molecular analysis of the oral microbiota of dental...
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Molecular Analysis of the Oral Microbiota of Dental Diseases
Eleni Kanasi
Department of Odontology, Oral Microbiology
Faculty of Medicine, Umeå University
Umeå 2008
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Copyright © Eleni Kanasi UMEÅ UNIVERSITY ODONTOLOGICAL DISSERTATION New Series No. 105 ISSN 0345-7532 ISBN 978-91-7264-657-5 Printed in Sweden by Print & Media Umeå 2008
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Σα βγεις στον πηγαιμό για την Ιθάκη,
να εύχεσαι νάναι μακρύς ο δρόμος,
γεμάτος περιπέτειες, γεμάτος γνώσεις.
Τους Λαιστρυγόνας και τους Κύκλωπας,
τον θυμωμένο Ποσειδώνα μη φοβάσαι,
τέτοια στον δρόμο σου ποτέ σου δεν θα βρεις,
αν μέν’ η σκέψις σου υψηλή, αν εκλεκτή
συγκίνησις το πνεύμα και το σώμα σου αγγίζει.
…Πάντα στον νου σου νάχεις την Ιθάκη.
Το φθάσιμον εκεί είν’ ο προορισμός σου…
Κι αν πτωχική την βρεις, η Ιθάκη δεν σε γέλασε.
Έτσι σοφός που έγινες, με τόση πείρα,
ήδη θα το κατάλαβες η Ιθάκες τι σημαίνουν.
ΙΘΑΚΗ, Κ. Π. Καβάφης, 1911
As you set out for Ithaka
hope the voyage is a long one,
full of adventure, full of discovery.
Laistrygonians and Cyclops,
angry Poseidon - don't be afraid of them:
you'll never find things like that on your way
as long as you keep your thoughts raised high,
as long as a rare excitement stirs your spirit and your body.
…Keep Ithaka always in your mind.
Arriving there is what you are destined for…
And if you find her poor, Ithaka won't have fooled you.
Wise as you will have become, so full of experience,
you will have understood by then what these Ithakas mean.
ITHAKA, Constantine Petrou Cavafy, 1911 Translated by Edmund Keeley/Philip Sherrard
In memory of my grandfather Spyridona Iatridi
Dedicated to Irini Iatridou-Kanasi, Spyridona Kanasi,
Thoma Thomou
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Abstract Traditionally, bacterial culture has been used for bacterial detection, allowing study of living microorganisms. Molecular methods are rapid and allow simultaneous identification of numerous species and uncultivated phylotypes. The objective of this doctoral thesis was to investigate the role of the oral microbiota, including poorly characterized and uncultivated bacteria, in dental caries and periodontitis, by comprehensive molecular, clinical, and statistical methods. The microbiota of 275 pre-school children (75 with caries and 200 caries-free) was examined by whole genomic DNA probes, 16S rDNA cloning and sequencing, and PCR. Streptococcus mutans, exhibiting a combined association with Streptococcus sobrinus, was significantly associated with Early Childhood Caries (ECC). Plaque from children with Severe Early Childhood Caries (S-ECC) was diverse with 138 identified and 107 unidentified taxa, which possibly included novel phylotypes. Other species/phylotypes associated with childhood caries included Lactobacillus gasseri (p<0.01), Lactobacillus fermentum, Actinomyces israelii, and Actinomyces odontolyticus (all p<0.05, ECC), Veillonella parvula (p<0.01), Veillonella atypica (p<0.05), and Veillonella sp. HOT-780 (p<0.01, S-ECC). Lactobacillus acidophilus and Lactobacillus reuteri, both used as probiotic therapy species, were detected more frequently in caries-free children than those with ECC. Fastidious periodontal species, including Parvimonas micra, Aggregatibacter actinomycetemcomitans, Eubacterium brachy, Filifactor alocis (all p <0.05), and Porphyromonas gingivalis (p<0.01), were also more frequently detected in children with dental caries than in caries-free children. Other variables associated with ECC were race, dental visit, snacking (all p<0.05), and visible dental plaque (p<0.01). The oral microbiota of early periodontitis in young adults (N=141) was analyzed by whole genomic and oligonucleotide DNA probes, and PCR. Species detected more frequently in early periodontitis than periodontal health included Treponema denticola, F. alocis, Porphyromonas endodontalis, Bacteroidetes sp. HOT-274 (oral clone AU126), and A. odontolyticus (p<0.01) by oligonucleotide DNA probes, and P. gingivalis (p<0.001) and T. forsythia (p=0.03) by PCR. Subgingival samples exhibited a higher prevalence of periodontitis-associated species than samples from tongue surface, including A. actinomycetemcomitans, T. denticola, T. forsythia (all p<0.05), and uncultivated TM7, Treponema, and Actinobaculum clones (all p<0.05). P. gingivalis (p<0.01) by PCR was associated with periodontal disease progression. Early periodontitis was associated with older age (p=0.01), male gender (p=0.04), and cigarette smoking (p=0.05). The role of bacterial subgroups in periodontitis was examined by studying the serotypeability of 313 genotyped clinical A. actinomycetemcomitans isolates (189 subjects). A total of 95 strains (30 subjects) remained non-serotypeable, although PCR revealed presence of the serotype- specific genes. The absence of the immunodominant serotype-specific antigen was confirmed by immunoblot assays. No major DNA rearrangement in the studied serotype-specific gene clusters was found. In summary, detection of previously cultured species and uncultivated phylotypes revealed the diversity of the oral microbiota in dental diseases and health already early in life. Bacterial species have insufficiently characterized subgroups that may have attributes to evade the host response. Molecular approaches used in this study enable comprehensive, culture-independent characterization of the oral microbiome that may in the future lead to identification of diagnostic bacterial profiles for dental diseases.
Key words: Early Childhood Caries, Early Periodontitis, 16S rDNA cloning and sequencing, whole genomic DNA probes, oligonucleotide DNA probes, PCR, diversity, molecular, Aggregatibacter actinomycetemcomitans, serotypes
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Table of Contents List of Papers.............................................................................................................. 6 Introduction………………………………………………………………………………………………… 7 Genomic Era of Microbiological Identification……………………………………………. 7 Oral Microbiota…………………………………………………………………………………..…… 8 Dental Plaque – Colonization Sites and Diversity………………………………….. 9 Dental Diseases………………………………………………………………………………………... 10 Bacterial Etiology of Dental Diseases – Hypotheses………………………………. 11 Dental Caries – Caries Development and Risk Factors…………………………… 11 Dental Caries in Children………………………………………………………………….... 12 Microbiota of Dental Caries in Children……………………………………………….. 13 Periodontal Diseases and Risk Factors…………………………………………………. 15 Microbiota of Periodontal Health and Disease………………………………………. 16 Molecular Microbiological Identification of Oral Bacteria…………………………….. 18 Hypothesis and Aims…………………………………………………………………………………….. 20 Materials and Methods………………………………………………………………………………….. 21 Study Populations and Clinical Measurements…………………………………………….. 21 Microbial Analysis…………………………………………………………………………………….. 23 DNA-DNA Hybridization…………………………………………………………………….. 23 Polymerase Chain Reaction……………………………………………………………….... 24 Cloning and Sequencing………………..……………………………………………………. 25 Antigen Identification……………..………...……………………………………………………… 26 Statistical Analysis…………………………………………………………………………………….. 26 Results and Discussion………………………………………………………………………………….. 28 Oral Microbiota in Patients with Childhood Caries…………………………................ 28 Mutans and Non-mutans Streptococci………………………………………………….. 30 Lactobacillus Species………………………………………………………………………….. 31 Other Species…………………………………………………………………………………….. 32 Periodontitis-associated Bacteria…………………………………………………………. 34 Socio-demographic, Dietary, and Clinical Factors………………………………….. 34 Oral Microbiota of Early Adult Periodontitis……………………………………………….. 36 Microbiota of Early Periodontitis and Periodontally Healthy Subjects…….. 36 Subgingival and Tongue Surface Microbiota………………………………………….. 38 Progressing Chronic Periodontitis………………………………………………………… 38 Socio-demographic Factors in Early Periodontitis………………………………….. 40 Aggregatibacter actinomycetemcomitans Lack of Serotype Antigen Expression………………………………………………………………………………………………… 40 Summary and Conclusions……………………………………………………………………………... 42 Acknowledgments………………………………………………………………………………………….. 44 References…………………………………………………………………………………………………….. 46 Papers I-V………………………………………………………………………………………… Appendix I-V
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List of Papers
The original papers that comprise this doctoral thesis are listed below and will be
referred to by Roman numerals in the text.
I. Kanasi E, Johansson I, Lu SC, Kressin NR, Nunn ME, Kent R Jr, Tanner
ACR. Oral Microbiota of Pre-School Children in Pediatrician’s Offices. J
Dent Res 2008. Re-submitted for publication (after revision).
II. Kanasi E, Kent R Jr, Barbuto S, Moore A, Hughes CV, Pradhan N,
Dewhirst FE, Tanner ACR. Molecular Analysis of the Oral Microbiota of
Severe Early Childhood Caries. Manuscript.
III. Tanner ACR, Paster BJ, Lu SC, Kanasi E, Kent R Jr, Van Dyke T, Sonis ST.
Subgingival and Tongue Microbiota during Early Periodontitis. J Dent Res
2006; 85: 318-323
IV. Tanner ACR, Kent R Jr, Kanasi E, Lu SC, Paster BJ, Sonis ST, Murray LA,
Van Dyke TE. Clinical Characteristics and Microbiota of Progressing Slight
Chronic Periodontitis in Adults. J Clin Periodontol 2007; 34: 917-930
V. Kanasi E, Doğan B, Karched M, Thay B, Oscarsson J, Asikainen S. Lack of
Serotype Antigen Expression in Aggregatibacter actinomycetemcomitans.
Manuscript.
Copyright permission was kindly granted by the publishers of the included material
in the thesis.
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Introduction
Genomic Era of Microbiological Identification
Historically, bacteria have been isolated by culture methods and classified according
to their phenotypic features, including cellular characteristics and metabolic profiles.
Bacterial cultivation made it possible to work with living microorganisms, examine
their physiology, antimicrobial resistance, and pathogenicity. In medical
microbiology, commensal and pathogenic microorganisms were isolated in pure
cultures using artificial media in well-defined conditions. This approach was fruitful
and essential for defining the field of microbiology. Despite the extensive microbial
cultivation, however, it has been estimated that >99% of microorganisms found in
nature cannot be cultured using standard techniques (Hugenholtz et al., 1998).
Moreover, researchers have been for years biased towards evaluations of single
microorganisms in planktonic states, which rarely reflects the complexity of in vivo
biofilms (Kolter, 2005).
In the 1970s, DNA-DNA hybridization was introduced for differentiating bacterial
species, where homology greater than 70% between isolates defined the same species
(Medini et al., 2008). Ten years later, the introduction of the 16S rRNA
macromolecule to facilitate determination of phylogenetic relationships (Lane et al.,
1985), marked a new genomic era for microbiologists. The 16S rRNA molecule was
chosen for its universal distribution, high information content, large enough size
(1,500 bp) for informatics purposes, and inclusion of highly conserved primer
binding sites, as well as hypervariable regions which could provide species-specific
sequences (Janda & Abbott, 2007). Following the improvement and availability of
DNA techniques, the field of molecular microbiology has greatly expanded.
One of the major advances of molecular microbiology was recognizing the
prokaryotic diversity. Novel phylotypes are continuously being identified from
different environments. Multidisciplinary approaches, including statistical and
mathematical models, are used to manage the vast amount of data produced (Medini
et al., 2008). The field of metagenomics has also been created to address whole
community genomes and facilitate examination of the diversity of cultured and
uncultivated microorganisms (Stein et al., 1996). The extent of prokaryotic diversity
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is a complicated issue which has divided the scientific community into two opposing
sides; those who believe that the diversity is large (Hugenholtz et al., 1998) and those
who claim that fewer novel sequences are obtained annually suggesting that most
taxa have been identified (Hagstrom et al., 2002). Differences could be in part due
to varying methodologies, differing microbial environments, and sample sizes (Curtis
et al., 2006).
Organization complexity of microbial communities has also been a major recent
research focus. Bacterial biofilms, matrix-enclosed populations of bacteria which
adhere to surfaces and to each other, are diverse and exhibit intricate architectural
characteristics, such as nutrient channels (Costerton et al., 1999). Molecular genetics
are used in biofilm research to identify genes and regulatory pathways required for
biofilm development (Davey & O'Toole G, 2000). Sessile bacterial cells forming
biofilms differ from plaktonic cells observed in suspended culture in terms of protein
and genomic expression (Sauer & Camper, 2001; Sauer et al., 2002). Gene
expression involved for instance in motility, intra- and inter-species communication,
and antibiotic resistance, differs in bacteria growing as biofilms compared with those
in planktonic lifeform (Davey & O'Toole G, 2000; Gjersing et al., 2007; Kolter &
Greenberg, 2006; Whiteley et al., 2001). Understanding bacterial biofilm formation
and regulation can be pivotal from a clinical viewpoint, since multiple chronic
infections in the human body, such as osteomyelitis, endocarditis, cystic fibrosis
pneumonia, otitis media, periodontitis, and dental caries, involve biofilms (Costerton
et al., 1999).
Oral Microbiota
The human oral cavity is a diverse environment with hard and soft tissues
comprising a total area of 215 cm2 bathed in saliva (Collins & Dawes, 1987). The oral
environment is thus optimal for microorganisms to grow as biofilms, similar in their
architecture and characteristics to biofilms in nature (Auschill et al., 2001). Specific
to oral biofilms is the ability of sessile oral bacterial cells to tolerate short-term
abundance of external nutrient supply and to withstand nutrient restraint (Carlsson
& Johansson, 1973). Nutrient deprived biofilm cells after reactivation have been
shown to exhibit low reactivity in vitro, suggesting that slower reactivation of these
cells might be a survival strategy (Chavez de Paz et al., 2008). Bacterial cells in oral
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biofilms interact by various recognized ways including co-aggregation (Kolenbrander
et al., 2006), metabolic exchange, cell-cell communication (Li et al., 2002), and
exchange of genetic material (Roberts et al., 2001). These mechanisms benefit
bacterial survival and can make dental biofilms difficult therapeutic targets in dental
diseases.
Dental Plaque – Colonization Sites and Diversity
Oral biofilms are far from uniform in their composition. The healthy oral cavity was
shown by culture to harbor mainly Actinomyces, Streptococcus species and Rothia
dentocariosa (Moore et al., 1987). By 16S rDNA cloning and sequencing, the oral
cavity of healthy subjects exhibited a specific bacterial flora according to the sample
site and the individual, which was not associated with the dental disease microbiota
(Aas et al., 2005).
Supragingival dental plaque has been extensively studied from initial colonization of
bacteria on tooth surfaces to dental caries development. Initial colonizers in the
dental biofilm seem to be dominated by Streptococcus species (Diaz et al., 2006;
Dige et al., 2007; Kilian & Rolla, 1976; Socransky et al., 1977). Other “pioneering”
genera observed are Gram-positive Actinomyces, Gemella, Granulicatella, and
Rothia species, as well as Gram-negative Neisseria, Prevotella, and Veillonella
species. Dental plaque on tooth surfaces from healthy volunteers consists mainly of
Gram-positive organisms with coccoid cells (Listgarten, 1976). Epithelial cells seem
to also be present in the initial biofilms (Kolenbrander et al., 2006; Loe et al., 1965).
The degrading host cells could serve as nutrient depots for the bacteria (Diaz et al.,
2006).
Subgingival plaque forms from apical migration of the plaque growing on the tooth
surface (Theilade & Theilade, 1985). Histological sections of human subgingival
plaque have revealed two zones of microorganisms. One zone of bacteria is clearly
attached to the tooth surface and the other is lining the epithelial surface of the
pocket (Listgarten, 1976; Socransky & Haffajee, 2002). Spirochetes and Gram-
negative bacteria have been found to dominate the subgingival biofilm (Listgarten,
1976). Bacteria detected by immunohistochemical and DNA hybridization methods
in the deeper sections of the epithelial surface biofilms include asaccharolytic
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obligate anaerobes Porphyromonas gingivalis, Tannerella forsythia (previously
Bacteroides forsythus, Tannerella forsythensis) and Treponema denticola species
that are recognized periodontal pathogens (American Academy of Periodontology,
1996; Dibart et al., 1998; Kigure et al., 1995).
Bacterial analysis of soft tissue surfaces by whole genomic DNA probes, revealed
marked differences in bacterial composition and proportions in the soft tissues
sampled, and the tongue microbiota exhibited great similarity with bacteria in saliva
(Mager et al., 2003). The tongue microbiota has been of special interest, since the
tongue can harbor high biomasses of colonizing bacteria, including black-pigmented
and other species able to produce volatile sulfur compounds associated with halitosis
(Claesson et al., 1990; Persson et al., 1990; Spencer et al., 2007). It has also been
proposed that the tongue could serve as a reservoir of pathogenic microorganisms
and thus contribute to dental disease development (Faveri et al., 2006; Kazor et al.,
2003; Tanner et al., 2002). Reports have indicated that halitosis could be an oral
issue in both presence and absence of dental diseases, thus leaving the role of the
tongue microbiota in dental disease development unsolved (Bosy et al., 1994; De
Boever & Loesche, 1995; Hughes & McNab, 2008).
Dental Diseases
Bacterial infection is a necessary factor for the etiology of dental diseases. However,
both dental caries and periodontal diseases (gingivitis and periodontitis) are
multifactorial in their pathogenesis. Despite decades of scientific research and
health education, they still affect a major portion of the population.
According to the World Health Organization (WHO), the prevalence of dental caries
worldwide among adults is nearly 100% of the population [Decayed Missing Filled
Teeth index (DMFT) ≥14 teeth] in most industrialized countries, with a lower
prevalence in developing countries (Petersen et al., 2005). Regarding children,
dental caries affects 60-90% of school-aged children in industrialized countries with
the highest severity in 12-year old children in the Americas (DMFT= 3.0) followed by
Europe (DMFT= 2.6) (Bratthall, 2005; Petersen et al., 2005).
Gingivitis is universally present in adults and approximately 53% of dentate adults in
the United States have a life-experience of periodontal attachment loss of >3 mm
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(Albandar, 2002). In children, gingivitis is also highly prevalent with a tendency to
increase from infancy to puberty, but periodontitis is rare and the prevalence of
aggressive periodontitis cases varies between 0.1% and 2.6% (Jenkins & Papapanou,
2001). Incipient periodontitis in children, however, may lead to periodontitis in
adulthood (Bimstein, 1991; Kamma et al., 2000; Tanner et al., 2002).
Bacterial Etiology of Dental Diseases - Hypotheses
Different hypotheses have been proposed for the bacterial etiology of dental diseases.
These theories have developed through the improved understanding of dental
biofilm composition and organization. They also reflect the development of
appropriate laboratory methods to identify these communities. Investigators from
1880 to 1930 suggested that possible etiological factors of periodontitis were amebae,
spirochetes, fusiforms, and streptococci (Socransky & Haffajee, 1994). The initial
hypothesis (1950) established for the microbial etiology of dental disease was the
“non-specific plaque hypothesis”, in which dental diseases were caused by a
concerted effect of the total oral plaque microflora (Theilade, 1986). Subsequently,
the “specific plaque hypothesis” was proposed to suggest that only a few species from
the resident microbiota were responsible for dental diseases, which although present
in dental health were detected less frequently and in lower levels than in disease
conditions (Loesche, 1976; Socransky, 1977). Finally, the “ecological plaque
hypothesis”, a modification of the specific plaque hypothesis, proposed that change
in the local ecosystem could promote growth of certain dental plaque
microorganisms and lead to their increased numbers and dental disease
development in disease susceptible individuals (Marsh, 2003).
Dental Caries – Caries Development and Risk Factors
In 1890, the “father” of oral microbiology, W. D. Miller suggested that oral bacteria
are able to metabolize dietary carbohydrates and convert them into acid (Levine,
1977). The invariable role of bacterial infection combined with high sucrose diet in
dental caries was demonstrated in experiments on hamsters (Gibbons & Keyes,
1969). A byproduct of acidogenic bacterial metabolism, lactic acid, lowers the pH
locally and leads to enamel dissolution (Higuchi et al., 1969). Clinically, the initial
loss of tooth mineral is reversible by remineralization, for instance by topical fluoride
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treatment, decrease of sugar consumption, reduction of cariogenic bacteria or of
bacterial ability to produce acid. However, if acidic challenge continues disease may
progress into the adjacent dentin and the pulpal tissue causing endodontic infections
(Figdor & Sundqvist, 2007) and possibly leading to necrotic dental pulps.
Apart from bacteria and fermentable carbohydrates, additional risk factors for dental
caries include behavioral factors such as poor oral hygiene (Axelsson & Lindhe,
1981a; Axelsson & Lindhe, 1981b; Axelsson et al., 2004), reduced salivary secretion
and buffering capacity (Almstahl & Wikstrom, 1999), and genetic predisposition
(Jonasson et al., 2007; Stenudd et al., 2001). Previous caries experience and
presence of visible plaque have been identified as reliable predictors of increased
caries risk in children (Alaluusua & Malmivirta, 1994). Caries risk assessment
packages, such as Cariogram and Caries-risk Assessment Tool (CAT), have been
designed for use in clinical environments to identify patients needing dental caries
prophylaxis (American Academy of Pediatric Dentistry, 2005; Petersson & Bratthall,
2000).
Dental Caries in Children
Childhood dental caries is a major public health issue and is the most common
chronic disease in children (Edelstein & Douglass, 1995). Despite the availability of
prophylactic measures and treatment, approximately a third of pre-school children
are affected by dental caries in the United States and Sweden (Beltran-Aguilar et al.,
2005; Stecksen-Blicks et al., 2008; Wendt et al., 1992).
Caries in primary teeth of young children has been termed “baby bottle tooth decay”,
“nursing caries” and “night bottle mouth” (Harris et al., 2004). The need for
development and adoption of standardized definitions and criteria for the diagnosis
of dental caries in primary teeth has been apparent for years. In 1997, new
definitions for childhood caries were proposed at a National Institutes of Health
(NIH) Workshop for diagnosis and research purposes (Drury et al., 1999). Later
these guidelines were also adopted by the American Academy of Pediatric Dentistry
(AAPD) (Reference Manual 2004-2005, www.aapd.org) and have been widely used
in clinical studies in recent years. These definitions for Early Childhood Caries (ECC)
and Severe Early Childhood Caries (S-ECC) are explained below (Table 1).
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Table 1. National Institutes of Health workshop definitions of Early Childhood Caries (ECC) and Severe Early Childhood Caries (S-ECC) (Drury et al. 1999)
Age (months)
Early Childhood Caries (ECC)
Severe Early Childhood Caries (S-ECC)
0-35 1 or more dmf surfaces † 1 or more smooth dmf surfaces † 36-47 1 or more dmf surfaces † 1 or more cavitated, filled or missing
(due to caries) smooth surfaces in primary maxillary anterior teeth or dmfs score ≥ 4
48-59 1 or more dmf surfaces † 1 or more cavitated, filled or missing (due to caries) smooth surfaces in primary maxillary anterior teeth or dmfs score ≥ 5
60-71 1 or more dmf surfaces † 1 or more cavitated, filled or missing (due to caries) smooth surfaces in primary maxillary anterior teeth or dmfs score ≥ 6
† Any carious lesion, non-cavitated decayed (d1) or cavitated decayed (d2), missing tooth due to caries (m), or filled surface (f). Includes primary teeth only.
Microbiota of Dental Caries in Children
Mutans Streptococci
There is extensive evidence associating Streptococcus mutans and Streptococcus
sobrinus (mutans streptococci) with dental caries. Specifically in children with ECC,
the mutans streptococci can dominate the cultivable plaque microbiota associated
with the lesions or the carious material itself (van Houte et al., 1982).
Mutans streptococci use sucrose to produce extracellular glucan, a water insoluble
polysaccharide, which enables the bacteria to attach to the tooth surface and also
protects them from external factors such as mechanical disruption, salivary
clearance, and antimicrobial substances (Tinanoff et al., 2002). Equally important is
the ability of mutans streptococci to both produce acid (acidogenic) and survive in an
acidic environment (aciduric), properties that enable them to exhibit high
pathogenicity (Svensater et al., 1997). Finally, mutans streptococci can survive when
external carbohydrates are not present due to their ability to store intracellular
polysaccharide (Takahashi et al., 1991).
Pre-school children with high levels of salivary mutans streptococci have greater
caries prevalence and greater risk for new lesions compared with children with lower
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mutans streptococci levels (O'Sullivan & Thibodeau, 1996). Studies have also
indicated that the earlier mutans streptococci colonize the oral cavity of children, the
higher their caries experience is (Alaluusua & Renkonen, 1983). On the other hand,
several studies have failed to detect S. mutans in a significant portion of the children
who develop caries (Aas et al., 2008; Dasanayake & Caufield, 2002; Graves et al.,
1991), suggesting that additional species may play a significant role in dental caries
development in children.
Lactobacillus and Other Species
In children with dental caries Lactobacillus fermentum, Lactobacillus rhamnosus,
and Lactobacillus casei are prevalent in nursing caries lesions by culture methods
(Marchant et al., 2001). Lactobacillus species’ numbers in saliva seem to reflect the
amount of simple carbohydrate consumption by the host (Tanzer et al., 2001). Non-
mutans streptococci, Actinomyces, and Veillonella species are found in high amounts
in supragingival plaque and may comprise over one half of the cultivable flora.
Gram-positive species of other genera with acidogenic potential found less frequently
in plaque include Bifidobacterium, Clostridium, Eubacterium, Propionibacterium,
and Rothia species (van Houte, 1994).
The concept that enamel and dentin demineralization can be due to other
microorganisms apart from mutans streptococci and lactobacilli, has been the focus
of recent research. Svensäter and co-workers showed in adult participants that 50%
of the total cultured plaque microbiota from caries and health was able to grow at pH
5.5. At pH 5.0, Streptococcus species were the dominant group, but mutans
streptococci accounted for less than half of the viable streptococcal count (Svensater
et al., 2003).
Molecular techniques have aided in precise bacterial identification and enumeration
in dental caries (Munson et al., 2004). By 16S rDNA cloning and sequencing,
analysis of bacterial species in ECC identified 10 novel uncultivated phylotypes
(Becker et al., 2002). By oligonucleotide DNA probes, bacterial species associated
with caries in order of decreasing cell numbers were: Actinomyces gerencseriae,
Bifidobacterium species, S. mutans, Veillonella species, Streptococcus salivarius,
Streptococcus constellatus, Streptococcus parasanguinis, and L. fermentum (Becker
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et al., 2002). Another study, using both culture and molecular methods, indicated
that the taxa most often isolated from ECC lesions (pH 5.2) were Streptococcus
oralis, S. mutans, Actinomyces israelii, and Actinomyces naeslundii (Marchant et
al., 2001). Other bacteria identified by molecular approaches and that are
associated with childhood caries include non-mutans Streptococcus, Lactobacillus,
Actinomyces, Bifidobacterium and Veillonella species (Aas et al., 2008; Corby et al.,
2005). Further studies are needed to complete the knowledge regarding the array of
bacterial species and phylotypes associated with ECC and S-ECC.
Periodontal Diseases and Risk Factors
Periodontal diseases, gingivitis and periodontitis, are inflammatory processes in
tooth supporting tissues. They are frequently caused by the host defense factors
induced by bacterial infection (Darveau et al., 1997; Kornman et al., 1997). Gingivitis
involves an inflammatory response to dental plaque biofilms growing adjacent to the
gingival margin. Plaque-induced gingivitis is reversible, if optimal oral hygiene is
achieved, and the gingival tissue can return to its homeostatic state without
permanent damage (Loe et al., 1965). Periodontal connective tissue attachment is
lost in periodontitis, as opposed to gingivitis. Host defense against bacterial insult
leads to apical migration of the junctional epithelium at the base of the gingival
crevice. This process coupled with gingival edema, due to inflammatory response,
leads to deepened periodontal pockets. In addition, in periodontitis the surrounding
tissue (bone and connective tissue) of the teeth is destroyed in an attempt to limit the
inflammatory process (Page & Kornman, 1997). The presence and extent of
periodontal fiber loss can be determined by probing, which is the routinely used
clinical method to estimate the extent of tissue destruction in periodontitis.
Marginal alveolar bone loss can be diagnosed radiographically.
The classical Koch’s postulates for defining causative pathogens, have been amended
in the case of periodontitis to include an evaluation of the evidence concerning
association, elimination, host response, virulence factors, animal studies, and risk
assessment of each potential pathogen (Fredericks & Relman, 1996; Haffajee &
Socransky, 1994). Other factors associated with periodontitis, apart from bacterial
colonization, include: compromised immunity (Cainciola et al., 1977), cigarette
smoking (Haber et al., 1993), and diabetes mellitus (Kinane & Bouchard, 2008).
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Microbiota of Periodontal Health and Disease
Periodontally healthy sites exhibit a low cultivable bacterial load by individual sulcus
(102 to 103 microorganisms) with a predominantly Gram-positive microbiota,
including Streptococcus and Actinomyces species (Table 2) (Darveau et al., 1997;
Syed & Loesche, 1978; Tanner et al., 1996). Gingivitis is characterized by an
increased microbial load (104 to 105 microorganisms by periodontal sulcus) as well as
an increased prevalence of Gram-negative bacteria (15-50%) (Table 2) (Lai et al.,
1987; Moore et al., 1987; Slots, 1979).
The most extensively studied periodontal pathogen which is associated with
aggressive forms of periodontal destruction is Aggregatibacter (previously
Actinobacillus) actinomycetemcomitans. This small non-motile, capnophilic Gram-
negative coccobacillus has been identified as the main causative agent of aggressive
periodontitis in young individuals, but has also been found in adults (Asikainen,
1986; Asikainen et al., 1986; Slots et al., 1980; Tanner et al., 1979). A.
actinomycetemcomitans has been grouped into six serotypes (Kaplan et al., 2001;
Saarela et al., 1992; Zambon et al., 1983) and it has been suggested that certain
serotypes are associated more frequently with periodontitis than periodontal health.
Exemplifying this relationship, serotype c has been detected more frequently from
periodontally healthy individuals and serotypes a and b more frequently in
periodontitis (Asikainen et al., 1991; Dogan et al., 1999; Yang et al., 2005; Zambon et
al., 1983). Although differences are reported in A. actinomycetemcomitans serotype
distribution when taking geographic location and/or ethnicity into account (Dahlen
et al., 2002; Dogan et al., 2003), still 3-8% of strains have remained non-
serotypeable (Paju et al., 2000).
Gram-negative asaccharolytic obligate anaerobes P. gingivalis, T. forsythia, and T.
denticola (Socransky et al., 1988) have been also extensively associated with
periodontitis. P. gingivalis has been detected in association with periodontal lesions
(Dahlen et al., 1992; Loesche et al., 1985; Moore et al., 1983) and possesses an
arsenal of virulence factors that can potently activate the host response (Holt &
Ebersole, 2005; Kuramitsu, 2003). T. forsythia was first described at the Forsyth
Institute and soon became a recognized periodontitis pathogen due to its frequent
detection from sites with destructive periodontitis and its strong association with
••••
17
increasing pocket depth (Gmur et al., 1989; Lai et al., 1987; Tanner et al., 1979;
Tanner & Izard, 2006). Finally, spirochetes, such as T. denticola, are also frequently
found in periodontally diseased sites subgingivally and decrease after successful
treatment (Simonson et al., 1988; Simonson et al., 1992; Socransky et al., 1969).
Table 2. Dominant bacterial species detected in periodontal health, gingivitis, and periodontitis. Species are ordered according to likelihood of detection by culture in each category starting from the most likely and proceeding to the least likely detected. (Adapted from Darveau et al., 1997) Periodontal Health Gingivitis Periodontitis Streptococcus oralis Streptococcus oralis Porphyromonas gingivalis Streptococcus sanguinis Streptococcus sanguinis Aggregatibacter
actinomycetemcomitans Streptococcus mitis Streptococcus mitis Tannerella forsythia Streptococcus gordonii Streptococcus intermedius Spirochetes Streptococcus mutans Capnocytophaga ochracea Treponema denticola Streptococcus anginosus Capnocytophaga gingivalis Prevotella intermedia Streptococcus intermedius Campylobacter gracilis Prevotella nigrescens Gemella morbillorum Prevotella loescheii Campylobacter rectus Actinomyces naeslundii Eubacterium nodatum Fusobacterium nucleatum
subspecies vincentii Actinomyces gerencseriae Actinomyces naeslundii Fusobacterium nucleatum
subspecies nucleatum Actinomyces odontolyticus Actinomyces israelii Selenomonas noxia Parvimonas micra Campylobacter concisus Selenomonas flueggeii Eubacterium nodatum Actinomyces odontolyticus Enteric species Capnocytophaga ochracea Fusobacterium nucleatum
subspecies nucleatum Filifactor alocis
Capnocytophaga gingivalis Eubacterium brachy Lactobacillus uli Campylobacter gracilis Eikenella corrodens Veillonella parvula Fusobacterium nucleatum subspecies polymorphum
Aggregatibacter actinomycetemcomitans
Other species that have been associated with periodontitis include Fusobacterium
nucleatum, Parvimonas micra, Prevotella intermedia, Prevotella nigrescens,
Campylobacter rectus, Eikenella corrodens, Selenomonas and Eubacteria species
(Haffajee & Socransky, 1994; Moore & Moore, 1994) and more recently Filifactor
alocis (Table 2) (Dahlen & Leonhardt, 2006; Hutter et al., 2003; Kumar et al.,
2006). Recently, molecular microbiological studies have expanded the series of
bacteria recognized in association with periodontal diseases to include uncultivated
and less often identified phylotypes (Kumar et al., 2006; Kuramitsu, 2003; Paster et
al., 2001).
18
Molecular Microbiological Identification of Oral Bacteria
Bacterial culture, particularly anaerobic culture, has been critical in the appreciation
of the diversity of the subgingival microbiota. Use of non-selective and selective
media and various atmospheric conditions, has aided in growth of multiple bacterial
species from the oral cavity. Not all bacteria, however, present in oral samples have
been cultured to date. The fact that certain microorganisms cannot be cultured and
that culture has recognized limitations including sample transportation, time
constraints, labor intensity, and high expense, has led to increasing use of molecular
detection and identification methods. DNA based methods are currently being
extensively used and renewed possibilities to study bacterial etiology of dental
diseases have risen. DNA methods, such as PCR, DNA-DNA hybridization, 16S
rDNA cloning and sequencing, and microarray analysis, allow simultaneous
identification of numerous previously known bacteria and/or novel phyla and
phylotypes (Table 3). These approaches providing the possibility of identification of
novel pathogens associated with dental diseases.
Table 3. Molecular techniques and culture as methods of bacterial identification in oral samples. A comparison of strengths, limitations and application areas. Method Strengths Limitations Applications Culture Cultured
representatives Time, cost, limited detection
Characterization, antibiotic resistance
PCR Rapid, sensitive
Contamination, non-viable detection
Species present in low levels
Whole genomic DNA probes
Sensitive, quantitative
Specificity, cultured species
Multiple species
Oligonucleotide DNA probes
Specific, uncultivated
Sensitivity, primer bias
Multiple species, phylotypes
DNA microarray Multiple species Variance, multiple comparisons
Genetics, bacterial profiles
16S rDNA cloning and sequencing
Uncultivated Time, cost, few samples
Phylogeny, diversity
In the human oral cavity, 700 bacterial species and phylotypes have been recently
identified by molecular methods (Aas et al., 2005; Aas et al., 2008; Becker et al.,
2002; Chhour et al., 2005; Corby et al., 2005; Kazor et al., 2003; Kroes et al., 1999;
Paster et al., 2001; Preza et al., 2008). These molecular techniques open intriguing
possibilities for identification of novel bacterial phylotypes (Table 4).
••••
19
Table 4. Identification of novel bacterial phylotypes from dental caries or periodontitis in recent studies using 16S rDNA cloning and sequencing. Disease Subjects
(Samples) (N)
Total Species/ Phylotypes
(N)
Novel Phylotypes
(N)
Reference
Caries (ECC)† 2 (5) 68 10 Becker et al. 2002 Caries 5 (5) 79 31 Munson et al. 2004 Caries 10 (10) 75 2 Chhour et al. 2005 Caries (S-ECC)† 7 (22) 197 12 Aas et al. 2008 Caries† 21 (43) 245 6 Preza et al. 2008 Periodontitis† 31 (31) 347 215 Paster et al. 2001 Periodontitis† 30 (45) 274 6 Kumar et al. 2005 † Studies include control group Moreover, molecular methods also allow determining intra-species differences with
higher reproducibility than phenotypic methods that rely on gene expression. In
particular PCR-based methods, such as arbitrarily primed PCR (AP-PCR) and
denaturing gradient gel electrophoresis (DGGE), have been widely used. Several
studies have investigated the intra-species diversity of bacterial species associated
with dental caries, such as S. mutans, S. sobrinus (Mattos-Graner et al., 2001;
Saarela et al., 1996), and Actinomyces species (Ruby et al., 2003) and with
periodontitis, such as A. actinomycetemcomitans, P. gingivalis, P. intermedia, and
C. rectus (Asikainen et al., 1995; Asikainen et al., 1996; Matto et al., 1996).
Additionally, by PCR amplification of serotype-specific DNA sequences it is currently
possible to group A. actinomycetemcomitans strains into serotype categories
(Kaplan et al., 2001; Suzuki et al., 2001). These molecular techniques allow for more
comparable studies between laboratories on the associations of different bacterial
serotypes with dental diseases, due to the commercial availability of the reagents.
It has been estimated that half of the oral bacteria have not yet been cultured (Paster
et al., 2001). Although 50% successful cultivation of bacteria in the oral cavity by far
exceeds the 1% cultured on Earth (Hugenholtz et al., 1998), cultivation-based
approaches have provided an incomplete picture of the microbial diversity of dental
diseases and oral health. The conclusions drawn about bacteria associated with
dental diseases have been dependent on the available technology. Molecular
approaches provide the possibility of expanding the knowledge of the diverse array of
microorganisms present in the oral cavity. In order to be able to identify candidate
pathogens and beneficial species a comprehensive approach of the oral microbiota
involved in oral health and disease is needed.
20
Hypothesis and Aims The objective of this doctoral thesis was to investigate the role of the oral microbiota
in dental caries and periodontitis using comprehensive molecular, clinical, and
statistical methodology. The central hypothesis was that currently poorly
characterized, fastidious, and uncultivated bacteria are involved in dental diseases.
Specific Aims
This doctoral thesis aimed by use of molecular methods:
1. To identify cultured caries- and periodontitis-associated species and
determine their possible association with childhood caries.
2. To identify uncultivated species and novel phylotypes, determine their
possible association with childhood caries, and examine the bacterial diversity
of childhood caries.
3. To identify fastidious species and uncultivated phylotypes and determine their
possible association with early periodontitis and periodontitis progression.
4. To investigate the reason for non-serotypeability of a subset of A.
actinomycetemcomitans strains and their association with periodontitis.
••••
21
Materials and Methods
Study Populations and Clinical Measurements
All study designs (Table 5), protocols, and informed consents were examined and
approved by the Institutional Review Boards and/or the local ethical committees of
the institutions involved.
Table 5. Summary of study design, origin of study populations, number and age of participants, dental disease category, and laboratory methods used for bacterial identification. The studies are presented in the order attached and discussed in the thesis.
Study Design Population (N of subjects)
Age (yrs)
Dental Disease
Laboratory Method
I Cross-sectional Descriptive †
Boston, MA (N=195)
1-6 Caries (ECC)
Whole genomic probes, PCR
II Cross-sectional Case-control
Boston, MA (N=80)
2-6 Caries (S-ECC)
16S rDNA cloning and sequencing, PCR
III Cross-sectional Descriptive †
Boston, MA (N=141)
20-40 Early periodontitis
Oligonucleotide probes, multiplex PCR
IV Longitudinal Cohort
Boston, MA (N=117)
20-40 Progressing periodontitis
Oligonucleotide probes, Whole genomic probes, multiplex PCR
V Cross-sectional Experimental
Finland (N=152) Turkey (N=37)
13-74 Aggressive and chronic periodontitis
Immunodiffusion, SDS-PAGE, Western blot, PCR, AP-PCR
† Nested in a cohort
Childhood Caries (Study I and II)
Children with (N=35) and without (N=160) ECC were recruited from pediatric
clinics, as part of a larger cohort study. Children with (N=40) and without (N=40)
S-ECC were recruited from dental clinics in a case-control study (Study II). Inclusion
criteria for both studies were that the child was up to 6 years old, medically healthy,
had not used antibiotics for the past 3 months, and that the parent or guardian was
willing to consent to the child’s clinical examination and microbial sampling.
Children were examined by trained dental hygienists or dentists. Presence and
status of teeth (Drury et al., 1999), plaque and gingival status were recorded.
Bacterial samples were collected from anterior, posterior teeth, and the dorsum of
the tongue. Children’s socio-demographic characteristics (Study I and II), diet, and
oral health practices (Study I) were collected.
22
Early Periodontitis (Study III and IV)
Periodontally healthy subjects (N=28) and patients with early periodontitis (N=113)
were recruited in this multicenter study (Study III). Recruitment methods and
inclusion criteria have been previously described in detail (Tanner et al., 2005).
Briefly, inclusion in the study was restricted to 20-40 year old subjects, who were
medically healthy, and without recent antibiotic use or previous periodontal therapy.
Clinical entry criteria included presence of at least 24 teeth, mean periodontal
attachment loss of <2 mm, and no generalized gingival recession. Information
concerning medical and dental histories, socio-demographic data, and tobacco use
was collected. Clinically, duplicate measurements of probing depth and periodontal
attachment level and single measurements of plaque index, gingival index, and
bleeding on probing were obtained from six sites on all teeth (excluding third
molars). Clinicians were trained and calibrated and exhibited intra-class correlations
of 0.87 for pocket depth and 0.85 for clinical attachment level (Tanner et al., 2005).
Monetary incentive was given to participants after completion of each clinical
measurement visit.
A subset of participants (N=117) was followed longitudinally every 6 months for 18
months (Study IV). Subjects exhibiting ≥ 1 site with > 1.5 mm inter-proximal clinical
attachment loss over 18 months of monitoring were labeled as “active”. “Inactive”
subjects exhibited no sites with > 1.5 mm inter-proximal clinical attachment loss
during the observation period.
Aggressive and Chronic Periodontitis (Study V)
Participants from Finland (N=152) and Turkey (N=37) were recruited to obtain 311
A. actinomycetemcomitans strains. Inclusion criteria were that patients were
medically healthy, had no recent use of antibiotics, and were willing to partake in the
study. The periodontal status of the subjects was determined clinically and
radiographically.
••••
23
Microbial Analysis
DNA-DNA Hybridization
Whole Genomic DNA Probe Assay
Plaque samples were analyzed in Studies I and IV by whole genomic DNA probes as
previously described (Socransky et al., 1994). Briefly, samples were denaturated,
neutralized, and fixed onto a membrane by ultraviolet (UV) irradiation. The two last
lanes on each membrane were used as a quantitative reference containing all probe
species mixed as a DNA standard for 105 and 106 bacterial cell equivalents. The
membrane containing the samples and standards was hybridized with digoxigenin-
labeled probes directed to the species of interest. Finally, anti-digoxigenin
antibodies, covalently bound to alkaline phosphatase, were used to visualize the final
product with standard chemiluminescence. Signals were detected using a Storm
Fluorimager system (Molecular Dynamics, Sunnyvale, CA) and subsequently
converted to absolute bacterial counts by comparison with the standards on the
membrane. The sensitivity of the assay was set to 104 bacterial cells by adjusting the
concentrations of the probes. The specificity of the assay was <10% cross-reactivity
for 97.4% of probes, <5% cross-reactivity for 93.5% of probes, and <1% cross-
reactivity for 82.1% of the probes (Socransky et al., 2004).
In Study I, samples were analyzed using whole genomic DNA probes to 74 species
(Study I, Table 2), including 44 acidogenic and aciduric bacterial species (Becker et
al., 2002; Marchant et al., 2001; Munson et al., 2004; Svensater et al., 2003) and 30
mainly Gram-negative bacterial species selected according to previously reported
associations with dental health or disease.
For the initial periodontitis population (Study IV) a set of 38 whole genomic DNA
probes was selected to include species identified from previous culture studies of
initial periodontitis (Tanner et al., 1998).
Oligonucleotide DNA Probe Assay
Samples were analyzed in Studies III and IV by 78 16S rDNA probes selected to
include cultured species and uncultivated phylotypes previously associated with
periodontitis (Kazor et al., 2003; Paster et al., 2001) in an assay as previously
described (Paster et al., 1998). Briefly, the oligonucleotide DNA probes (17-22 bases)
24
were cross-linked to a nylon membrane using UV irradiation. Two universal
standard probes were used for each membrane. DNA from samples was amplified
with two universal primers, one of which was labeled with digoxigenin. The
digoxigenin-labeled amplicon was subsequently hybridized to the capture probes and
anti-digoxigenin antibodies, covalently bound to alkaline phosphatase, were used to
visualize the final product by chemiluminescence. Signals were detected using a
Storm Fluorimager system and reactions were scored as species detected or not
detected.
Polymerase Chain Reaction
S. mutans and A. actinomycetemcomitans PCR
PCR for detection of S. mutans (Study I and II) and of A. actinomycetemcomitans
(Study II) from samples was used to detect these specific dental pathogens from
clinical samples. In Study I, samples were treated by Proteinase K (Dewhirst et al.,
2000) to inactivate proteases and the product was used as a template for the PCR. In
Study II, genomic DNA was extracted and purified from samples as previously
described (Li et al., 2007). A. actinomycetemcomitans PCR (Study II) was
performed in a nested format, by running a universal PCR first and then using the
template for the PCR as previously described (Ashimoto et al., 1996). To ensure
correct amplification products, all A. actinomycetemcomitans positive reactions
were sequenced.
A. actinomycetemcomitans Genotyping PCR
Arbitrarily primed PCR was performed (Study V) using the random oligonucleotide
sequence OPA-13 (5’-CAG CAC CCC AC-3’) to separate A. actinomycetemcomitans
strains into distinct genotypes as previously described (Asikainen et al., 1995; Dogan
et al., 1999; Paju et al., 2000).
A. actinomycetemcomitans Serotype-specific PCR
The gene sequences specific for A. actinomycetemcomitans serotypes a through f
were assayed (Study V) as previously described (Dogan et al., 2003; Kaplan et al.,
2001; Suzuki et al., 2001). Moreover, the entire serotype-specific gene clusters were
analyzed for serotype a, c, and f.
••••
25
P. gingivalis and T. forsythia Multiplex PCR
To improve detection of the two major periodontitis-associated pathogens
P. gingivalis and T. forsythia (Study III, IV) a multiplex PCR was performed based
on the PCR assay by Tran and Rudney (Tran & Rudney, 1999). The detection limit
was for P. gingivalis 102 and for T. forsythia 103.
Cloning and Sequencing
Genomic DNA was extracted from the samples using a previously described protocol
(Li et al., 2007). PCR amplification of the 16S rRNA gene, and cloning and
sequencing were performed (Study II) as previously described (Paster et al., 2001)
using universal PCR primers under standard conditions (Dewhirst et al., 1999).
Cloning of the PCR-amplified DNA was performed with a commercial kit (TOPO TA
Cloning kit, Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions.
Transformation was performed in competent One Shot TOP 10 E. coli cells
(Invitrogen) overnight on Luria-Bertani agar plates supplemented with kanamycin.
An X-galactose screen determined successful transformation.
Forty-eight to ninety-six transformed colonies were used as PCR templates with
vector primers M13F and M13R (TOPO TA Cloning kit, Invitrogen, CA). Colonies
with the correct insert size (1,600 bases) were cycle-sequenced (Big Dye Terminator
cycle sequencing kit with AmpliTaq DNA polymerase FS, Perkin-Elmer, Waltham,
MA) with 533 reverse primer (Paster et al., 2001). After a final clean-up, reactions
were sequenced using an ABI 3100 DNA Sequencer.
Sequences were edited using Sequencher (Gene Codes Corporation, Ann Arbor, MI),
aligned, and checked individually for chimeric inserts. Sequences were compared
using BLAST (Basic Local Alignment Search Tool) to a reference set of sequences in
the Human Oral Microbiome Database, with a threshold for species differentiation
>98% identity (Dewhirst et al., 2008). Neighbor-joining phylogenetic trees were
created from full 16S rRNA Human Oral Microbiome Database reference sequences
by DNASTAR MegAlign software using the CLUSTAL V alignment method.
26
Antigen Identification
SDS-PAGE and Western Immunoblotting
For A. actinomycetemcomitans serotype antigen detection, SDS-PAGE and Western
immunoblotting were performed (Study V) as previously described (Karched et al.,
2008). Whole cell preparations in phosphate buffer solution (PBS) were used and
selected preparations were treated with Proteinase K. Both rabbit antisera against A.
actinomycetemcomitans serotypes a-f and human sera were used. Immunoreactive
bands were detected by chemiluminescence.
Immunodiffusion Assay
Serotype-specific rabbit antisera against whole cells of A. actinomycetemcomitans
serotypes a through e were used as antibodies as reported previously (Saarela et al.,
1999), with the addition of serotype f (Study V). Antigens were prepared from each
A. actinomycetemcomitans strain by autoclaving whole cell suspensions and using
the supernatant in the immunodiffusion assay.
Statistical Analysis
Univariate Analysis
Study I: Student’s t-test was used to compare differences in means, Chi-square test
for differences in proportions between the ECC and the caries-free group, and
McNemar’s test for species differences in paired samples. A p-value ≤0.05 was
considered significant. Results were adjusted for multiple comparisons using the
false discovery rate (α=0.1), which is a non-conservative approach to control for false
positive results using the proportion of false positives rather than the chance of any
false positives (Benjamini & Hochberg, 1995). Statistical analyses were performed
using SPSS® software version 11.
Study II: Species and phylotypes were grouped into phyla and the number of
distinct identification in each phylum by Human Oral Taxon number (HOT) was
compared between S-ECC and caries-free children (Dewhirst et al., 2008). Taxa that
could not be differentiated by 500 base 16S rRNA sequence comparison were
grouped together for analysis. Student’s t-test (clinical and socio-demographic
variables) and Kruskall-Wallis test (bacterial variables) were used to determine
differences in means between S-ECC and caries-free children. The Chi-square test
••••
27
was used to test differences in proportions of socio-demographic variables. A p-value
≤0.05 was considered significant. Results were also adjusted for multiple
comparisons using the false discovery rate (α=0.05) (Benjamini & Hochberg, 1995).
Statistical analyses were performed using SPSS® software.
Study III: McNemar’s test was used to evaluate differences in species prevalence in
tongue and subgingival samples. Similarities in species detection in tongue and
subgingival samples were evaluated by Chi-square test for association. Comparison
of species prevalence across clinical groups was evaluated by Mantel-Haenszel Chi-
square for trend. Statistical analyses were performed using SPSS® and SAS®
software.
Study IV: Student’s t-test was used to evaluate differences between active and
inactive subjects with respect to age and baseline clinical variables. Analysis of
covariance adjusting for baseline values was used for clinical variable comparison
(mean values over 18 months) across the groups. Baseline clinical variables and
subsequent change were evaluated by Spearman’s rank correlation. Clinical
associations of active and inactive disease were compared with the Mantel-Haenszel
Chi-square for trend. For microbial analysis, species were compared between active
and inactive groups by Chi-square test for the oligonucleotide DNA probes and by
Student’s t-test for the whole genomic DNA probes. The Chi-square test was also
used to evaluate the PCR data. Statistical analyses were performed using SPSS® and
SAS® software.
Multifactorial analysis
Study I: Regression analysis was used to evaluate the extent of caries in the children
with age and plaque, and their interactions using SPSS® software. Multivariate
analysis was performed using partial least squares (PLS) modeling (SIMCA P,
Umetrics, Umeå, Sweden) to find positive and negative bacterial associations with
caries. PLS is a linear model that detects correlations between independent and
outcome variables and generates a Variable Importance in Projection (VIP) value
which if >1 is influential and if ≥1.5 is highly influential.
Study IV: Logistic regression was used to evaluate clinical measurements at
baseline, P. gingivalis and T. forsythia PCR detection and subsequent loss of
attachment using SAS® software.
28
Results and Discussion
Oral Microbiota in Patients with Childhood Caries (Study I and II)
Bacterial samples from 275 children (75 with caries and 200 caries-free) were
examined (Study I and II). By whole genomic DNA probes, 74 cultured species were
identified including acidogenic, aciduric, and periodontitis-associated species (Study
I, Table 2). By 16S rDNA cloning and sequencing, 3802 clones yielded identifications
representing 138 bacterial taxa (77 cultured named species, 11 cultured unnamed
species, and 50 uncultivated phylotypes) (Study II, Figure 1). An additional 107
clones (39 from caries and 68 from caries-free children) remained unidentified and
may include novel phylotypes. Full 16S rRNA analysis of the unidentified clones will
be needed to determine novelty of species.
Species detected (N=37) by whole genomic DNA probes (Study I) and 16S rDNA
cloning and sequencing analysis (Study II) in children with and without dental caries
are presented in Table 6. The table also indicates whether these species were
detected more frequently in children with ECC and S-ECC than caries-free children
and whether the detection differed significantly between the comparison groups
(caries versus caries-free). The species detected by both assays represent half of the
species assayed by the probe assay (37/74, 50%) and included both Gram-positive
and Gram-negative species. Species assayed by the whole genomic DNA probes were
pre-selected, whereas 16S rDNA cloning and sequencing species/phylotype detection
was random. Significant associations for bacterial species detection in children with
caries compared to caries-free children were observed in 13 different species by the
two molecular methods in the two populations (Table 6). The only species exhibiting
a significant invert association with the caries and caries-free group by the two
methods was Streptococcus intermedius associated with ECC (p<0.05) (Study I,
Figure 1), but not with S-ECC (p<0.05) (Study II, Figure 3a). By culture methods,
although S. intermedius dominated children’s dental plaque from initial caries, it did
not significantly differ from caries-free sites (Svensater et al., 2003).
••••
29
Table 6. Species detected (N=37) by both whole genomic DNA probes (Study I) and 16S rDNA cloning and sequencing (Study II). The table indicates whether species detection was more frequent from children with dental caries [Early Childhood Caries (ECC, Study I) or Severe Early Childhood Caries (S-ECC, Study II)] compared to caries-free controls. Significant differences of species between caries and caries-free children are also indicated.
Species Detected by Both Assays Whole Genomic DNA Probes
16S rDNA Cloning and Sequencing
ECC p<0.05 a S-ECC p<0.05 b Streptococcus mutans + c * d - e ns f
Streptococcus sobrinus + ns + na g
Streptococcus oralis + ns + ns Streptococcus mitis /pneumonia + ns + ns Streptococcus sanguinis + ns - * Streptococcus parasanguinis + ns + ns Streptococcus cristatus + ns - * Streptococcus salivarius + ns + na Streptococcus gordonii + ns - ns Streptococcus intermedius + * - * Streptococcus constellatus + ns + ns Streptococcus anginosus + ns + ns Lactobacillus vaginalis + * - na Lactobacillus fermentum + * - na Lactobacillus gasseri + * - ns Gemella morbillorum + ns + ns Actinomyces israelii + * - na Actinomyces naeslundii + ns - na Eubacterium saburreum + ns - ns Eubacterium brachy + * + ns Parvimonas micra + * - ns Prevotella melaninogenica + ns + na Prevotella nigrescens + * - na Capnocytophaga gingivalis + ns - * Capnocytophaga ochracea + ns + na Capnocytophaga sputigena + ns + na Aggregatibacter actinomycetemcomitans + * + na Neisseria mucosa/ flava /sicca + ns - ns Eikenella corrodens + ns + ns Kingella oralis + ns + ns Campylobacter concisus + ns - ns Campylobacter showae + ns + na Campylobacter gracilis + ns - ns Veillonella parvula /dispar + * + ns Selenomonas sputigena + ns + ns Selenomonas noxia + ns - ns Selenomonas flueggei + ns + na
a Chi-square test, b Kruskall-Wallis test, c + = Species detected more frequently from caries group (ECC Study I, S-ECC Study II), d * = p<0.05, e - = Species detected more frequently from caries-free group, f ns= not significant, g na = not available (detected in one group)
Mutans and Non-mutans Streptococci
Studies examining dental caries often focus on presence and association of mutans
streptococci, which is a collective name for S. mutans and S. sobrinus. In the present
studies, S. mutans was associated with dental caries in children both with ECC
(Study I) and with S-ECC (Study II). S. mutans was detected from molar plaque
samples by PCR in high percentages of children in both study populations: 69% in
ECC (p<0.001, compared to caries-free children) and 73% in S-ECC (p<0.01,
compared to caries-free children) (Figure 1). It has been previously reported by
culture that 35% of 8-15 month old children harbored S. mutans, with 14% of the
children by 10 months and 60% by 15 months of age (Karn et al., 1998). Moreover,
S. mutans has been shown to exceed 30% of the cultured microbiota in ECC
(Berkowitz et al., 1984; Marchant et al., 2001; van Houte et al., 1982). S. mutans,
however, was not associated with S-ECC by 16S rDNA cloning and sequencing (Study
II, Figure 3a) and was detected at a mean detection frequency of 1% by child. The
infrequent detection of S. mutans by 16S rDNA cloning and sequencing analysis in
subjects with dental caries has been previously reported in both childhood and adult
caries, as well as root caries (Aas et al., 2008; Chhour et al., 2005; Preza et al.,
2008). This could suggest that S. mutans represents a subset of the caries-associated
microbiota (Loesche & Syed, 1973) and additional species could be responsible for
caries development. However, methodological limitations cannot be excluded.
Figure 1. S. mutans detection by PCR in children with ECC (Study I) and S-ECC (Study II). Panel A: S. mutans PCR detection by sampling site. Significantly higher detection of S. mutans from children with ECC than caries-free children was observed from molar and incisor teeth (p<0.001), but not from the tongue. Panel B: S. mutans detection was associated with S-ECC in molar teeth (p<0.01).
30
A. B.
••••
31
S. sobrinus was only associated with ECC together with S. mutans (Study I, Table 2)
and was found in only one child (S-ECC) by 16S rDNA cloning and sequencing (Study
II). Detection of S. sobrinus together with S. mutans by PCR has been previously
associated with higher caries prevalence than for S. mutans alone in 3-5 year old
children (Okada et al., 2005; Seki et al., 2006). Moreover, a previous report using
16S rDNA cloning and sequencing as well as oligonucleotide DNA probe analysis has
also shown that S. sobrinus is present in lower levels than S. mutans in children and
not associated with childhood caries (Becker et al., 2002).
Concerning non-mutans streptococci, S. mitis/pneumoniae and Streptococcus sp.
HOT-071 were detected more frequently from S-ECC children than caries-free
children (Study II, Figure 3a). Although these species did not exhibit a significant
association with childhood caries in Study II, they are of interest since they were
previously associated with childhood caries by oligonucleotide DNA probes (Becker
et al., 2002). By 16S rDNA cloning and sequencing Streptococcus sp. HOT-071 was
detected from carious root surfaces of adults (Preza et al., 2008). Further studies are
needed to determine the role of this unnamed species in dental caries.
Several non-mutans streptococcus species that were detected more frequently in
caries-free children than children with S-ECC (Study II, Figure 3a) included
Streptococcus cristatus (p<0.05), Streptococcus sanguinis (p<0.05), S. intermedius
(p<0.05), and Abiotrophia defectiva (p<0.01) [A. defectiva previously classified as
an atypical Streptococcus species (Kawamura et al., 1995)]. These findings are
consistent with molecular studies of the microbiota in caries-free children of pre-
school age (Aas et al., 2008; Becker et al., 2002; Corby et al., 2005). S. sanguinis, S.
intermedius, and A. defectiva have also been identified as part of the commensal oral
bacterial flora by 16S rDNA cloning and sequencing from samples of dentally healthy
adults (Aas et al., 2005).
Lactobacillus Species
Lactobacillus species exhibiting association with childhood caries were Lactobacillus
gasseri (p<0.01), L. fermentum (p<0.05), and Lactobacillus vaginalis (p<0.05)
(Study I, Figure 1). Significant associations with the two first species have been
found by use of 16S rDNA cloning and sequencing and oligonucleotide DNA probe
32
analysis in childhood caries (Aas et al., 2008; Corby et al., 2005). Although
L. vaginalis has been found in the saliva of caries-active women (Caufield et al.,
2007), it is usually considered to be part of the normal vaginal flora (Thies et al.,
2007). Based on culture methods, Lactobacillus species can be transiently detected
in the infant oral cavity, but do not persist to colonize (Carlsson & Gothefors, 1975).
In S-ECC, L. fermentum, L. gasseri, L. vaginalis, and Lactobacillus paracasei were
detected in low frequencies by child (0.06-0.12%) and were detected more frequently
in caries-free children (Study II). No conclusions can be, however, drawn about the
role of these species in S-ECC due to the low detection frequency.
Noteworthy was the finding that both Lactobacillus acidophilus and Lactobacillus
reuteri were found to be consistently influential by multifactorial analysis (VIP>1.0)
in children with low decayed filled teeth index (dft), exhibiting an inverse
relationship with dental caries (Study I). On restricting the analysis to children with
visible plaque, L. acidophilus (VIP>1.5) became highly negatively influential on
caries extent (dft) and L. reuteri, L. rhamnosus, and Lactobacillus plantarum
exhibited negative influential associations with caries (VIP 1.0-1.4) (Study I, Figure
2). L. acidophilus and L. reuteri have been used for probiotic enteric therapy in
infants (Lee et al., 2007; Savino et al., 2007). All four species have also been tested
as oral anti-caries agents in children (Twetman & Stecksen-Blicks, 2008), and are
consistently reported as beneficial species not associated with dental caries.
Other Species
A. israelii (p<0.05) and Actinomyces odontolyticus (p<0.05) were detected more
frequently from children with caries than caries-free children by whole genomic DNA
probes (Study I, Figure 1). Both species were highly influential with caries extent
(higher dft scores) by multifactorial analysis (VIP>1.0) in all ages. When restricting
the analysis to children with visible dental plaque, A. israelii remained highly
influential with caries extent (VIP>1.0) and A. odontolyticus was borderline
influential (VIP=0.9) (Study I, Figure 2). A previous study focusing on the
predominant cultured microbiota of interapproximal surfaces in children found that
A. israelii was the most predominant of all Actinomyces species and that
A. odontolyticus was the most prevalent species below the contact area of the teeth
••••
33
(Babaahmady et al., 1997). A. israelii has been found more frequently associated
with dental plaque in children than with caries lesions by culture (Marchant et al.,
2001). A. israelii has been previously detected in children younger than 18 months
using the whole genomic DNA probe assay (Tanner et al., 2002). These findings
have not been previously reported by oligonucleotide DNA probes (Aas et al., 2008;
Corby et al., 2005; Tang et al., 2003). This possibly reflects a limitation of PCR
based methods to amplify representatives of the Actinobacterium phylum, which
contains Gram-positive species with high 16S rRNA G+C content. For detection of
Actinomyces species, the whole genomic DNA probe technique used in Study I was a
more preferable approach than molecular methods utilizing amplification of the 16S
rRNA gene (such as oligonucleotide DNA probes or 16S rDNA cloning and
sequencing).
Veillonella parvula (p<0.01) (Study I, Figure 1), Veillonella atypica (p<0.05), and
Veillonella sp. HOT-780 (p<0.01) (Study II, Figure 3a) were associated with dental
caries in the childhood populations examined. Frequent association with Veillonella
species and caries, especially childhood caries, have been shown both by culture
(Marchant et al., 2001) and by molecular methods (Becker et al., 2002). Veillonella
species have also been shown to dominate the oral microbiota of all stages of caries
cavity formation in both primary and secondary teeth (Aas et al., 2008). Although
Veillonella species are weak acid producers and probably not involved in the
demineralization process of enamel or dentin, they may act as acid scavengers using
lactate as a carbon source (Egland et al., 2004).
In terms of detection frequency and mean detection of bacteria in childhood caries,
the most frequently detected species (>60% of the children) included Streptococcus
and Actinomyces species, R. dentocariosa, F. alocis, and V. parvula (Study I, Table
2, Figure 1). Moreover, significantly higher mean levels were observed in children
with ECC compared to caries-free children for A. naeslundii (p<0.001), A.
odontolyticus (p<0.001), A. israelii (p<0.05), A. gerencseriae (p<0.05), V. parvula
(p<0.001), and F. nucleatum subspecies polymorphum (p<0.05) (Study I, data not
shown). A. odontolyticus (p<0.05), A. israelii (p<0.05), and V. parvula (p<0.01)
were detected significantly more frequently in children with caries than in caries-free
children (Study I, Figure 1). By 16S rDNA cloning and sequencing the majority of
34
clones detected by child included Veillonella, Streptococcus, and Capnocytophaga
species and novel phylotypes that were detected as a lower percentage of the
individual microbiota, possibly explaining the lack of association with childhood
caries (Study II, Figure 3a).
Periodontitis-associated Bacteria
Study I included a DNA probe panel for detection of additional bacterial species
associated with periodontal diseases (Study I, Table 2). Fastidious periodontal
pathogens found more frequently in children with caries than in caries-free children
included A. actinomycetemcomitans (p<0.05), P. micra (p<0.05), P. gingivalis
(p<0.01), Eubacterium brachy (p<0.05), and F. alocis (p<0.05) (Study I, Figure 1).
Presence of periodontal pathogens in young children has been previously reported by
culture in intact teeth and by DNA probes in dental caries (Kamma et al., 2000;
Tanner et al., 2002). E. brachy and F. alocis are species not frequently reported in
children but have been associated with periodontitis in adults both by culture, DNA
probes, and 16S rDNA cloning and sequencing (Dahlen & Leonhardt, 2006; Hill et
al., 1987; Kumar et al., 2006).
A. actinomycetemcomitans by 16S rDNA cloning and sequencing was present in 15%
of children by PCR (Study II, Figure 3b) and was not associated with S-ECC (Study
II, Figure 3a). The detection frequency of the species was similar to that previously
described in young children by culture and PCR in Europe and the United States
(Alaluusua & Asikainen, 1988; Ashimoto et al., 1996). Notably, in children and
adolescents originating from Africa, the prevalence of the species (69%) and of
particular clones of the species exhibiting higher pathogenic potential was higher
(Haubek et al., 1996; Haubek et al., 2008). The use of PCR for both
A. actinomycetemcomitans and S. mutans improved detection of these species
present in low levels of the microbiota.
Socio-demographic, Dietary, and Clinical Factors
Clinical and background variables other than microbial composition associated with
childhood caries included race (p<0.05), previous dental visit (p<0.05), cracker,
chip, and cereal consumption (all p<0.05), and visible plaque (p<0.001) (Study I,
Table 1). Black and Asian children exhibited a higher prevalence of dental caries
••••
than children of other races as has also been observed previously in the United States
(Beltran-Aguilar et al., 2005; Shiboski et al., 2003). Snacking has been previously
associated with ECC, and especially snacking on high starch foods, like chips and
crackers that can be retained on the tooth surfaces (Kashket et al., 1996). Previous
dental visit was associated with presence of dental caries, since families with children
who experience dental pain are those who seek dental treatment. Finally, presence of
visible dental plaque, which exhibited the highest association with childhood caries
(Study I, Table 1), has been previously strongly associated with childhood caries
(Mohebbi et al., 2006; Wennhall et al., 2002) and serves as a predictor for future
caries development (Alaluusua & Malmivirta, 1994) in young children.
Age and presence of plaque (Study I) exhibited a linear relationship and a significant
interaction effect on caries levels (dft) (βage*plaque=0.495, p<0.05) (Figure 2).
Nonetheless, species associated with ECC and caries-free children in comparisons of
all children and children with visible plaque were similar (Study I, Figure 2).
Figure 2. Extent of dental caries (dft: decayed filled teeth index) in relation to child age. The scatter plot of dft versus age in the presence of plaque illustrates that age and plaque exhibited a significant interaction effect on caries extent (βage*plaque=0.495, p<0.05).
35
36
Oral Microbiota of Early Adult Periodontitis (Study III and IV)
The oral microbiota associated with early adult periodontitis was examined in
Studies III and IV. A total of 141 individuals participated in Study III comprised of
28 periodontally healthy subjects, 71 with Early Periodontitis 1 (subjects with mean
attachment level ≤1.5 mm and at least one site ≥ 2 mm attachment loss), and 42 with
Early Periodontitis 2 (subjects with mean attachment level >1.5 mm and mean
attachment loss ≥ 2 mm) (Study III, Table 1). One hundred seventeen individuals
were followed over 18 months and 19% (22/117) exhibited active periodontal
breakdown by losing >1.5 mm clinical attachment in one or more interapproximal
sites during the observation time (Study IV).
Use of oligonucleotide DNA probes (Study III, IV) allowed identification of
phylotypes with no cultured representatives, such as TM7 and Obsidian Pool
phylotypes (Hugenholtz et al., 1998), and fastidious species, such as treponemes and
F. alocis (Study III, Figures 1, 2 and Study IV, Figure 4).
Microbiota of Early Periodontitis and Periodontally Healthy Subjects
The most frequently detected species (>60% of the subjects) included Streptococcus,
Fusobacterium, and Granulicatella species (Study III, Figure 2). Interestingly, most
of the uncultivated phylotypes identified were detected in >20% of subjects (Study
III, Figure 2).
Species detected more frequently in early periodontitis than in periodontally healthy
subjects included T. denticola, F. alocis, Porphyromonas endodontalis, Bacteroides
sp. HOT-274 (oral clone AU126), F. nucleatum, and A. odontolyticus (p<0.01) by
oligonucleotide DNA probes (Study III, Figure 2) and P. gingivalis (p<0.001) and T.
forsythia (p=0.03) by PCR (Study III, Figure 3). In previous studies, T. denticola, F.
alocis, and Bacteroides sp. HOT-274 (oral clone AU126) have been detected more
frequently in periodontitis by 16S rDNA cloning and sequencing than in health
(Paster et al., 2001). Moreover, by means of oligonucleotide DNA probes the
previously reported species with the addition of P. endodontalis were associated with
periodontitis (Kumar et al., 2003). F. alocis and P. endodontalis, both strict
anaerobes have likely been underrepresented by culture of periodontitis samples,
due to their fastidious growth requirements. It has been recently proposed to include
•••
37
these two species in the panel of species used for periodontitis diagnostic purposes
(Dahlen & Leonhardt, 2006).
The significant association of P. gingivalis with early periodontitis compared to oral
health (p<0.001) (Study III, Figure 3) and with periodontal disease progression
compared to stability (p<0.001) (Study IV, Figure 7) in these two studies was
notable. In previous culture studies of initial periodontitis, our group had not found
an association of P. gingivalis with periodontitis (Tanner et al., 1996). Few studies
have examined early or incipient periodontitis in adults. The literature, thus, mainly
supports identification of the species in more advanced and chronic periodontitis
both by culture and molecular methods (Hamlet et al., 2001; Slots, 1986; Zambon et
al., 1981).
T. forsythia detection by PCR was only associated with early periodontitis at baseline
(p=0.03) (Study III, Figure 3) and not at follow-up analysis (p=0.07) (Study IV,
Figure 7). Tanner and co-workers have previously detected T. forsythia in
association with initial periodontitis by culture (Tanner et al., 1996). In a
longitudinal prospective study of adults using the same PCR method as the current
study, persistent detection of T. forsythia was predictive of progression of
periodontal attachment loss (Tran et al., 2001).
A. actinomycetemcomitans was not significantly associated with initial periodontitis
(Study III) or with periodontitis progression (Study IV), although it was detected at
24% from subjects exhibiting and at 21% from subjects not exhibiting attachment
loss (Study IV, Figure 4). Detection frequency was similar at baseline (Study III)
and corresponded to detection frequency by culture (27%) of 18-25 year old men with
minimal attachment loss (Muller et al., 1996). Since the methods used did not allow
for specific serotype identification of A. actinomycetemcomitans, the strains
identified could also have been of serotypes previously associated with periodontal
health (Asikainen et al., 1995; Zambon et al., 1983). Thus, it might be pivotal to
identify specific clones or serotypes of bacteria associated with either periodontal
health or disease.
38
Subgingival and Tongue Surface Microbiota
There were distinct differences by oligonucleotide DNA probes in species detection
between bacterial samples from subgingival sites and the tongue surface (Study III,
Figure 1). Species detected more frequently from the tongue surface were mainly
Gram-positive Streptococcus, Gemella and Granulicatella species (Study III, Figure
1). Subgingival samples exhibited a higher prevalence of periodontitis-associated
species than samples from tongue surface, including A. actinomycetemcomitans, T.
denticola, T. forsythia, F. alocis, and P. endodontalis (all p<0.05), and uncultivated
phylotypes, including TM7, Treponema and Actinobaculum clones (all p<0.05)
(Study III, Figure 1). However, several species detected more frequently in
periodontitis patients than healthy controls in previous studies, also exhibited higher
detection frequency from tongue surfaces in the present study; for example
Eubacterium sulci (Han et al., 1991), Camplylobacter concisus (Kamma & Nakou,
1997; Macuch & Tanner, 2000), Megasphera (Kumar et al., 2003; Kumar et al.,
2006), and Obsidian Pool phylotypes (Kumar et al., 2003; Li et al., 2006).
Although P. gingivalis (by PCR) was detected equally frequently from the subgingival
and the tongue surface samples, T. forsythia was detected more frequently
subgingivally (p<0.001) (Study III, Figure 3). Similarly Dahlén and co-workers,
identified P. gingivalis as a species that could significantly discriminate between
sites with and without periodontitis even from the dorsum of the tongue (Dahlen et
al., 1992). However, examining our overall results, most Gram-negative species were
detected with a higher frequency from subgingival than tongue plaque samples
(Study III, Figure 1) using oligonucleotide DNA probes. Similarly, in the study of
childhood caries (Paper I) the detection frequency of all species assayed was lower in
plaque samples taken from the dorsum of the tongue than the tooth surfaces utilizing
the whole genomic DNA probes.
Progressing Chronic Periodontitis
Gram-positive species Streptococcus infantis, S. sanguinis, and S. parasanguinis,
and the Gram-negative F. nucleatum (all p<0.03) were the species detected
significantly at a higher frequency from early periodontitis subjects exhibiting
attachment loss during follow-up than from “inactive” subjects (Study IV, Figure 4).
These finding are consistent with the fact that subgingival plaque forms from apical
•••
39
migration of the plaque growing on the tooth surface supragingivally (Theilade &
Theilade, 1985). In incipient periodontitis it could be expected that the associated
Gram-negative microbiota has not yet been established.
Detection of uncultivated phylotypes (Study IV, Figure 4) such as the Obsidian Pool,
TM7, Actinobaculum, Megasphera, Eubacterium, Porphyromonas, Bacteroidetes,
and Treponema clones, although not statistically associated with periodontal
destruction activity, exemplify how complex and diverse the subgingival microbiota
is, which is not always apparent from culture studies. Higher detection frequency of
periodontitis-associated species including P. micra and F. alocis by whole genomic
DNA probes and T. forsythia and P. gingivalis by PCR (p=0.0073) was observed in
subjects that exhibited attachment loss compared to those that did not (Study IV,
Figures 5 and 7).
The fact that few bacterial species were associated with early periodontitis or disease
progression (Study III, IV) could be due to low bacterial numbers in shallow pockets.
In addition, during the follow-up period, supragingival plaque was removed before
subgingival plaque sampling (Study III) and subjects were offered professional
cleaning after each sampling. This could also have affected the number of bacteria
and quality of the subgingival biofilm analyzed during the follow-up, leading to low
DNA-DNA hybridization detection of the species of interest. It has been previously
observed that professional supragingival cleaning of teeth can have an effect on
gingival inflammation and subgingival bacterial composition; however, results are
contradictory (Goodson et al., 2004; McNabb et al., 1992; Westfelt et al., 1998;
Ximenez-Fyvie et al., 2000).
Finally, the detection of the assayed species was also evaluated according to tooth
type sampled (Study IV, Figure 6). Most of the species assayed by whole genomic
DNA probes were detected at higher levels from incisors of subjects exhibiting
periodontal attachment loss over the observation time compared to inactive subjects
(Study IV). Exemplifying this association, C. rectus from incisors and canines
(p≤0.05) and E. saphenum from incisors (p≤0.05) were more frequently found in
subjects with active attachment loss (Study IV, Figure 6). Both C. rectus and E.
saphenum have been associated with periodontitis (Kumar et al., 2003; Moore &
Moore, 1994).
40
Socio-demographic Factors in Early Periodontitis
In the cross-sectional analysis of the clinical data at baseline most participants were
Caucasian (65%) and had never smoked (68%) (Study III, Table). A positive
association between early periodontitis and the following socio-demographic
variables was observed: older age (p=0.01), male gender (p=0.04), and cigarette
smoking (p=0.05) (Study III, Table 1). These findings were no longer significant
after the 18 month follow-up (Study IV, Table 1). A possible explanation could be
due to bias by “loss to follow-up”, meaning that the subjects that stayed in the study
and were observed over time were more uniform in their socio-demographic
characteristics, compared to the subjects that did not continue to participate.
Aggregatibacter actinomycetemcomitans Lack of Serotype
Antigen Expression (Study V)
In an attempt to study the role of bacterial subgroups in periodontitis, the
serotypeability of clinical A. actinomycetemcomitans isolates recovered from various
periodontal conditions was investigated (Study V). A. actinomycetemcomitans was
detected in all populations studied in this thesis (Study I-IV) and because of its
previously found association with periodontitis both in adults and younger subjects,
the association of subgroups of this species with periodontitis was investigated.
Serotype identification using PCR in addition to immunodiffusion exhibited perfect
concordance when immunodiffusion assay serotypeable strains were analyzed (Paper
V, Table 2). Non-serotypeable strains (N=95) by immunodiffusion assay could be
identified to serotypes a, b, c, or f by PCR analysis, showing that the strains carried
serotype-specific genes (Study V, Table 3). Western blot analysis using respective
antisera, antiserum against a non-serotypeable strain, and sera of patients with non-
serotypeable A. actinomycetemcomitans strains confirmed the lack of expression of
serotype-specific antigen in these strains (Study V, Figures 1, 2, and 3). Analyses of
the serotype-specific gene cluster and sequencing of the whole serotype a gene
cluster did not reveal any differences or any obvious DNA rearrangement between
serotypeable and non-serotypeable strains.
•••
41
It appeared that the non-serotypeable A. actinomycetemcomitans strains did not
include novel serotypes, but the reason for non-serotypeability was lack of expressing
the serotype antigen. Most of these strains were detected more frequently from
patients with chronic rather than aggressive forms of periodontitis. Furthermore,
most of the strains belonged to a distinct genetic lineage (serotypes c) (Paper V,
Table 3). The serotype-specific polysaccharide of A. actinomycetemcomitans as the
immunodominant antigen of the species (Page et al., 1991; Sims et al., 1991; Wilson
& Schifferle, 1991) has been suggested to play a significant role in the co-aggregation
of the species with other Gram-negative species and to mediate subgingival biofilm
formation (Fujise et al., 2008; Kolenbrander et al., 1989; Rupani et al., 2008; Suzuki
et al., 2006). Thus, lack of its expression would seem disadvantageous for the
species, due to impaired biofilm formation. On the other hand, a benefit of the lack
of serotype antigen expression could be a competitive advantage against the
antibody-based host response over strains expressing this antigen. Whether these
strains transiently or permanently express their lack of serotype expression was not
investigated. It could be possible that the strains refrain from expression in certain
phases of the biofilm formation or of host response.
42
Summary and Conclusions
A comprehensive assessment of children’s oral microbiota was achieved in this thesis
through use of extensive molecular methods and examination of numerous children.
In terms of species identification, 16S rDNA cloning and sequencing and whole
genomic DNA probes complemented each other. Clonal analysis enabled an
equitable identification of a diverse microbiota of 138 taxa, half of which have not
been previously cultivated. In addition, 107 of 3909 clones remained unidentified
and may include novel phylotypes. The DNA probe assay identified and quantified a
selection of 74 cultured species. Out of these, 37 species were not detected by clonal
analysis and certain species, such as Actinomyces species, were underrepresented.
The association of bacterial species with dental caries in children also differed
according to molecular methods used. From the species detected by the DNA probe
analysis of children’s dental plaque, 23% (17/74) were associated with childhood
caries, including Streptococcus, Actinomyces, and Lactobacillus species, as well as
certain periodontal pathogens. These findings confirmed and extended
bacteriological findings regarding children’s caries-related microbiota previously
observed by molecular methods and culture. By clonal analysis, three out of the ten
most prevalent species/phylotypes in children with caries were uncultivated or
unnamed. Although clonal analysis might not be the optimal method for revealing
associations of oral microbes with dental disease or health, the associations observed
were clear and likely reflect the distribution of the predominant microbiota. Finally,
the single-species PCR increased detection sensitivity of S. mutans present in low
levels of the microbiota and enabled determining its association with dental caries in
the children. Depending on the choice of the molecular method used, the microbiota
of children can be approached from different viewpoints, focusing on the detection
frequency of specific recognized pathogens, species quantification, and/or detection
frequency of unnamed/uncultivable (even novel) phylotypes.
The microbiota of the early phase of adult periodontitis has been rarely studied,
although incipient periodontal disease could be considered an informative model for
studying initial microbial changes from health and during periodontitis progression.
In this thesis, microbiological analysis of initial periodontitis was performed by
•••
43
oligonucleotide and whole genomic DNA probes, and PCR. Generally, similarities
were observed in the microbiota of initial periodontitis and previously well-
characterized advanced periodontitis, with regards to detection of fastidious species
such as T. denticola, F. alocis, and P. endodontalis and a strong association with P.
gingivalis and T. forsythia. In addition, the detection of uncultivated Treponema,
Actinobaculum, and Bacteroidetes phylotypes, as well as environmental species with
no cultured representatives to date reveals the complexity of early adult periodontitis
and warrants further investigation. Methodologically, the use of different molecular
methods complemented each other also in the analysis of the microbiota associated
with periodontitis. The PCR technique increased detection sensitivity of specific
species and revealed their “hidden” associations with early periodontitis not
observed with the other methods employed. The whole genomic DNA probes
provided quantification of previously cultured species and the oligonucleotide DNA
probes allowed screening of cultured and uncultivated phylotypes.
Besides bacterial diversity at species/phylotype level, different genotypes and
phenotypes among strains of a species add complexity to the oral microbiota, as
exemplified by the present study on A. actinomycetemcomitans. The reason for non-
serotypeability of a number of A. actinomycetemcomitans strains was not the
detection of novel serotypes or unrecognized clonal types as determined by
genotyping, but lack of serotype antigen expression, despite that the strains carried
serotype-specific genes. That the strains were mainly found in chronic periodontitis
suggests that the serotype antigen deficient phenotype may provide a competitive
advantage against humoral immune response over strains expressing this antigen.
Various molecular methods used in this thesis shed light on different subsets of oral
bacterial populations, giving a variable perspective on their members. Until recently,
identification of oral pathogens and their association with dental diseases has not
been based on the microbiome of the oral cavity, but rather on the sporadic focus on
specific species usually previously isolated by culture. Identification of the complete
array of bacteria found in dental disease or health, through screening methods such
as 16S rDNA cloning and sequencing, is pivotal and can in the future lead to
identification of diagnostic bacterial profiles of dental diseases by more rapid
molecular methods such as microarray analysis.
44
Acknowledgements
This thesis was an active collaboration between the Divisions of Oral Microbiology and Cariology, Department of Odontology, Faculty of Medicine, Umeå University, Umeå, Sweden, and the Department of Molecular Genetics, The Forsyth Institute, Boston, Massachusetts, USA. Financial support was received from the Faculty of Medicine, Umeå University and from the National Institute of Dental and Craniofacial Research /National Institutes of Health, Bethesda, Maryland, USA.
I would like to express my deepest gratitude to my supervisors who supported me through this unique scientific journey.
Professor Sirkka Asikainen (primary supervisor), Head of the Division of Oral Microbiology, Umeå University. Thank you for giving me the privilege to collaborate with you, for your unbent scientific values, your everlasting belief in me, and your energy and enthusiasm.
Professor Anne C. R. Tanner (supervisor), Department of Molecular Genetics, The Forsyth Institute. Thank you for the smooth introduction to the American reality, for always treating me as an equal collaborator, and for teaching me everything from microbiology to proper tea drinking.
Professor Ingegerd Johansson (supervisor), Division of Cariology, Umeå University. Thank you for establishing the Umeå-Forsyth collaboration, for keeping me balanced, and for teaching me how work can balance one’s personal life.
Professor Nicklas Strömberg (examinator), Division of Cariology, Umeå University. Thank you for being an academic inspiration.
I am indebted to my advisor Professor Chester W. Douglass, Chair of the Department of Oral Health Policy and Epidemiology, Harvard School of Dental Medicine, for introducing me to the field of Oral Epidemiology and Public Health and for encouraging my interdisciplinary approach to dental research.
I extend my gratitude to Professor Floyd E. Dewhirst, Department of Molecular Genetics, The Forsyth Institute, for guiding me through the “omes” of research.
My heartfelt thanks to Dr. Ralph Kent Jr., Department of Biostatistics, The Forsyth Institute, for helping me appreciate the world of statistics.
I am deeply grateful to Dr. Daniel J. Smith, Department of Immunology, for his support, valuable scientific advice and guidance, as well as my Training Grant (T32) committee: Dr. Sigmund S. Socransky, Dr. Max Goodson, Dr. Henry Margolis, Dr. Richard Pharo, and Prof. Floyd E. Dewhirst. Thank you for all your scientific input.
I also wish to thank the Forsyth family, especially Dr. Philip P. Stashenko (President of the Forsyth Institute), Professor Bruce J. Paster (Head of Molecular Genetics), Professor Martin A. Taubman (Head of Immunology), Dr. Jacques Izard, Dr. Mary Ellen Davey, and compatriot Dr. Nikos S. Soukos (Head of Applied Molecular Photomedicine). A warm thank you to Lora A. Murray.
A special thank you and admiration extended to Professor Donald Hay.
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45
At Harvard School of Dental Medicine, I would like to extend my sincere gratitude to Dean Bruce Donoff, Professor Steven Sonis, Dr. Athanasios Zavras, Dr. Taru Kinnunen, Dr. Kaumudi Joshipura, and Dr. Peggy Timothy. Thank you for your guidance and help over the past three years. Thank you Ilona and Veronic.
At Boston University Goldman School of Dental Medicine, I would like to warmly thank collaborators and mentors Professor Thomas Van Dyke, Professor Raul Garcia, Dr. Christopher Hughes, Professor Nancy Kressin, Professor Michelle Henshaw, Dr. Martha Nunn, and Dr. Jennifer Soncini. At Tufts University School of Dental Medicine, I wish to thank Professor Catherine Hayes, Dr. Carole A. Palmer, and Dr. N. Pradhan. A special thanks goes to our international collaborator Professor William Wade, thank you for your valuable guidance.
I would like to extend a special thank you to those who supported me in my first steps in research, Dr. Sotirios Kalfas, Dr. Rolf Claesson, Dr. Anders Johansson, Dr. Georgios Belibasakis, and Dr. Lennart Hänström.
I want to express my deep gratitude and admiration to Professor Göran Sundqvist for teaching me to enjoy oral microbiology and for always caring together with Eva.
Dr. Patricia Corby and Dr. Pernilla Lif Holgerson; no thank you is enough.
I want to thank the Asikainen family especially Professor Başak Doğan, Dr. Maribasappa Karched, Dr. Ahu Uraz, Dr. Jan Oscarsson, Dr. Maria Hedberg, Kjell Eneslätt, Elisabeth Granström, and Bernard Thay. Thank you to Sari Korva.
I want to thank the Tanner family especially Dr. Charles Shulin Lu, Alexis Moore, and Jennifer J. Mathney and all students joining the group over the years.
Thank you to some special laboratory partners and friends Margaretha Jonsson, Maj-Britt Edlund, Christine Roth, Inger Sjöström (Umeå) and Sara Barbuto, Susan Boches, Tahani Boumenna (Boston).
Special thanks to fellow PhD students Monik Jimenez, Dr. Sona Tumanyan, Dr. Priyaa Shanmugham, Dr. Peyman Kelk, Dr. Pamela Hasslöf, Dr. Zivko Mladenovic.
Special thanks on a personal level to my very dear friends Angeliki Lambrou (imminent PhD), Dr. Eirini Iliaki, Dr. Wen Kou (imminent PhD), Dr. Rupali Bhalerao, Dr. Areti Pentedeka, Evangelos Liaras (imminent PhD), Dr. Ioannis Chrisanthou, Vicky Papadimitriou, Ioanna Lambropoulou, and Olympia Kortsari.
Thanks to all my family, friends, and family friends; especially my aunt Sapfo.
To my parents (my favorite dentists); this thesis is dedicated to you for having the dream for me, before me, and helping me realize it. You are my most precious influence.
To Thomas; you never let me feel alone, you always listen. Thank you for your endless love and support; thank you for inspiring me scientifically and otherwise.
Umeå, October 31, 2008
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