molecular oral microbiology molecular oral microbiology

Subgingival microbiome in smokers and non-smokers in Korean chronic periodontitis patients J.-H. Moon1,2, J.-H. Lee1,2 and J.-Y. Lee1 1 Department of Maxillofacial Biomedical Engineering, School of Dentistry, Institute of Oral Biology, Kyung Hee University, Seoul, Korea 2 Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul, Korea

Correspondence: Jin-Yong Lee, Department of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701, Korea Tel./fax: +82-2-960-2838; E-mail: [email protected] Keywords: Korean; periodontitis; pyrosequencing; smoking; subgingival microflora Accepted 28 September 2014 DOI: 10.1111/omi.12086

SUMMARY Smoking is a major environmental factor associated with periodontal diseases. However, we still have a very limited understanding of the relationship between smoking and subgingival microflora in the global population. Here, we investigated the composition of subgingival bacterial communities from the pooled plaque samples of smokers and non-smokers, 134 samples in each group, in Korean patients with moderate chronic periodontitis using 16S rRNA gene-based pyrosequencing. A total of 17,927 reads were analyzed and classified into 12 phyla, 126 genera, and 394 species. Differences in bacterial communities between smokers and non-smokers were examined at all phylogenetic levels. The genera Fusobacterium, Fretibacterium, Streptococcus, Veillonella, Corynebacterium, TM7, and Filifactor were abundant in smokers. On the other hand, Prevotella, Campylobacter, Aggregatibacter, Veillonellaceae GQ422718, Haemophilus, and Prevotellaceae were less abundant in smokers. Among species-level taxa occupying > 1% of whole subgingival microbiome of smokers, higher abundance (≥ 2.0-fold compared to non-smokers) of seven species or operational taxonomic units (OTUs) was found: Fusobacterium nucleatum, Neisseria sicca, Neisseria oralis, Corynebacterium matruchotii, Veillonella dispar, Filifactor alocis, and Fretibacterium AY349371. On the

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 227–241

other hand, lower abundance of 11 species or OTUs was found in smokers: Neisseria elongata, six Prevotella species or OTUs, Fusobacterium canifelinum, Aggregatibacter AM420165, Selenomonas OTU, and Veillonellaceae GU470897. Species richness and evenness were similar between the groups whereas diversity was greater in smokers than non-smokers. Collectively, the results of the present study indicate that differences exist in the subgingival bacterial community between smoker and non-smoker patients with chronic moderate periodontitis in Korea, suggesting that cigarette smoking considerably affects subgingival bacterial ecology.

INTRODUCTION Evidence indicates that chronic periodontitis results from the presence of complex microbial communities in the subgingival sulcus (Socransky & Haffajee, 2005; Shchipkova et al., 2010) and that smoking is a major environmental factor associated with the development of extensive and severe periodontal diseases (Tomar & Asma, 2000; Johnson & Hill, 2004; Mullally, 2004; Shchipkova et al., 2010). Clinical findings have led to our understanding of the role of smoking in the pathogenesis of periodontal disease. Increased 227

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probing depth, alveolar bone loss, tooth mobility and tooth loss have been reported to be more severe in €m & Floderussmokers than non-smokers (Bergstro Myrhed, 1983; Feldman et al., 1983, 1987; Bolin €m, 1986; Bergstro €m et al., 1986; Osterberg & Mellstro € & Eliasson, 1987; Ahlqwist et al., 1989; Bergstrom, €m et al., 1991). 1989; Goultschin et al., 1990; Bergstro There are numerous mechanisms by which smoking may affect host–parasite interactions in the oral cavity. Among these mechanisms are diminished cell-mediated and humoral immune responses (MacFarlane et al., 1992; Tew et al., 1996), favoring infection with microbial pathogens (Bundred et al., 1992; Bateson, 1993; Epstein et al., 1993), hampering antimicrobial therapy (Bateson, 1993), and increased resistance to antibiotics (Witteman et al., 1993). Meanwhile, there have been conflicting reports on whether or not smoking has an effect on the periodontal microflora. Some studies reported that cigarette smoking increased the likelihood of subgingival infection with certain periodontal pathogens (Zambon et al., 1996; Haffajee & Socransky, 2001), whereas others did not find significant differences between smokers and non-smokers in the composition of the subgingival microflora (Preber et al., 1992; Stoltenberg et al., 1993; Renvert et al., 1998; Darby et al., €m et al., 2001; Apatzidou et al., 2005). 2000; Bostro In the earlier investigations, the subgingival microbiome of smokers was examined by traditional methods such as culturing and targeted DNA-based assays such as polymerase chain reaction (PCR), real-time PCR, and DNA–DNA hybridization (Zambon et al., 1996; Shiloah et al., 2000; Haffajee & Socransky, 2001; van Winkelhoff et al., 2001; Van der Velden et al., 2003; Darby et al., 2005). The apparently contradictory results of the earlier studies may be due to different methods of identifying bacteria that are targeted. In fact, the closed-ended or selective approaches are inadequate for a comprehensive examination of subgingival microbiome, which is a highly complex ecosystem, because subgingival microbiome is largely uncultivated, and targeted molecular approaches are limited to the examination of only previously known species (Shchipkova et al., 2010). The adoption of 16S ribosomal RNA gene (rDNA) amplification by PCR, followed by cloning and sequencing, has allowed more comprehensive broadrange investigation of oral bacterial communities (Siqueira et al., 2012). In 2010, Shchipkova et al. 228

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explored the subgingival microbiome of current and never-smokers with periodontitis in the USA using 16S cloning and sequencing for bacterial identification. In the study, as-yet-uncultivated organisms as well as novel and previously unsuspected species were identified and counted. The microbial profile of smokers with periodontitis is distinct from that of nonsmokers, and more differences were observed in abundance than in prevalence of species (Shchipkova et al., 2010). More recently, new methods of high-throughput DNA sequencing, regarded as nextgeneration DNA sequencing technologies, have been introduced in oral microbiology research. Next-generation DNA sequencing technologies are less laborintensive than automated Sanger sequencing and do not require cloning of template DNA into bacterial vectors (Nowrousian, 2010; Siqueira et al., 2012). In a study performed in the Netherlands using subgingival plaque samples from 28 Caucasians and two non-Caucasians, differences were found in the composition of the subgingival microbiome between smokers and non-smokers, as revealed by pyrosequencing (Bizzarro et al., 2013). Despite recent advances in the field, we still have a very limited understanding of the relationship between smoking and subgingival microflora in the global population, as racial/ethnic and geographical differences exist in the subgingival microbiome (Haffajee et al., 2004; Rylev & Kilian, 2008; Kim et al., 2009). In the absence of data from East Asia on this aspect, the objective of the current study was to investigate the differences in the composition of subgingival microbiome between smokers and non-smokers in Korean periodontitis patients using 16S rRNA genebased pyrosequencing analysis. MATERIALS AND METHODS Patient recruitment, inclusion criteria and exclusion criteria Moderate periodontitis sites are regarded to be very similar ecological niches with a narrower range of the probing pocket depth (PPD) and clinical attachment loss (CAL) scale than moderate-to-severe sites. Smokers have greater severity of periodontal destruction compared with non-smokers (Tonetti, 1998), hence moderate sites of smokers may eventually, but more quickly, lead to more severe periodontal © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 227–241

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destruction, showing a distinct profile of subgingival microbial community shift in the transition to severe periodontitis. Therefore, the intermediate stage of periodontitis is more appropriate for examining the effect of smoking on the subgingival microbiome. In the present study, moderately diseased sites were selected for subgingival plaque sampling. This study was approved by the Ethics Committees of Kyung Hee University School of Dentistry (approval number: KHUSD IRB1009-02). Subjects for this study were recruited from the Department of Periodontology, Kyung Hee University Dental Hospital, Republic of Korea, and requested to sign an informed consent form to participate in the study. All participants were affected by chronic periodontitis and had at least 24 teeth, including at least four molars, and had, in different quadrants, at least eight sites with pocket depths of ≥ 4 mm, and six sites with attachment level of > 2 mm. Patients were excluded if > 30% of their total sites were graded as severely affected (PPD ≥ 7 mm); if they were pregnant; if they were a mixed ethnic background; if they had a known systemic condition that could influence the periodontal condition; if they had received antibiotic therapy within the past 3 months. Smoking status and tobacco exposure Measurement of smoking by self-report has been shown to be a valid method for estimating smoking habits (Petitti et al., 1981; Kamma et al., 1999). Hence, smoking status and tobacco exposure were assessed by questionnaire and expressed in packyears: calculated as the number of cigarettes smoked per day multiplied by the number of years the patient had smoked, divided by 20 (a standard pack of cigarettes). A patient was defined as a smoker if he was currently smoking and had been smoking five or more cigarettes a day for at least 10 years and as a nonsmoker if he had never smoked or quit smoking longer than 10 years before intake. Clinical measurements, sample collection and DNA isolation The following clinical measurements were recorded: plaque index (PI) (presence/absence), % bleeding on probing (BOP), PPD, and CAL. Supragingival plaque was removed with a Gracey curette, after which © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 227–241

Subgingival microbiome of Korean smokers

subgingival plaque samples were taken with a fresh sterile curette from four subgingival sites on the first or the second molars. The samples were placed in 100 ll of sterile PBS and stored frozen at
20°C until use. The samples were excluded if the teeth had the possibility of an endodontic infection, such as deep caries, secondary decay, old restoration, pulp necrosis, a narrow and deep pocket probing, and incomplete endodontic treatment (Lim et al., 2014). The samples were also excluded if the teeth had periapical radiolucency and deep pocket depths because the teeth may have an infected root canal system with chronic apical periodontitis (Abbott & Salgado, 2009; Verma et al., 2011). A total of 134 teeth with PPD of 4–6 mm and CAL of 3–5 mm were selected from 57 smokers; 134 teeth from 36 age- and sexmatched non-smokers served as controls. Bacterial DNA was extracted from the samples with a Wizard Genomic DNA Purification Kit (Promega, Madison, WI), quantified using a Nanodrop ND-1000 (NanoDrop Technologies, Montchanin, DE), and analyzed with the Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA). PCR amplification and pyrosequencing Amplification by PCR was performed using primers targeting from V1 to V3 regions of the 16S rDNA with the extracted bacterial genomic DNA. For bacterial amplification, barcoded primers of 9F (50 -CCTATCC CCTGTGTGCCTTGGCAGTC-TCAG-AC-AGAGTT T GATCMTGGCTCAG-30 ; underlining sequence indicates the target region primer) and 541R (50 -CCATCTCATCCCTGCGTGTCTCCGAC-TCAG-XAC-ATTACCGCGGCTGCTGG-30 ; where ‘X’ indicates the unique barcode for each pooled sample (http:// www.ezbiocloud.net/resource). The amplifications were carried out under the following conditions: initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 s, primer annealing at 55°C for 30 s, and extension at 72°C for 30 s, with a final elongation at 72°C for 5 min. The PCR product was confirmed by using 2% agarose gel electrophoresis and visualized under a Gel Doc system (BioRad, Hercules, CA). The amplified products were purified with the QIAquick PCR purification kit (Qiagen, Valencia, CA). An equimolar mixture containing the amplicons from two different pooled samples was prepared. The mixed amplicons were clonally amplified 229

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on capture beads by emulsion PCR, and the enriched DNA beads were deposited into a PicoTiter plate. Pyrosequencing of the emulsion PCR-amplified DNA fragments was carried out at Chunlab, Inc. (Seoul, Korea), with a GS Junior Sequencing system (Roche, Branford, CT) according to the manufacturer’s instructions. Data analysis The basic analysis was conducted according to the previous descriptions in other studies (Chun et al., 2010; Hur et al., 2011; Kim BS et al., 2012). Obtained reads from the two different samples were sorted by the unique barcodes of each PCR product. The sequences of the barcode, linker, and primers were removed from the original sequencing reads. Any reads containing two or more ambiguous nucleotides, low-quality score (average score < 25), or reads shorter than 300 bp were discarded. Potential chimera sequences were detected by the bellerophone method, which is comparing the BLASTN search results between forward half and reverse half sequences (Huber et al., 2004). After removing chimera sequences, the taxonomic classification of each read was assigned against the EzTaxon-e database (http://eztaxon-e.ezbiocloud.net) (Kim OS et al., 2012 ), which contains 16S rDNA sequences of type strains that have valid published names and representative species level phylotypes of either cultured or uncultured entries in the GenBank database with complete hierarchical taxonomic classification from the phylum to the species. The richness and diversity of the samples were determined by abundance-based coverage estimation and Shannon diversity index at the 3% distance. Random subsampling was conducted to equalize read size of samples for comparison of different read sizes among samples. To compare operational taxonomic units (OTUs) between the samples, shared OTUs were obtained with the exclusive or (XOR) analysis of CLCOMMUNITY program (Chunlab Inc., Seoul, Korea). The diversity indices and species richness were calculated using Taxonomy-Dependent Clustering–Taxonomy-Based Clustering (Jeon et al., 2013). RESULTS AND DISCUSSION It has been known that considerable variation exists in the human microbiome between individuals 230

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sampled within the same microenvironment, such as oral cavity, skin, airway, gastrointestinal tract and urogenital tract/vagina (Xie et al., 2014). Large differences in subgingival bacterial composition have been reported between patients and even within different pocket locations in the same patients (Dzink et al., 1988). Moreover, plaque samples collected from the same subgingival area at different time-points within the same individual did display some community composition differences (Xie et al., 2014). Meanwhile, in the analysis of extremely complex microbial communities such as soils and sediments, a common sampling strategy is the combining of multiple samples obtained from various locations within the area of interest into a single, putatively representative, homogeneous sample (Manter et al., 2010). For the assessment of taxon richness, individual plaque sampling may be a better choice. By pooling individual samples, sample-specific taxa may, however, become rare through a dilution effect (Manter et al., 2010). Nevertheless, it has been suggested that the homogenized samples by pooling can adequately capture the heterogeneity and dominant microbial community, especially when estimates of total phylotype richness are not the primary research objective (Kang & Mills, 2006). Moreover, sample pooling minimizes the variability between samples and provides an impression of the overall community structure (Ellingsøe & Johnsen, 2002). The objective of this study was to explore the differences in the composition of subgingival bacterial communities between smokers and non-smokers in Korean chronic periodontitis patients using an open-ended pyrosequencing technique. To obtain an impression of the subgingival bacterial community affected by smoking, especially focusing on abundant taxa, which are important and active ones (Engel et al., 2012), and key important shift in response to smoking, all the samples from the same group were pooled to obtain a single, putatively representative, homogeneous subgingival plaque. A total of 134 teeth with PPD of 4–6 mm and CAL of 3–5 mm were selected from 57 smokers while 134 teeth from 36 age- and sexmatched non-smokers served as controls. As shown in Table 1, all the patients had similar clinical and demographic characteristics except for tobacco exposure. No significant differences in the periodontal conditions were observed between the sample sites of the groups. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 227–241

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Subgingival microbiome of Korean smokers

Table 1 Clinical and demographic characteristics of participants (mean  standard deviation)

Characteristic

Smokers (n = 57)

Age (years) 50.3 Gender (% male) 100 Tobacco exposure Packs/year ( SD) 20.8 Years ( SD) 22.2 Full mouth periodontal profile No. of teeth 25.5 % PPD = 4–6 mm 69.1 % PPD ≥ 7 mm 10.0 % BOP 54.9 % PI 73.1 Profile of sample sites No. of sites sampled 134 Mean PPD (mm) 4.9 Mean CAL (mm) 4.2

 13.0

 8.6  11.5     

1.8 17.6 8.9 18.7 10.2

 0.6  0.8

Non-smokers (n = 36)

P-value

53.6  11.5 100

ns na < 0.01 < 0.01

0 0 26.0 68.1 11.7 47.7 69.0

    

2.0 20.5 9.2 20.0 8.9

134 5.0  0.7 4.3  0.9

ns ns ns ns ns na ns ns

BOP, bleeding on probing; CAL, clinical attachment level; na, not applicable; ns, not significant; PI, plaque index; PPD, probing pocket depth. Significant difference in tobacco exposure was seen between the two groups (two-sample t-test).

There are many taxonomic classification algorithms available (Liu et al., 2008) and several reference databases that can be used with any given algorithm. Taxonomic classification of the thousands/millions of 16S rRNA gene sequences generated in microbiome studies is often achieved using a naive Bayesian classifier (for example, the Ribosomal Database Project II [RDP] classifier), which is attributable to its favorable trade-offs among automation, speed, and accuracy (Wang et al., 2007; Cole et al., 2009; Werner et al., 2012). The Human Oral Microbiome Database (HOMD; http://www.homd.org.) provides more than 600 prokaryote species that are present in the human oral cavity based on a curated 16S rRNA gene-based provisional naming scheme (Chen et al., 2010). In our preliminary data analysis, all taxonomic classifications were assigned using naive Bayesian algorithm developed for the RDP classifier, HOMD, and EzTaxon-e database. We found good agreement between HOMD and EzTaxon-e classifications down to the level of genus whereas RDP showed insufficient resolution to classify the GN02 and Synergistetes (data not shown). As EzTaxon-e database has manually annotated sequences at the species level (Chun et al., 2007; Hsiao et al., 2012), covering © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 227–241

representatives of yet uncultured phylotypes (Kim OS et al., 2012), we finally analyzed the pyrosequencing reads using EzTaxon-e database. More than 17,900 amplicons from the V1 to V3 hypervariable regions of the 16S rRNA gene were sequenced, of which 10,181 reads passed quality control (Table 2). A total of 12 phyla, 126 genera and 394 species-level taxa were detected in the samples. Differences in bacterial communities between smokers and non-smokers with moderate chronic periodontitis were observed at all phylogenetic levels. The vast majority of the sequences (94.5%) belonged to one of the following six phyla: Bacteroidetes, Fusobacteria, Proteobacteria, Firmicutes, Spirochaetes, and Actinobacteria (Fig. 1, upper panel). Of the six common phyla, Bacteroidetes was more abundant in non-smokers while Fusobacteria sequences were found at higher levels in smokers. Meanwhile, Synergistetes occupied < 1% of the subgingival microflora of non-smokers, but was found at higher levels in smokers (Fig. 1, lower panel). In contrast to an earlier view that the oral microbiome consists of large numbers of uncultivated species (Paster et al., 2001), overall 79% of the sequences were classified into cultivated phylotypes according to a 16S database of the core human oral microbiome (http://microbiome. osu.edu). At the genus level, the predominant genera were Fusobacterium (16.9%), Prevotella (15.3%), Neisseria (9.9%), Porphyromonas (8.0%), Capnocytophaga (7.8%), Leptotrichia (4.7%), Treponema (4.4%), Selenomonas (4.4%), Campylobacter (2.7%), Fretibacterium (2.1%), Aggregatibacter (1.9%), Streptococcus (1.9%), Corynebacterium (1.8%), Veillonella (1.6%), Haemophilus (1.1%), Paludibacter (1.0%),

Table 2 Sequencing information and taxonomic assignment for the bacteria in subgingival plaque of smokers and non-smokers No. of reads (%) Rank

Similarity range

Smokers

Non-smokers

Species Genus Family Order Class Phylum Unidentified Total

x ≥ 97% 97 > x ≥ 94 > x ≥ 90 > x ≥ 85 > x ≥ 80 > x ≥ 75% > x

4175 138 30 13 4 0 0 4360

5499 237 64 21 0 0 0 5821

94% 90% 85% 80% 75%

(95.75) (3.16) (0.68) (0.29) (0.09) (0.00) (0.00) (100)

(94.46) (4.07) (1.09) (0.36) (0.00) (0.00) (0.00) (100)

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Figure 1 Difference between smokers and non-smokers at the level of phylum. Upper and lower panels show the abundance of cultivated and uncultivated phylotypes in common and rare phyla. Overall, 79% of the sequences were classified into cultivated species. More uncultivated sequences were observed in current-smokers.

Actinomyces (0.9%), Filifactor (0.9%), Dialister (0.9%), and Tannerella (0.8%), occupying about 90% of the subgingival microflora from the diseased site of Korean patients with moderate chronic periodontitis. The details of the differences between the two groups, smokers and non-smokers, at the level of genus are shown in Fig. 2. Smokers showed relatively higher occurrence (≥ 1.5-fold compared with non-smokers) of Fusobacterium, Fretibacterium, Streptococcus, Veillonella, Corynebacterium, TM7, and Filifactor (Fig. 2A). Among genera occupying < 1% of each group, Tannerella, Lautropia, Rothia, Eubacterium, Granulicatella, Gemella, Parvimonas Desulfobulbus, Bacteroides, Leptotrichiaceae, and Mogibacterium were more abundant (≥ 1.5-fold) in smokers (Fig. 2B). On the other hand, Prevotella, Campylobacter, Aggregatibacter, Veillonellaceae GQ422718, Haemophilus, Prevotellaceae, Cardiobacterium, Neisseriaceae FJ976399, and Porphyromonadaceae were less abundant in smokers. Figure 3 shows the details of the differences between smokers and non-smokers at the level of species. For convenience sake, bacteria of highoccurrence (> 1%) and low-occurrence (< 1%) were separately examined. Among species-level taxa occupying > 1% of the whole subgingival microbiome of smokers, higher abundance (≥ 2.0-fold compared with non-smokers) of seven species or OTUs was found 232

(Fig. 3A): Fusobacterium nucleatum, Neisseria sicca, Neisseria oralis, Corynebacterium matruchotii, Veillonella dispar, Filifactor alocis, and Fretibacterium AY349371. On the other hand, lower abundance (≥ 2.0-fold) of 11 species or OTUs was found in smokers: Neisseria elongata, six Prevotella species or OTUs, Fusobacterium canifelinum, Aggregatibacter AM420165, Selenomonas OTU, and Veillonellaceae GU470897. Among species-level taxa occupying < 1% of whole subgingival microbiome of smokers, 70 species including six Streptococcus species or OTUs, five Fretibacterium species or OTUs, five Neisseria species, four Actinomyces species or OTUs, four Veillonella species or OTUs, four Eubacterium species, three TM7 OTUs, and two Capnocytophaga species were more abundant (≥ 2-fold) in smokers (Fig. 3B). Meanwhile, 42 species including 14 Prevotella species or OTUs and six Selenomonas species or OTUs were less abundant (≥ 2-fold) in smokers (Fig. 3C). Our finding that genus Fusobacterium, consistently associated with periodontitis (Socransky et al., 1998; Kumar et al., 2005), is abundant in smokers is in good agreement with previous studies performed in the Netherlands (Bizzarro et al., 2013) and the USA (Shchipkova et al., 2010). Fusobacterium species including F. nucleatum are not traditionally regarded as the most important periodontal pathogens. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 227–241

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Subgingival microbiome of Korean smokers

A

B

Figure 2 Difference between smokers and non-smokers at the level of genus. The graphs indicate genera showing differences in relative abundance greater than 1.5-fold between smokers and non-smokers as compared with each other. (A) Genera that occupied > 1% of the subgingival microflora of smokers or non-smokers. (B) Genera that occupied < 1% of the subgingival microflora of smokers and non-smokers.

However, it is widely accepted that F. nucleatum plays an important role in the formation of the subgingival biofilm, acting as a bridge between the biofilm bacteria, and has local immunosuppressive capability (Signat et al., 2011). Furthermore, Fusobacterium is known to be one of the most abundant genera found in periodontal disease (Griffen et al., 2012) and is closely associated with periodontal attachment loss (Bizzarro et al., 2013).Taken together, these findings suggest that genus Fusobacterium and especially F. nucleatum may be one of the major determinants of subgingival bacterial community shift in response to smoking in different ethnic groups. Neisseria species have not been regarded as significant disease- or health-associated bacteria (Griffen et al., 2012). Bizzarro et al. (2013) reported that the © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 227–241

genus Neisseria occupied 1% of the subgingival microbiome of Dutch patients with moderate-to-severe periodontitis. In the study, no difference was found for the abundance of Neisseria species between smokers and non-smokers (Bizzarro et al., 2013). In contrast, we observed that genus Neisseria was one of the predominant genera occupying 9.9% of the subgingival microflora from the diseased site of Korean patients with moderate chronic periodontitis. Interestingly, N. elongata was abundant in non-smokers while five other Neisseria species, including N. oralis and N. sicca, were associated with smoking (Fig. 3). The reason for the dissimilarity between the studies is not clear, but microbial differences may be due to genetic background, diet, racial/ethnic or cultural background of the subjects (Haffajee et al., 2005). 233

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A

B

C

Figure 3 Difference between smokers and non-smokers at level of species. The graphs indicate species-level taxa showing differences in relative abundance greater than 2.0-fold between smokers and non-smokers as compared with each other. (A) Species that occupied > 1% of the subgingival microflora of smokers or non-smokers. (B) and (C) Species that occupied < 1% of the subgingival microflora of smokers and non-smokers.

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Further studies are needed to investigate the global distributions and the role of oral Neisseria taxa in the oral microbial ecosystem, and the association between smoking and the bacteria. Molecular methods of bacterial identification have facilitated the ability to identify previously overlooked bacteria associated with periodontal disease. One such organism is Filifactor alocis, a Gram-positive anaerobic rod (Moffatt et al., 2011). Filifactor alocis has emerged as an organism that increases in number in diseased periodontal sites in comparison with healthy sites (Kumar et al., 2003, 2006; Dahlen & Leonhardt, 2006; Wade, 2011). In addition, it has been reported that smoking cessation led to a decrease in the levels of Filifactor alocis (Delima et al., 2010). In the present study, we observed a strong association of Filifactor alocis with smoking. We also found that smokers have relatively higher proportions of four Eubacterium species including Eubacterium nodatum, which has been reported to be strongly associated with periodontitis (Haffajee et al., 2006, 2008; Griffen et al., 2012). Although Filifactor alocis and E. nodatum were found in lower proportions than F. nucleatum, differences in the relative abundance between non-smokers and smokers of the two species were 3.0-fold and 5.3-fold, respectively, which were greater than that of F. nucleatum (Fig. 3). Of particular interest to us is the question how smoking increases the occurrence of Filifactor alocis and E. nodatum in the subgingival plaque. Further studies are needed to elucidate mechanisms underlying the association between smoking and the bacteria. Synergistetes is a novel bacterial phylum consisting of numerous non-cultivated phylotypes (Belibasakis et al., 2013), and the human-originated oral Synergistetes are divided principally into cluster A and cluster B (Vartoukian et al., 2009). Increasing lines of evidence demonstrate that this phylum, especially cluster A, not B, is associated with periodontal diseases (Belibasakis et al., 2013; You et al., 2013) and is found at higher prevalence, numbers, and proportions in saliva from patients with periodontitis than in non-periodontitis subjects (Belibasakis et al., 2013). To date, only one species of cluster A oral Synergistetes has been cultivated: Fretibacterium fastidiosum (Vartoukian et al., 2013). It is noteworthy that a recent project of subgingival microbiome revealed that a higher proportion of uncultivated species is associated with periodontal disease condition (Griffen et al., © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 227–241

Subgingival microbiome of Korean smokers

2012). Here, we found that not-yet-cultivated Synergistetes phylotypes especially five Fretibacterium OTUs including Fretibacterium fastidiosum were associated with smoking. Although our understanding of Synergistetes is rudimentary, it seems probable that relative abundance of certain Synergistetes taxa belonging to cluster A in smokers may be indicative of future disease severity. The phylogeny, global distributions, and disease associations of oral Synergistetes taxa require further detailed investigation. TM7 is a bacterial division originally described in natural environmental habitats such as soil, freshwater and seawater, deep-sea sediments, groundwater and hot springs (Brinig et al., 2003; Kumar et al., 2003). In humans, TM7 has been detected in subgingival plaque, and studies have shown an association of the TM7 bacterial division with periodontitis (Brinig et al., 2003; Kumar et al., 2003; Ouverney et al., 2003). In the present study, we observed an association of TM7 with smoking at all phylogenetic levels. As the TM7 division of bacteria has been suggested to play an important role in the early stages of inflammatory mucosal processes by modifying growth conditions for competing bacterial populations (Kuehbacher et al., 2008), higher abundance of TM7 bacterial division in smokers may be related to future disease severity. Another interesting finding was that, smoker periodontitis patients exhibited higher levels of Veillonella and Streptococcus (Fig. 2), species that are known to be abundant in health-associated biofilms (Paster et al., 2001; Kumar et al., 2005, 2006). This finding is not consistent with the results from previous studies performed in the Netherlands (Bizzarro et al., 2013) and the USA (Shchipkova et al., 2010). However, the parallel relationship observed between levels of streptococci and Veillonella is not surprising in view of the fact that veillonellae use short-chain acids that are secreted by gram-positive facultatives such as streptococci (Mikx & Van der Hoeven,1975) and have been shown to colonize tooth surfaces only in the presence of streptococci (McBride & Van der Hoeven,1981). Higher levels of streptococci, especially Streptococcus sanguinis, in smokers also seem to be related to a proportional increase of Corynebacterium matruchotii (Fig. 3) since S. sanguinis forms a corncob complex together with C. matruchotii, leading to biofilm initiation and development (Sbordone & Bortolaia, 2003). Notably, the growth of Streptococcus mutans and 235

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S. sanguinis was accelerated in the vicinity of cigarette smoke (Zonuz et al., 2008). Moreover, Streptococcus has been known to act as antagonistic strains against periodontopathogens, such as Porphyromonas gingivalis and Prevotella intermedia, preventing colonization of this niche by the pathogens (Van Hoogmoed et al., 2008). All of these observations make it tempting to speculate that a proportional increase of the genus Streptococcus in smokers may not only be related to higher abundance of Veillonella and C. matruchotii but also contribute to a lower proportion of Prevotella, including P. intermedia, in the whole microbiome. Despite consistent reports linking members of streptococci and veillonellae with healthy condition, our results implicate that the association of these bacteria with periodontal disease in smokers remains somewhat inconclusive. Therefore, further investigation is needed to clarify the system-level mechanisms underlying changes in oral microbiome ecology in periodontal disease in response to smoking. Oxygen tension (pO2) in periodontal pocket, a major environmental determinant of the subgingival microflora, is generally lower in smokers than in

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non-smokers (Hanioka et al., 2000). The microaerophilic Capnocytophaga species have been found in pockets with a pO2 greater than 15 mmHg (Loesche et al., 1983). Hence, our observation that two Capnocytophaga species were less abundant in smokers is not surprising and consistent with findings of earlier investigations (Shchipkova et al., 2010; Bizzarro et al., 2013). In the present study, the richness of total subgingival plaque bacterial communities of smokers and non-smokers was estimated by rarefaction analysis. The shapes of the rarefaction curves (Fig. 4A) indicate that species richness is similar between these two groups. Depending on the cut-off used in sequence differences between OTUs (3%), the estimates of richness of subgingival plaque bacterial communities ranged between 849 and 1040 phylotypes in smokers and from 899 to 1092 phylotypes in non-smokers. The ecological organization of the communities was calculated using the Shannon’s diversity and the Simpson’s evenness indices. The microbiota from smokers showed statistically significantly higher diversity than non-smokers, but their microbiota

A

B

Figure 4 Species richness, diversity, and evenness in smokers and non-smokers. The shapes of the rarefaction curves (A) indicate that species richness is similar between these two groups. The microbiota from smokers showed statistically significantly higher diversity and evenness similar to non-smokers (B).

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evenness was similar to that of non-smokers (Fig. 4B). Some studies found that lower taxonomic diversity in particular ecological environments including the oral cavity reflects poor health of the environments (Kumar et al., 2012; Bizzarro et al., 2013). However, this finding is not consistent with a recent study showing that diversity was higher in periodontitis than in health (Griffen et al., 2012). Further detailed investigation is required to validate the diversity–disease relationship. As observed in Korean patients in this study, it has been reported previously that the genus Fusobacterium is more abundant in smokers than non-smokers in other ethnic groups and national-origin populations, such as North American and Dutch patients (Shchipkova et al., 2010; Bizzarro et al., 2013). In addition, other putative smoking-associated bacteria such as Fretibacterium, Eubacterium, and TM7 were found in the Korean periodontitis patients for the first time. Direct comparison of results from different studies may not be reasonable because of the different clinical sampling and laboratory analysis techniques that were applied. Nevertheless, all the results clearly indicate that smoking considerably affects subgingival bacterial ecology and make it tempting to speculate that the differences in the subgingival bacterial community between smoker and non-smoker patients vary depending on race and ethnicity. Taken together, the results of the present study indicate that considerable differences exist in the subgingival bacterial communities between smoker and non-smoker patients with chronic moderate periodontitis in Korea, suggesting that cigarette smoking considerably affects subgingival bacterial ecology. This work confirms previous findings that certain disease-associated phylotypes (e.g. Fusobacterium) are more common in smokers and provides a much broader picture of the bacterial community associated with smoking, thereby suggesting additional putative smoking-associated species (e.g. Fretibacterium, Filifactor alocis, and E. nodatum) that deserve more attention. Our results may have potential limitations because the number of participants was different between groups and the measurement of smoking status was self-reported. Further comparative studies using the samples with and without pooling will be needed to investigate whether the apparent differences between groups are the result of a consistent shift in each © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 227–241

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sample or of very large shifts in a small subpopulation of samples. Continued sampling and sequencing efforts focusing on subgroups of the population (effect of ethnicity, clinical characteristics, and the extent and timing of smoking exposure) will also need to be performed to obtain a better understanding of the effect of smoking on the oral microbial community. ACKNOWLEDGEMENTS This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0009233). REFERENCES Abbott, P.V. and Salgado, J.C. (2009) Strategies for the endodontic management of concurrent endodontic and periodontal diseases. Aust Dent J 54: S70–S85. Ahlqwist, M., Bengtsson, C., Hollender, L., Lapidus, L. and Osterberg, T. (1989) Smoking habits and tooth loss in Swedish women. Community Dent Oral Epidemiol 17: 144–147. Apatzidou, D.A., Riggio, M.P. and Kinane, D.F. (2005) Impact of smoking on the clinical, microbiological and immunological parameters of adult patients with periodontitis. J Clin Periodontol 32: 973–983. Bateson, M.C. (1993) Cigarette smoking and Helicobacter pylori infection. Postgrad Med J 69: 41–44. € Emingil, G. and Bostanci, €rk, V.O., Belibasakis, G.N., Oztu N. (2013) Synergistetes cluster A in saliva is associated with periodontitis. J Periodont Res 48: 727–732. €m, J. (1989) Cigarette smoking as risk factor in Bergstro chronic periodontal disease. Community Dent Oral Epidemiol 17: 245–247. €m, J. and Eliasson, S. (1987) Cigarette smokBergstro ing and alveolar bone height in subjects with a high standard of oral hygiene. J Clin Periodontol 14: 466–469. €m, J. and Floderus-Myrhed, B. (1983) Co-twin Bergstro control study of the relationship between smoking and some periodontal disease factors. Community Dent Oral Epidemiol 11: 113–116. €m, J., Eliasson, S. and Preber, H. (1991) CigaBergstro rette smoking and periodontal bone loss. J Periodontol 62: 242–246. Bizzarro, S., Loos, B.G., Laine, M.L., Crielaard, W. and Zaura, E. (2013) Subgingival microbiome in smokers and non-smokers in periodontitis: an exploratory study

237

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using traditional targeted techniques and a next-generation sequencing. J Clin Periodontol 40: 483–492. Bolin, A., Lavstedt, S., Frithiof, L. and Henrikson, C.O. (1986) Proximal alveolar bone loss in a longitudinal radiographic investigation. IV. Smoking and some other factors influencing the progress in individuals with at least 20 remaining teeth. Acta Odontol Scand 44: 263–269. €m, L., Bergstro €m, J., Dahle n, G. and Linder, L.E. Bostro (2001) Smoking and subgingival microflora in periodontal disease. J Clin Periodontol 28: 212–219. Brinig, M.M., Lepp, P.W., Ouverney, C.C., Armitage, G.C. and Relman, D.A. (2003) Prevalence of bacteria of division TM7 in human subgingival plaque and their association with disease. Appl Environ Microbiol 69: 1687–1694. Bundred, N.J., Dover, M.S., Coley, S. and Morrison, J.M. (1992) Breast abscesses and cigarette smoking. Br J Surg 79: 58–59. Chen, T., Yu, W.H., Izard, J., Baranova, O.V., Lakshmanan, A. and Dewhirst, F.E. (2010) The Human Oral Microbiome Database: a web accessible resource for investigating oral microbe taxonomic and genomic information. Database (Oxford). http://www.homd.org (Aug, 2014) Chun, J., Lee, J.H., Jung, Y. et al. (2007) EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol 57: 2259–2261. Chun, J., Kim, K.Y., Lee, J.H. and Choi, Y. (2010) The analysis of oral microbial communities of wild-type and toll-like receptor 2-deficient mice using a 454 GS FLX Titanium pyrosequencer. BMC Microbiol 10: 101. Cole, J.R., Wang, Q., Cardenas, E. et al. (2009) The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 37 (Database issue): D141–D145. Dahlen, G. and Leonhardt, A. (2006) A new checkerboard panel for testing bacterial markers in periodontal disease. Oral Microbiol Immunol 21: 6–11. Darby, I.B., Hodge, P.J., Riggio, M.P. and Kinane, D.F. (2000) Microbial comparison of smoker and non-smoker adult and early-onset periodontitis patients by polymerase chain reaction. J Clin Periodontol 27: 417–424. Darby, I.B., Hodge, P.J., Riggio, M.P. and Kinane, D.F. (2005) Clinical and microbiological effect of scaling and root planing in smoker and non-smoker chronic and aggressive periodontitis patients. J Clin Periodontol 32: 200–206.

238

J.-H. Moon et al.

Delima, S.L., McBride, R.K., Preshaw, P.M., Heasman, P.A. and Kumar, P.S. (2010) Response of subgingival bacteria to smoking cessation. J Clin Microbiol 48: 2344–2349. Dzink, J.L., Socransky, S.S. and Haffajee, A.D. (1988) The predominant cultivable microbiota of active and inactive lesions of destructive periodontal diseases. J Clin Periodontol 15: 316–323. Ellingsøe, P. and Johnsen, K. (2002) Influence of soil sample sizes on the assessment of bacterial community structure. Soil Biol Biochem 34: 1701–1707. Engel, M., Behnke, A., Bauerfeld, S. et al. (2012) Sample pooling obscures diversity patterns in intertidal ciliate community composition and structure. FEMS Microbiol Ecol 79: 741–750. Epstein, J.B., Freilich, M.M. and Le, N.D. (1993) Risk factors for oropharyngeal candidiasis in patients who receive radiation therapy for malignant conditions of the head and neck. Oral Surg Oral Med Oral Pathol 76: 169–174. Feldman, R.S., Bravacos, J.S. and Rose, C.L. (1983) Association between smoking different tobacco products and periodontal disease indexes. J Periodontol 54: 481– 487. Feldman, R.S., Alman, J.E. and Chauncey, H.H. (1987) Periodontal disease indexes and tobacco smoking in healthy aging men. Gerodontics 3: 43–46. Goultschin, J., Cohen, H.D., Donchin, M., Brayer, L. and Soskolne, W.A. (1990) Association of smoking with periodontal treatment needs. J Periodontol 61: 364–367. Griffen, A.L., Beall, C.J., Campbell, J.H. et al. (2012) Distinct and complex bacterial profiles in human periodontitis and health revealed by 16S pyrosequencing. ISME J 6: 1176–1185. Haffajee, A.D. and Socransky, S.S. (2001) Relationship of cigarette smoking to the subgingival microbiota. J Clin Periodontol 28: 377–388. Haffajee, A.D., Bogren, A., Hasturk, H., Feres, M., Lopez, N.J. and Socransky, S.S. (2004) Subgingival microbiota of chronic periodontitis subjects from different geographic locations. J Clin Periodontol 31: 996–1002. Haffajee, A.D., Japlit, M., Bogren, A., Kent, R.L. Jr, Goodson, J.M. and Socransky, S.S. (2005) Differences in the subgingival microbiota of Swedish and USA subjects who were periodontally healthy or exhibited minimal periodontal disease. J Clin Periodontol 32: 33–39. Haffajee, A.D., Teles, R.P. and Socransky, S.S. (2006) Association of Eubacterium nodatum and Treponema denticola with human periodontitis lesions. Oral Microbiol Immunol 21: 269–282.

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 227–241

J.-H. Moon et al.

Haffajee, A.D., Socransky, S.S., Patel, M.R. and Song, X. (2008) Microbial complexes in supragingival plaque. Oral Microbiol Immunol 23: 196–205. Hanioka, T., Tanaka, M., Takaya, K., Matsumori, Y. and Shizukuishi, S. (2000) Pocket oxygen tension in smokers and non-smokers with periodontal disease. J Periodontol 71: 550–554. Hsiao, W.W., Li, K.L., Liu, Z., Jones, C., Fraser-Liggett, C.M. and Fouad, A.F. (2012) Microbial transformation from normal oral microbiota to acute endodontic infections. BMC Genom 13: 345. Huber, T., Faulkner, G. and Hugenholtz, P. (2004) Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20: 2317–2319. Hur, M., Kim, Y., Song, H.R., Kim, J.M., Choi, Y.I. and Yi, H. (2011) Effect of genetically modified Poplars on soil microbial communities during the phytoremediation of waste mine tailings. Appl Environ Microbiol 77: 7611– 7619. Jeon, Y.S., Chun, J. and Kim, B.S. (2013) Identification of household bacterial community and analysis of species shared with human microbiome. Curr Microbiol 67: 557– 563. Johnson, G.K. and Hill, M. (2004) Cigarette smoking and the periodontal patient. J Periodontol 75: 196–209. Kamma, J.J., Nakou, M. and Baehni, P.C. (1999) Clinical and microbiological characteristics of smokers with early onset periodontitis. J Periodontal Res 34: 25–33. Kang, S. and Mills, A.L. (2006) The effect of sample size in studies of soil microbial community structure. J Microbiol Methods 66: 242–250. Kim, T.S., Kang, N.W., Lee, S.B., Eickholz, P., Pretzl, B. and Kim, C.K. (2009) Differences in subgingival microflora of Korean and German periodontal patients. Arch Oral Biol 54: 223–229. Kim, B.S., Kim, J.N., Yoon, S.H., Chun, J. and Cerniglia, C.E. (2012) Impact of enrofloxacin on the human intestinal microbiota revealed by comparative molecular analysis. Anaerobe 18: 310–320. Kim, O.S., Cho, Y.J., Lee, K. et al. (2012) Introducing Eztaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62: 716–721. Kuehbacher, T., Rehman, A., Lepage, P. et al. (2008) Intestinal TM7 bacterial phylogenies in active inflammatory bowel disease. J Med Microbiol 57: 1569–1576. Kumar, P.S., Griffen, A.L., Barton, J.A., Paster, B.J., Moeschberger, M.L. and Leys, E.J. (2003) New bacterial species associated with chronic periodontitis. J Dent Res 82: 338–344.

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 227–241

Subgingival microbiome of Korean smokers

Kumar, P.S., Griffen, A.L., Moeschberger, M.L. and Leys, E.J. (2005) Identification of candidate periodontal pathogens and beneficial species by quantitative 16S clonal analysis. J Clin Microbiol 43: 3944–3955. Kumar, P.S., Leys, E.J., Bryk, J.M., Martinez, F.J., Moeschberger, M.L. and Griffen, A.L. (2006) Changes in periodontal health status are associated with bacterial community shifts as assessed by quantitative 16S cloning and sequencing. J Clin Microbiol 44: 3665–3673. Kumar, P.S., Mason, M.R., Brooker, M.R. and O’Brien, K. (2012) Pyrosequencing reveals unique microbial signatures associated with healthy and failing dental implants. J Clin Periodontol 39: 425–433. Lim, J.H., Lee, J.H. and Shin, S.J. (2014) Diagnosis and treatment of teeth with primary endodontic lesions mimicking periodontal disease: three cases with long-term follow ups. Restor Dent Endod 39: 56–62. Liu, Z.Z., DeSantis, T.Z., Andersen, G.L. and Knight, R. (2008) Accurate taxonomy assignments from 16S rRNA sequences produced by highly parallel pyrosequencers. Nucleic Acids Res 36: e120. Loesche, W.J., Gusberti, F., Mettraux, G., Higgins, T. and Syed, S. (1983) Relationship between oxygen tension and subgingival bacterial flora in untreated human periodontal pockets. Infect Immun 42: 659–667. MacFarlane, G.D., Herzberg, M.C., Wolff, L.F. and Hardie, N.A. (1992) Refractory periodontitis associated with abnormal polymorphonuclear leukocyte phagocytosis and cigarette smoking. J Periodontol 63: 908–913. Manter, D.K., Weir, T.L. and Vivanco, J.M. (2010) Negative effects of sample pooling on PCR-based estimates of soil microbial richness and community structure. Appl Environ Microbiol 76: 2086–2090. McBride, B.C. and Van der Hoeven, J.S. (1981) Role of interbacterial adherence in colonization of the oral cavities of gnotobiotic rats infected with Streptococcus mutans and Veillonella alcalescens. Infect Immun 33: 467–472. Mikx, F.H. and Van der Hoeven, J.S. (1975) Symbiosis of Streptococcus mutans and Veillonella alcalescens in mixed continuous cultures. Arch Oral Biol 20: 407–410. Moffatt, C.E., Whitmore, S.E., Griffen, A.L., Leys, E.J. and Lamont, R.J. (2011) Filifactor alocis interactions with gingival epithelial cells. Mol Oral Microbiol 26: 365–373. Mullally, B.H. (2004) The influence of tobacco smoking on the onset of periodontitis in young persons. Tob Induc Dis 2: 53–65. Nowrousian, M. (2010) Next-generation sequencing techniques for eukaryotic microorganisms: sequencing-based solutions to biological problems. Eukaryot Cell 9: 1300– 1310.

239

Subgingival microbiome of Korean smokers

€m, D. (1986) Tobacco smoking: Osterberg, T. and Mellstro a major risk factor for loss of teeth in three 70-year-old cohorts. Community Dent Oral Epidemiol 14: 367–370. Ouverney, C.C., Armitage, G.C. and Relman, D.A. (2003) Single-cell enumeration of an uncultivated TM7 subgroup in the human subgingival crevice. Appl Environ Microbiol 69: 6294–6298. Paster, B.J., Boches, S.K., Galvin, J.L. et al. (2001) Bacterial diversity in human subgingival plaque. J Bacteriol 183: 3770–3783. Petitti, D.B., Friedman, G.D. and Kahn, W. (1981) Accuracy of information on smoking habits provided on selfadministered research questionnaires. Am J Public Health 71: 308–311. €m, J. and Linder, L.E. (1992) OccurPreber, H., Bergstro rence of periopathogens in smoker and non-smoker patients. J Clin Periodontol 19: 667–671. n, G. and Wikstro €m, M. (1998) The cliniRenvert, S., Dahle cal and microbiological effects of non-surgical periodontal therapy in smokers and non-smokers. J Clin Periodontol 25: 153–157. Rylev, M. and Kilian, M. (2008) Prevalence and distribution of principal periodontal pathogens worldwide. J Clin Periodontol 35(8 Suppl): 346–361. Sbordone, L. and Bortolaia, C. (2003) Oral microbial biofilms and plaque-related diseases: microbial communities and their role in the shift from oral health to disease. Clin Oral Invest 7: 181–188. Shchipkova, A.Y., Nagaraja, H.N. and Kumar, P.S. (2010) Subgingival microbial profiles of smokers with periodontitis. J Dent Res 89: 1247–1253. Shiloah, J., Patters, M.R. and Waring, M.B. (2000) The prevalence of pathogenic periodontal microflora in healthy young adult smokers. J Periodontol 71: 562– 567. Signat, B., Roques, C., Poulet, P. and Duffaut, D. (2011) Fusobacterium nucleatum in periodontal health and disease. Curr Issues Mol Biol 13: 25–36. ^cßas, I.N. (2012) Siqueira, J.F. Jr, Fouad, A.F. and Ro Pyrosequencing as a tool for better understanding of human microbiomes. J Oral Microbiol 4: 10743. Socransky, S.S. and Haffajee, A.D. (2005) Periodontal microbial ecology. Periodontol 2000 38: 135–187. Socransky, S.S., Haffajee, A.D., Cugini, M.A., Smith, C. and Kent, R.L. Jr (1998) Microbial complexes in subgingival plaque. J Clin Periodontol 25: 134–144. Stoltenberg, J.L., Osborn, J.B., Pihlstrom, B.L. et al. (1993) Association between cigarette smoking, bacterial pathogens, and periodontal status. J Periodontol 64: 1225–1230.

240

J.-H. Moon et al.

Tew, J.G., Zhang, J., Quinn, S. et al. (1996) Antibody of the IgG2 subclass, Actinobacillus actinomycetemcomitans and early onset periodontitis. J Periodontol 67: 317–322. Tomar, S.L. and Asma, S. (2000) Smoking-attributable periodontitis in the United States: findings from NHANES III. National Health and Nutrition Examination Survey. J Periodontol 71: 743–751. Tonetti, M.S. (1998) Cigarette smoking and periodontal diseases: etiology and management of disease. Ann Periodontol 3: 88–101. Van der Velden, U., Varoufaki, A., Hutter, J.W. et al. (2003) Effect of smoking and periodontal treatment on the subgingival microflora. J Clin Periodontol 30: 603– 610. Van Hoogmoed, C.G., Geertsema-Doornbusch, G.I., Teughels, W., Quirynen, M., Busscher, H.J. and Van der Mei, H.C. (2008) Reduction of periodontal pathogens adhesion by antagonistic strains. Oral Microbiol Immunol 23: 43–48. Vartoukian, S.R., Palmer, R.M. and Wade, W.G. (2009) Diversity and morphology of members of the phylum “Synergistetes” in periodontal health and disease. Appl Environ Microbiol 75: 3777–3786. Vartoukian, S.R., Downes, J., Palmer, R.M. and Wade, W.G. (2013) Fretibacterium fastidiosum gen. nov., sp. nov., isolated from the human oral cavity. Int J Syst Evol Microbiol 63: 458–463. Verma, P.K., Srivastava, R., Gupta, K.K. and Srivastava, A. (2011) Combined endodontic – periodontal lesion: a clinical dilemma. J Interdiscip Dentistry 1: 119–124. Wade, W.G. (2011) Has the use of molecular methods for the characterization of the human oral microbiome changed our understanding of the role of bacteria in the pathogenesis of periodontal disease? J Clin Periodontol 38(Suppl 11): 7–16. Wang, Q., Garrity, G.M., Tiedje, J.M. and Cole, J.R. (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73: 5261–5267. Werner, J.J., Koren, O., Hugenholtz, P. et al. (2012) Impact of training sets on classification of high-throughput bacterial 16s rRNA gene surveys. ISME J 6: 94– 103. van Winkelhoff, A.J., Bosch-Tijhof, C.J., Winkel, E.G. and van der Reijden, W.A. (2001) Smoking affects the subgingival microflora in periodontitis. J Periodontol 72: 666–671. Witteman, E.M., Hopman, W.P., Becx, M.C. et al. (1993) Short report: smoking habits and the acquisition of metro-

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 227–241

J.-H. Moon et al.

nidazole resistance in patients with Helicobacter pylorirelated gastritis. Aliment Pharmacol Ther 7: 683–687. Xie, G., Lo, C.C., Scholz, M. and Chain, P.S. (2014) Recruiting human microbiome shotgun data to site-specific reference genomes. PLoS ONE 9: e84963. You, M., Mo, S., Watt, R.M. and Leung, W.K. (2013) Prevalence and diversity of Synergistetes taxa in periodontal health and disease. J Periodont Res 48: 159–168. Zambon, J.J., Grossi, S.G., Machtei, E.E., Ho, A.W., Dunford, R. and Genco, R.J. (1996) Cigarette smoking

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increases the risk for subgingival infection with periodontal pathogens. J Periodontol 67: 1050– 1054. Zonuz, A.T., Rahmati, A., Mortazavi, H., Khashabi, E. and Farahani, R.M. (2008) Effect of cigarette smoke exposure on the growth of Streptococcus mutans and Streptococcus sanguis: an in vitro study. Nicotine Tob Res 10: 63–67.

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