of reads classified at the species level (Al-Hebshi et al., 2015).”

“…bacteria may be linked or serve as biomarkers for certain systemic diseases, such as pancreatic cancer (Farrell et al., 2012), diabetes type II (Demmer et al, 2015), pediatric Crohn’s Disease (Docktor et al., 2012), heart disease (Leishman et al., 2010), and low weight, preterm birth (Shira…”

“With the development of teeth, an increase in the presence of genera such as Leptotrichia and Campylobacter is observed and along with colonization by additional species such as Prevotella denticola and members of the Fusobacterium and Selenomonas genera (Kononen et al., 1994).”

“…to identify ~290 oral bacterial species and has been used in several disease related and oral microbiome characterization studies (Colombo et al., 2009, Colombo et al., 2012, Olson et al., 2011, Lif Holgerson et al., 2011, Duran-Pinedo et al., 2011, Torlakovic et al., 2012, Belstrom et al., 2015).”

“Restriction fragment length polymorphism (RFLP) was used to digest homologous DNA sequences and variation in the resulting fragment length was then used as a tool for genome mapping (Deng et al., 2008).”

“…be linked or serve as biomarkers for certain systemic diseases, such as pancreatic cancer (Farrell et al., 2012), diabetes type II (Demmer et al, 2015), pediatric Crohn’s Disease (Docktor et al., 2012), heart disease (Leishman et al., 2010), and low weight, preterm birth (Shira Davenport, 2010).”

“…first pioneering microorganisms to colonize the oral cavity with Streptococcus salivarius found mostly on the tongue dorsum and in saliva, Streptococcus mitis on the buccal mucosa and Streptococcus sanguinis on the teeth (Gibbons & Houte, 1975, Socransky & Manganiello, 1971, Smith et al., 1993).”

“This instrument is sensitive enough to detect a single fluorescently labeled nucleotide (Korlach et al., 2008, Levene et al., 2003, Lundquist et al., 2008) and is purportedly able to generate ~10,000 bp reads.”

“Over time, other microbial communities take over including Prevotella melaninogenica, Fusobacterium nucleatum, Veillonella, Neisseria and nonpigmented Prevotella (Kononen et al., 1992).”

“Another version of checkerboard hybridization was a reverse-capture protocol that utilized labeled 16S rDNA PCR products that were hybridized to 16S rDNA taxa-specific oligonucleotide probes bound to the membrane (Paster et al., 1998, Socransky et al., 1994).”

“Early studies of refractory periodontal disease demonstrated a lack of typical periodontal pathogens (Magnusson et al., 1991).”

“…to identify ~290 oral bacterial species and has been used in several disease related and oral microbiome characterization studies (Colombo et al., 2009, Colombo et al., 2012, Olson et al., 2011, Lif Holgerson et al., 2011, Duran-Pinedo et al., 2011, Torlakovic et al., 2012, Belstrom et al., 2015).”

“Since 31% of the known oral taxa still cannot be grown in vitro, bacterial culture is still important in microbiology (Vartoukian et al., 2010).”

“A more recent study implicated Actinetobacter baumannii, an important nosocomial pathogenthat is notoriously antibiotic resistant, as a risk factor for refractory periodontitis (Richards et al., 2015).”

“Periodontal disease has been shown to predispose individuals to cardiovascular disease through its ability to induce chronic inflammation (Valtonen, 1991, Syrjanen, 1990).”

“, 2016a), and in biofilm models in response to sucrose induced dysbiosis (Rudney et al., 2015).”

“…be linked or serve as biomarkers for certain systemic diseases, such as pancreatic cancer (Farrell et al., 2012), diabetes type II (Demmer et al, 2015), pediatric Crohn’s Disease (Docktor et al., 2012), heart disease (Leishman et al., 2010), and low weight, preterm birth (Shira Davenport, 2010).”

“…techniques such as denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) separated DNA of the same size, which in turn could be sequenced for identification purposes (Anderson & Cairney, 2004, Deng et al., 2008, Nishigaki et al., 2000).”

“Using the 16S rRNA approach, several predominant species that had been previously associated with odontogenic infections were detected (Flynn et al., 2012).”

“In contrast, using newer molecular techniques such as HOMIM, Colombo et al. 2009 were able to demonstrate that refractory periodontitis differed from treatable periodontitis by having a higher frequency of putative periodontal pathogens as listed above and in Table 1.”

“…to identify ~290 oral bacterial species and has been used in several disease related and oral microbiome characterization studies (Colombo et al., 2009, Colombo et al., 2012, Olson et al., 2011, Lif Holgerson et al., 2011, Duran-Pinedo et al., 2011, Torlakovic et al., 2012, Belstrom et al., 2015).”

“Additionally, the archaeal genus Methanobrevibacter, which feeds on metabolites from other gut microbes and produces methane, is typically found in the human colon and is highly active; along with other less dominant methanogenic archaea, these organisms drive bacterial metabolism by removing hydrogen from the local e…”

“There are about 700 taxa, of which one-third cannot be grown in vitro [88,89].”

“54,55 Some of these microorganisms can produce acids by metabolizing food carbohydrates with a pH reduction in the plaque within 1–3 min.”

“odontal sulcus, dental plaque, tongue, buccal mucosa, and saliva (9).”

“It has been proposed that oral microbiome play a causal role in the dynamic equilibrium with the immune-inflammatory response of the host (13, 14).”

“While bac‐ teria such as Aggregatibacter actinomycetemcomitans, Tannerella forsythia, and certain Prevotella and Treponema species have been implicated in periodontitis [4,11], specific bac‐ terial combinations are recognized as better indicators of disease, with the most well‐ known being the “red complex” consisting…”

“Its acquisition occurs from the first minutes of life, in contact with microbiota from the skin, vagina and mouth of the mother [28].”

“16S rDNA reads were BLASTed against a customized BLAST program (ProbeSeq for HOMINGS) developed at the Forsyth Institute, Cambridge, USA [27] and assigned taxonomically at Table 1.”

“The oral microbiome is incredibly complex, with 200 predominant bacterial species and 700 predominant taxa, with various interactions between different bacterial species [16].”

“The saliva microbiota has been associated with oral and general health status (Krishnan et al., 2017).”

“Studies of children with and without caries have found higher abundance of Streptococcus mutans in affected children [28] and higher abundance of Streptococcus cristatus in young children has been linked to tooth decay later in childhood [7], while Porphyromonas catonoiae and Neisseria flavescens were more abundant in…”

“As part of the HOMD, the Human Microbiome Project (HMP) provides genomic sequences for around 400 oral bacterial taxa that account for the 58% of known species [6].”

“However, the field is calling for functional investigation of the bacteria within the biofilm microenvironment and of the associated host oral immune functions to elucidate the ultimate impact of hostmicrobe interaction on dysbiosis and tissue destruction (Wade, 2011; Krishnan et al., 2017).”

“The oral microbiome, including that of plaque biofilm associated with caries, however, is complex as determined from cultural [25,26] and molecular [27] methods and comprises many different species [28].”

“of microbes from dental biofilms may not be well represented in saliva alone, particularly dental caries-causing bacteria.(124,125) Nonetheless, a growing body of evidence suggests that alterations in the oral microbiome have potential in sentinel diagnostic and prognostic applications.”

“by groups of species that live harmlessly in the mouth in very low numbers [19].”

“About 50 to 100 billion bacteria have been identified in the oral cavity and 600 prevalent taxa at the species level, with distinct subsets predominating different habitats [25].”

“Thus, the salivary microbiota is believed to be a compilation of bacteria shed from oral surfaces [3,4], which is why salivary microbiota may poten-”

“Previous reports suggest that the average adult has about 50–100 billion bacteria in the oral cavity, including as many as 500–700 bacterial species (31, 32).”

“Current methods to study the oral microbiome include cultures and microscopy, gel-based techniques, polymerase chain reactions methods, DNA microarrays and Next Generation Sequencing (NGS) techniques as part of the Human Microbiome Project that enabled a better understanding of how the microbiome impacts human health…”

“Over the past two decades, the advent of low-cost, genetic sequencing has allowed for investigation into the unexplored world of the human oral microbiome and its contribution to systemic disease [6].”

“The oral cavity is considered an important source of alterations that manifest at distant body sites, including the neural system [27].”

“The oral microbiome is an essential player that induces a dynamic equilibrium with the immune-inflammatory response of the host [12].”

“Notably, less than one-third of these taxa have been successfully cultured in vitro, highlighting the challenges of fully characterizing the microbial diversity in the oral cavity [22].”

“Therefore, NRB play an essential role in the NO 3 The microbial community of the OC consists of more than 700 prokaryotic taxa and 50–100 billion bacteria (including various NRB) [71–73].”

“Together, the pioneer traditional studies and the more advanced sequencing analyses have identified the presence of approximately 700 predominant taxa in the oral microbiome, of which approximately one-third are ‘yet-to-becultivated’ (Krishnan et al., 2017).”

“Most intriguing, recent evidence raises the possibility that changes in salivary microbiome composition may have potential as biomarkers for detecting HNCs 13 – 17 .”

“Streptococcus salivarius electively colonizes the oral mucosa and is over presenting in saliva, acting as an opportunistic pathogen that produce polysaccharides in the presence of fructose, increasing in this way salivary pH [26].”

“As saliva does not harbor an endogenous microbiota, the salivary microbiota is believed to be a compilation of bacteria shed from oral surfaces (Kilian et al. 2016; Krishnan et al. 2017).”

“Importantly, 63% of species in the human oral microbiome have been sequenced, including several uncultivated and recently-cultivated strains implicated in oral health and disease ( Krishnan et al., 2017 ; Siqueira Jr and Roˆc¸as, 2013 ).”

“The bacterial taxa colonizing the oral cavity contribute to oral health and oral diseases [33].”

“This is consistent with previous studies showing that the salivary microbiome primarily consists of bacteria shed from the surface of the mouth, especially from the tongue and throat (Segata et al., 2012; Krishnan et al., 2016).”

“Enteroco cus faecalis, another species included in this study, has a du l r le, since can act as commensal or as a pathogen, and this last role was associated with its presence in the oral cavity [14,22,37].”

“The oral microbiome harbors 50– 100 billion bacteria in the oral cavity, and it has attracted increased attention by researchers and clinicians for its accessibility compared with microbiomes at other sites (Krishnan et al., 2017).”

“mutans is not the only pathogenic bacterium related to the etiology of dental caries [13].”

“The analysis of the oral microbiota in various clinical conditions allows one to draw a specific disease risk profile for each patient and to set treatment options as personalized as possible [15,16].”

“, the supragingival and subgingival margins) [39].”

“When the flora is stable and healthy, it serves as a protective barrier, but when one or more of these factors are altered, the bacteria existing synergistically with its host can shift in composition, resulting in opportunistic infection (2).”

“In the oral cavity, Brevundimonas diminuta was detected in refractory periodontitis (Krishnan et al., 2017).”

“A perturbed balance in the oral microbiome during infancy could disrupt the equilibrium of this oral ecosystem, carrying increased risk of oral and potentially systemic diseases [9,10]; this is certainly true of the gut microbiome [11,12].”

“Methods for analyzing microorganisms present in the mouth have evolved rapidly over recent decades with the switch from culturing to DNA detection and sequencing [6].”

“The oral microbiota has multiple stages of maturation, acquiring Streptococcus as pioneer colonizers before being populated by other organisms (Table 2) [81].”

“The oral microbiota is mainly composed of bacteria, viruses, fungi, protozoa, and archaea [11].”

“The currently developed culture-independent approaches enable us to identify, classify, and characterize the uncultivable microbiological genera, as summarized in Table 1 and Figure 1 (Liu et al., 2012; Lagier et al., 2015; Krishnan et al., 2017; Benn et al., 2018; Verma et al., 2018).”

“Interest-ingly, pecies from the genus Atopobium have been previously related to carious lesions (Krishnan et al., 2017).”

“Metagenomic analyses can provide functional information that reveals the underlying mechanisms linking microbiome interactions and diseases [21].”

“The oral cavity is home to approximately 700 predominant bacterial taxa that only partly have been cultivated in vitro (24).”

“Thus, oral cavity is colonized by 50 to 100 billion bacteria [1], and they are responsible for many infectious diseases in the mouth, which include caries (tooth decay) and periodontitis (gum disease) [2,3].”

“Next-generation sequencing (NGS) has revolutionized the study of microbial diversity [15].”