Microbial Ecology in Health and Disease. 2009; 21: 183–192
ORIGINAL ARTICLE
Subgingival microflora in chronic obstructive pulmonary disease
INGA LEUCKFELD1,2, INGAR OLSEN1, ODD GEIRAN2,3, ØYSTEIN BJØRTUFT2,3 & BRUCE J. PASTER4,5 1Institute
of Oral Biology, Faculty of Dentistry, University of Oslo, 2Division of Cardiac and Respiratory Medicine and Surgery, Oslo University Hospital–Rikshospitalet, 3Faculty Division Rikshospitalet, University of Oslo, Oslo, Norway, 4Department of Molecular Genetics, Forsyth Institute, and 5Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, MA, USA
Abstract Objective: Recent reports have shown an association between periodontitis and chronic obstructive pulmonary disease (COPD). Our objective was to assess the subgingival microflora in relation to COPD status. Methods: Eighteen subjects were classified into four different groups according to clinical periodontal status and the diagnosis of COPD. DNA from subgingival plaque samples was extracted and the bacterial 16S rRNA genes were PCR amplified, cloned, and sequenced to determine species identity. Results: From a total of 1041 clones, 168 species or phylotypes were detected. Higher amounts of Actinomyces spp., Veillonella spp., and Campylobacter gracilis were detected in the subgingival microflora of periodontally healthy COPD subjects, as compared with controls. COPD status was found to have little influence on the subgingival microflora in periodontitis subjects. Conclusion: COPD status does seem to influence the healthy but not the diseased subgingival microflora in periodontitis. Although preliminary, these findings indicate that there may be host factors responsible for the increased susceptibility of COPD subjects to periodontal disease. Key words: Periodontitis, subgingival bacteria, 16S rRNA gene, sequence analysis
Introduction Chronic obstructive pulmonary disease (COPD) is a major global health problem. COPD is predicted to rise from its ranking as the eleventh most prevalent disease world wide in 2002 to the seventh, and from the fifth most common cause of death to the fourth, by 2030 (1). The hallmark of COPD, progressive airway obstruction, is associated with an abnormal inflammatory response of the lungs to noxious particles and gases. The main etiology of COPD is cigarette smoking and, in some cases, environmental pollutants. Other factors may be involved such as a genetic predisposition and bronchial hyperresponsiveness. Bacterial and viral infections may contribute to the airway inflammation in COPD and act as amplifiers of the established disease (2–4). It has been suggested that localized infectious diseases such as periodontitis (PD) may influence a number of systemic diseases (5–9). Bacteria from dental plaque may cause infection at a distant site by
hematogenous spread or periodontopathogenic bacteria may stimulate the release of proinflammatory cytokines and acute phase proteins and contribute to extraoral inflammation (10–12). Oral bacteria may also translocate to other mucosal surfaces, such as the lungs, to cause inflammation and infection. It has been reported that many of the microbes found in the lungs of hospitalized patients originate in dental plaque and in the oral flora of these patients (13–17). Recent reports have shown an association between PD and COPD (5,18,19). However, no causal relationship has been established. Whether the association is caused by a common genetic predisposition or whether PD is able to alter disease progression in COPD, either by supporting colonization of dental plaque with pulmonary pathogens or by periodontal pathogens and their products promoting airway inflammation and exacerbation, remains speculative. It has been estimated that the oral cavity harbors 700 bacterial species, of which approximately 400
Correspondence: Inga Leuckfeld, Department of Respiratory Medicine, Oslo University Hospital–Rikshospitalet, Sognsvannsveien 20, 0027 Oslo, Norway. Tel: +47 230 70013. Fax: +47 230 73917. E-mail:
[email protected] (Received 25 November 2008; accepted 25 June 2009) ISSN 0891-060X print/ISSN 1651-2235 online © 2009 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS) DOI: 10.3109/08910600903194412
184
I. Leuckfeld et al.
species are found in the subgingival plaque (20). Conventional cultivable methodology is insufficient for the identification of the full spectrum of the subgingival microflora, as there are several notyet-cultivated species or difficult-to-grow species involved in periodontitis. Approximately 50% of the oral bacteria have been identified based on culture-independent molecular methods (20,21). To date, there have been no reports describing the subgingival microflora in COPD subjects. Thus, the purpose of this study was to investigate the subgingival microflora in relation to COPD status in periodontal healthy and diseased subjects, using culture-independent molecular methods.
Material and methods Subject population Eighteen subjects admitted to the Department of Respiratory Medicine, Oslo University HospitalRikshospitalet, Norway were included. Subjects were classified into four different groups according to clinical periodontal status and COPD status according to their medical record and lung function testing by spirometry. The four groups were as follows: four patients with no COPD and no PD (referred to as the NCP-group), four patients with
COPD and no PD (COPD-group), five patients with COPD and PD (COPD-PD-group), and five patients with PD and no COPD (PD-group). COPD severity was classified according to the GOLD-classification (22), with the modification that lung function testing was performed without prior bronchodilatation. Clinical periodontal status was assessed by probing depth, bleeding on probing, and teeth mobility testing, and subjects who had at least four sites with probing depth ≥4 mm and bleeding on probing with or without tooth mobility were categorized as having periodontitis. No subjects had received periodontal treatment within the last 6 months or taken any antibiotics within the previous 2 months. No subject in the PD-group used systemic corticosteroids; in the other groups (NCP, COPD, and COPD-PD) one subject per group used a daily dose (5–10 mg) of prednisolone. The clinical and demographic data are shown in Table I. The study was approved by the local ethics committee and all study subjects signed the committee-approved informed consent. Microbiological sampling After removal of supragingival plaque and isolation of sample sites with cotton rolls, subgingival plaque was collected by inserting sterile paper points to the
Table I. Characteristics of subjects.
Age
Smoking status
Pack years*
COPD†
Bleeding on probing
NCP-group (non-COPD, non-PD) Female 57.0 Male 28.0 Male 47.0 Male 57.0
Former Former Former Former
18.0 13.0 25.0 16.0
0 0 0 0
Yes Yes No No
4 4 4 4
mm mm mm mm
No No No No
COPD-group (COPD, non-PD) Male 62.0 Female 52.0 Female 64.0 Male 67.0
Current Former Former Current
11.0 22.0 18.9 18.0
III IV IV II
No No Yes No
4 4 4 4
mm mm mm mm
No No No No
COPD-PD-group (COPD, PD) Female Male Female Female Male
69.0 65.0 65.0 63.0 49.0
Current Former Current Current Former
11.0 45.0 45.0 18.0 22.0
II II II II IV
Yes Yes Yes Yes Yes
6 4–6 4–6 4–6 4–6
mm mm mm mm mm
No Slight horizontal Moderate horizontal Horizontal + vertical Moderate horizontal
PD-group (non-COPD, PD) Female Female Male Male Male
39.0 57.0 65.0 66.0 68.0
Never Current Current Former Former
0.0 29.0 34.0 6.0 12.0
0 0 0 0 0
Yes Yes Yes Yes Yes
4 mm 4–6 mm ≥6 mm 4–6 mm 4 mm
No Slight horizontal Moderate horizontal Slight horizontal Slight horizontal
Gender
COPD, chronic obstructive pulmonary disease; PD, periodontitis. ∗Smoking exposure expressed as number of years smoking 20 cigarettes per day. †Severity based on prebronchodilatator spirometry.
Pocket depth
Tooth mobility
Oral microflora in COPD bottom of the pockets. The sampled healthy sites were chosen randomly; from diseased sites the deepest pockets were sampled. Two to seven pockets were sampled per subject. Samples were pooled and transferred to a tube with 200 μl TE-buffer (50 mM Tris, 1 mM EDTA, pH 7.6) and immediately frozen and kept at −80°C until further analysis. DNA extraction DNA was extracted from 40 μl of the TE-buffer with dental plaque using a commercial kit (QIAamp Mini Kit, Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions.
185
Sequencing of 16S rRNA genes PCR products were purified by adding 1 μl of shrimp alkaline phosphatase and 1 μl of exonuclease I to 7 μl of the PCR product according to the manufacturer’s instructions (Amersham Biosciences, GE Healthcare). Purified DNA was sequenced in both directions using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase FS (Applied Biosystems). The primers PB and PC (PB: 5-TAA CAC ATG CAA GTC GAA CG-3 and PC: 5-CCC ACT GCT GCC TCC CGT AG-3, respectively) were used for sequencing (23). Sequencing reactions were run on an ABI Prism 3100 DNA sequencer (Applied Biosystems).
PCR amplification of 16S rDNA
Data analyses of the 16S rRNA sequences
The 16S rRNA genes were amplified under standardized conditions using previously published universal bacterial primers (23). The primers were as follows: PA 5-AGA GTT TGA TCC TGG CTC AG-3 and PD 5-GTA TTA CCG CGG CTG CTG-3. PCR was performed in thin-walled tubes using the GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA, USA), with a master mix solution (HotStarTaq Master Mix, Qiagen) containing 2.5 units of Taq DNA polymerase, 1 PCR buffer with 1.5 mM MgCl2, 200 μM of each deoxynucleoside triphosphate, 0.2 μM of each primer, and 5 μl of the template DNA, in a total volume of 50 μl. Samples were denatured at 95°C for 15 min, followed by amplification consisting of a denaturation step at 95°C for 40 s, annealing at 58°C for 1 min, and elongation at 72°C for 1 min with an additional 5 s for each cycle. A total of 32 cycles was performed, followed by a final elongation step at 72°C for 10 min. Each set of experiments included negative and positive controls. The results of PCR amplification were examined by electrophoresis in a 1% agarose gel under ultraviolet light after ethidium bromide staining. Before cloning, amplicons were purified using the QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer’s instructions.
DNA sequences were assembled with the SEQUENCHER 4.6 program (Gene Codes Corp., Ann Arbor, MI, USA) and analyzed for the presence of chimeric sequences using the Chimera Check program from the Ribosomal Database Project II (hptt://rdp.cme. msu.edu/). After elimination of suspected chimeric sequences, the partial 16S rRNA sequences of approximately 500 bp were used to determine identity or nearest phylogenetic position. For identification of closest relatives, the consensus sequences were compared with 16S rRNA sequences of over 20 000 microorganisms in our database and over 250 000 sequences in the Ribosomal Database Project (24), EMBL (http://www.ebi.ac.uk/embl/), GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and DDBJ (http://www.ddbj.nig.ac.jp/search/top-e. html) nucleotide sequence databases. The evolutionary history was inferred using the neighbor joining method (25). The optimal tree with the sum of branch length 7.63835334 is shown in Figures 1 and 2. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Jukes-Cantor method (26) and are in the units of the number of base substitutions per site. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons (pairwise deletion option). There were a total of 1433 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 (27). TREECON software package for the Microsoft Windows environment was used for the construction and drawing of evolutionary trees (28).
Cloning procedures Cloning of the purified amplicons was performed using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The insert size of clones was verified by PCR using M13 primers (forward: 5-GTT TTC CCA GTC ACG AC-3, and reverse: 5-CAG GAA ACA GCT ATG AC-3, respectively), and clones with correct insert size were analyzed by sequencing.
Results A total of 1041 16S rRNA clones were sequenced, ranging from 49 to 93 clones per subject, mean 58 10. Overall 10 bacterial phyla were detected,
186
I. Leuckfeld et al.
Figure 1. Evolutionary relationships and distribution of the phyla Firmicutes, Fusobacteria, Synergistes, Chloroflexi, and Actinobacteria from clone libraries of all subjects in each sample group. GenBank accession numbers are given in brackets. The marker bar represents a 2% difference in nucleotide sequences. The prevalence of the different species is indicated by color code: blue, 1–5%; green, 5–20%; red, 20% of clones/subject.
Oral microflora in COPD
187
Figure 2. Evolutionary relationships and distribution of the phyla Bacteroidetes, Chlamydiae, TM7, Spirochaetales, and Proteobacteria from clone libraries of all subjects in each sample group. GenBank accession numbers are given in brackets. The marker bar represents a 5% difference in nucleotide sequences. The prevalence of the different species is indicated by color code: blue, 1–5%; green, 5–20%; red, 20% of clones/subject.
188
I. Leuckfeld et al.
represented by 168 different bacterial species; 98 (58%) cultivable species within known genera and 70 (42%) sequences from not-yet-cultivated phylotypes or species that are currently unrecognized. The number of different species or phylotypes detected within each sample group was similar, ranging from 13 to 25 per subject (Table II). The bacterial diversity and the different bacterial profiles at the subject level are presented in Figures 1 and 2. Bacterial profiles in subjects with no periodontitis (NCP- and COPD-group) The dominant phylogenetic group in the NCP and COPD sample group were the Firmicutes, consisting of nearly 50% of all the species detected (Table II). Granulicatella spp. were detected only in those two groups. Streptococcus mitis was the predominant Firmicutes detected in NCP subjects, whereas Veillonella parvula/dispar was most prevalent, and detected in highest levels, in the COPD-group. Actinobacteria, in our study, dominated by Actinomyces spp., were not detectable in the periodontitis group (PD-group) but were found in all other groups, with the highest levels in the COPD-group. The COPD-group was also characterized by a high prevalence and clone level of Campylobacter gracilis (Tables III and IV). The relatively high clone levels (but low prevalences) of Porphyromonas gingivalis and Tannerella forsythia in the NCP-group were found in the one female subject of the group with clinical bleeding on probing. Similarly, the high level of Fusobacterium nucleatum subsp. animalis in the COPD-group was associated with the subject in this group with the clinical sign of bleeding on probing (Tables I and IV). Bacterial profiles in subjects with periodontitis (COPD-PD- and PD-group) In the periodontitis groups (COPD-PD and PD), Firmicutes was found in lower clone levels but with a similar diversity, whereas Bacteroidetes showed a Table II. Number of clones and different species per sample group.
Sample groups∗ NCP COPD COPD-PD PD Total ∗See
No. of subjects
No. of clones analyzed
Total no. of species/ phylotypes
No. of species/ phylotypes per subject (mean)
4 4 5 5 18
207 274 283 277 1041
59 65 74 74 168
14–24 (19) 16–25 (19) 13–23 (18) 13–24 (21) 13–25 (19)
Table I for definition of sample groups.
higher diversity and tendency toward higher levels compared with the non-periodontitis groups (NCP and COPD) (Table III). There were no predominant Firmicutes except for a high prevalence and level of V. parvula Oral taxon 161 Clone AA050 and Streptococcus gordonii in periodontitis without COPD (PDgroup). Collectively speaking, species of Selenomonas were found in both periodontitis groups (COPD-PD and PD). Por. gingivalis, Porphyromonas endodontalis, Prevotella intermedia,T. forsythia, and Bacteroidetes spp. were predominant species from the Bacteroidetes family in both periodontitis groups (COPD-PD and PD). Synergistes and Chloroflexi were only detected in the two periodontitis groups (COPD-PD and PD). Species from the TM7 phylum were found in all sample groups but were represented by seven different species and at the highest levels in the PD-group compared with only one species in the other groups (NCP, COPD, and COPD-PD). Similarly, Treponema spp. and Leptotrichia spp. were more prevalent and detected in higher levels in the periodontitis group without COPD (PD-group) compared with the other sample groups (Table IV). Discussion To our knowledge, this is the first report describing the subgingival microbial profile in COPD subjects. The rationale for this study was based on a previous report demonstrating an association between periodontitis and COPD (18). The main finding of the present study was that the healthy subgingival microflora differed in relation to COPD status, whereas the diseased microflora in periodontitis subjects did not. The predominant bacterial phyla in all groups were Firmicutes and Bacteroidetes. A similar pattern of predominant phyla was found in COPD and non-COPD subjects with periodontitis, with comparable prevalence and clone levels detected for Bacteroidetes spp., Synergistes spp., Fusobacterium naviforme, Por. endodontalis, Por. gingivalis, Prev. intermedia, and T. forsythia. However, in COPD subjects with periodontitis, we found lower prevalences and levels of Strep. gordonii, Selenomonas spp., Leptotrichia spp., Treponema spp., and TM7 spp. as compared with periodontitis subjects without COPD. Granulicatella spp., Strep. mitis, and other Streptococcus spp. were found more frequently in the healthy periodontium in both COPD and non-COPD subjects. In the COPD subjects with a healthy periodontium, we detected higher prevalences and levels of Actinomyces spp., Camp. gracilis, and V. parvula/dispar as compared with all other groups. The diversity of bacterial species in human subgingival plaque in periodontal health and in periodontal diseases was determined previously (20,29) in
Oral microflora in COPD
189
Table III. Bacterial phyla identified in the sample groups∗. Total
NCP
Phylogenetic group
Taxa
% of total clones†
Taxa
Firmicutes Bacteroidetes Proteobacteria Actinobacteria Spirochaetales Fusobacteria TM7 Synergistes Chloroflexi Chlamydiae
65 38 17 15 11 10 7 3 1 1
38.1 34.9 9.5 2.9 1.6 8.8 2.2 1.5 0.2 0.2
22 14 12 4 3 3 1 – – –
COPD
% of clones‡ 47.1 30.1 15.5 1.9 1.5 2.9 1.0 – – –
COPD-PD
PD
Taxa
% of clones
Taxa
% of clones
Taxa
% of clones
27 14 9 8 1 4 1 – – 1
51.5 17.9 11.3 7.3 0.4 10.6 0.4 – – 0.7
28 20 6 6 4 5 1 3 1 –
25.8 48.8 8.1 2.1 1.8 7.8 2.8 2.5 0.4 –
28 18 6 – 6 6 7 2 1 –
30.7 41.2 4.7 – 2.9 12.6 4.3 3.2 0.4 –
∗See
Table I for definition of sample groups. †Total number of all clones 1041. ‡See Table II for number of clones per group.
similar 16S rRNA clonal analyses to those used in this study. Our observations of the most prevalent bacterial species in periodontitis and health are in general agreement with their findings. Socransky et al. (30), using whole genomic DNA probes in
checkerboard hybridization assays for 40 cultivable subgingival species, investigated over 13 000 plaque samples from 185 subjects, and defined 5 subgingival bacterial complexes associated with periodontal disease. The red complex consisting of Por. gingivalis,
Table IV. Distribution of major species in the sample groups∗. NCP Species Synergistes spp. Bacteroidetes spp. Selenomonas spp. Streptococcus gordonii Fusobacterium nucleatum subsp. animalis Fusobacterium naviforme Fusobacterium spp. Prevotella intermedia Prevotella oralis Prevotella spp. Porphyromonas endodontalis Porphyromonas gingivalis Porphyromonas spp. Tannerella forsythia TM7 spp. Treponema spp. Leptotrichia spp. Capnocytophaga spp. Neisseria spp. Streptococcus spp. Streptococcus mitis Veillonella parvula Oral taxon 161 Clone AA050 Veillonella parvula/dispar Veillonella spp. Campylobacter gracilis Actinomyces spp. Granulicatella spp. ∗See
COPD
COPD-PD
PD
% of clones†
n‡
% of clones
n‡
% of clones
n‡
% of clones
n‡
– – – – – 1.0 2.4 – – 11.2 1.0 11.2 13.1 2.4 1.0 1.5 1.0 2.9 5.8 27.2 21.8 3.4 4.4 8.7 1.5 1.9 2.9
– – – – – 2 3 – – 3 1 1 2 1 1 1 2 2 2 4 4 2 2 3 3 2 1
– 0.4 1.1 1.1 8.0 1.8 10.2 – 6.9 11.3 0.7 – 1.1 0.4 0.4 0.4 0.4 4.0 – 20.1 12.8 5.1 15.0 21.2 7.3 6.6 3.3
– 1 2 1 2 1 3 – 1 2 1 – 2 1 1 1 1 2 – 4 2 1 3 4 4 3 3
2.5 4.6 1.4 0.4 – 3.9 6.0 4.9 1.8 19.1 8.5 9.2 17.7 4.9 2.8 1.8 1.8 2.1 5.3 12.4 1.4 2.5 1.1 3.9 0.4 1.1 –
3 4 2 1 – 3 4 1 1 5 2 2 3 3 1 2 2 1 2 3 1 1 1 1 1 2 –
3.2 4.0 4.7 2.9 1.4 7.2 9.0 7.9 – 16.6 5.4 10.1 15.5 4.0 4.3 2.9 3.6 0.7 1.1 5.4 – 8.3 0.4 8.7 1.1 – –
3 2 2 3 1 5 5 1 – 4 2 2 4 2 4 5 4 2 1 5 – 4 1 4 2 – –
Table I for definition of sample groups. †See Table II for number of clones per group. ‡Number of subjects with species. For number of subjects per group see Table II.
190
I. Leuckfeld et al.
T. forsythia, and Treponema denticola was found to be most strongly related to clinical parameters of periodontal disease. The oral cavity may serve as a reservoir for respiratory pathogens responsible for pneumonia in highrisk patients (13–17). It has also been shown that the dorsum of the tongue serves as a potential reservoir for bacterial species involved in ventilatorassociated pneumonia (VAP) (31). In acute exacerbation of COPD (AECOPD), approximately half of the exacerbations are caused by bacterial infections (32–35), and it has been suggested that the periodontal pockets may serve as reservoirs for potential pathogens. Apart from one subject with Haemophilus influenzae and one subject with Chlamydia, we detected none of the bacterial species mostly associated with AECOPD (e.g. H. influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, and Staphylococcus spp.) in the COPD subjects. Most bacteria live in a stable relationship on the oral and nasal mucosa. The composition of the bacterial flora inhabiting these sites is highly site- and host-specific, suggesting that host factors are important determinants for colonizing microbes. In chronic periodontal disease, the likely etiological factors are the microorganisms involved, local environmental factors other than bacteria (calculus, defective dental restorations, prosthetic appliances, diet, smoking, etc.), and the host defense. We detected higher prevalence and levels of Actinomyces spp., C. gracilis, and V. parvula/dispar in the periodontally healthy COPD subjects. A possible explanation might be host factors associated with COPD allowing the predominance of those bacteria in the subgingival plaque. However, when periodontitis was established and local host defense deteriorated, we were unable to detect a clear difference of the microbial profile related to COPD status. There are other possible explanations for our findings. The group of subjects without COPD or PD (NCP) consisted of only former smokers, whereas the other groups were represented by former and current smokers, although one periodontitis subject (PD) never smoked, which may have affected the microbial colonization profiles. However, the effect of smoking status on the subgingival microbiota is not clear. Some studies have indicated higher levels of certain species in smokers (36–38), others have failed to detect differences in the microbiota between subjects with different smoking status (39–42). In the lower airways, current smoking both in COPD and asymptomatic smokers, and severe airflow limitation have been identified as risk factors for lower airway bacterial colonization (43–45). In this study, with
only a limited number of subjects investigated, Actinomyces spp., C. gracilis, and V. parvula/dispar were prevalent in both former and current smokers in the periodontally healthy COPD subjects. This is a preliminary study with only a small number of subjects investigated. It is interesting that COPD subjects with a clinical healthy periodontium seem to harbor different bacterial species as compared with non-COPD subjects, but we cannot imply any specific association of those species with COPD disease itself. Veillonella spp. and Actinomyces spp. are considered part of the normal oral microflora. However, they have been found in increased levels in periodontal diseased sites and, similar to C. gracilis, they may precede the occurrence of more periodontopathogenic bacteria (e.g. Por. gingivalis, T. forsythia, and Trep. denticola) (30). It is also noteworthy that Veillonella spp. and Actinomyces spp. are among the most frequent anaerobic bacterial species reported from pulmonary samples. In a study of clinical isolates of Actinomyces, several were from respiratory specimens and some were reported from patients with COPD (46). In pulmonary infections in cystic fibrosis patients and VAP, high incidences of anaerobes were detected and the main organisms reported were Actinomyces,Veillonella, Prevotella, Propionibacterium, and Fusobacterium spp. (47,48). In another study on VAP, species detected were typical VAP pathogens, such as H. influenzae, Strep. pneumoniae, Staphylococcus aureus and Ps. aeruginosa, but also many other putative pathogens, some of which have not yet been cultivated, were found. It was suggested that the diversity of bacterial pathogens in VAP is far more complex than previously believed (31). In conclusion, we found a similar subgingival microflora in periodontitis subjects regardless of COPD status. Species considered putative pulmonary pathogens do not seem to colonize the subgingival plaque in COPD subjects, whereas higher prevalences and levels of Actinomyces spp., Veillonella spp., and C. gracilis were found in COPD subjects with a healthy periodontium. Although the data are preliminary, these findings suggest that host factors in COPD may be responsible for the increased susceptibility of COPD subjects to periodontal disease. Acknowledgements This study was supported by research funds from AstraZenica AS, Boehringer Ingelheim Norway KS, and Pfizer AS. The sponsors had no involvement in any part of this study. I.L. was supported by the Faculty of Dentistry, University of Oslo and B.J.P.
Oral microflora in COPD was supported by NIH grant DE011443. The authors report no conflict of interest. They alone are responsible for the content and writing of the paper.
17.
18.
References 1. Mathers CD, Loncar D. Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med. 2006; 3:e442. 2. Hurst JR, Perera WR, Wilkinson TMA, Donaldson GC, Wedzicha JA. Systemic upper and lower airway inflammation at exacerbation of chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2006;173:71–8. 3. Murphy TF. The role of bacteria in airway inflammation in exacerbations of chronic obstructive pulmonary disease. Curr Opin Infect Dis. 2006;19:225–30. 4. Platz J, Beisswenger C, Dalpke A, Koczulla R, Pinkenburg O, Vogelmeier C, et al. Microbial DNA induces host defence reaction of human respiratory epithelial cells. J Immunol. 2004;173:1219–23. 5. Hayes C, Sparrow D, Cohen M, Vokonas PS, Garcia RI. The association between alveolar bone loss and pulmonary function: the VA dental longitudinal study. Ann Periodontol. 1998;3:257–61. 6. Li X, Kolltveit KM, Tronstad L, Olsen I. Systemic diseases caused by oral infection. Clin Microbiol Rev. 2000;13: 547–58. 7. López NJ, Da Silva I, Ipinza J, Gutiérrez J. Periodontal therapy reduces the rate of preterm low birth weight in women with pregnancy-associated gingivitis. J Periodontol. 2005;76:2144–53. 8. Mattila KJ, Valtonen VV, Nieminen M, Huttunen JK. Dental infection and the risk of new coronary events: prospective study of patients with documented coronary artery disease. Clin Infect Dis. 1995;20:588–92. 9. Taylor GW, Burt BA, Becker MP, Genco RJ, Shlossman M, Knowler WC, et al. Severe periodontitis and risk for poor glycemic control in patients with non-insulin-dependent diabetes mellitus. J Periodontol. 1996;67:1085–93. 10. Bahrani-Mougeot FK, Paster BJ, Coleman S, Ashar J, Barbuto S, Lockhart PB. Diverse and novel oral bacterial species in blood following dental procedures. J Clin Microbiol. 2008;46:2129–32. 11. D’Aiuto F, Parkar M, Andreou G, Suvan J, Brett PM, Ready D, et al. Periodontitis and systemic inflammation: control of the local infection is associated with a reduction in serum inflammatory markers. J Dent Res. 2004;83:156–60. 12. Noack B, Genco RJ, Trevisan M, Grossi S, Zambon JJ, De Nardin E. Periodontal infections contribute to elevated systemic C-reactive protein level. J Periodontol. 2001;72: 1221–7. 13. Didilescu AC, Skaug N, Marica C, Didilescu C. Respiratory pathogens in dental plaque of hospitalised patients with chronic lung diseases. Clin Oral Invest. 2005;9:141–7. 14. Fourrier F, Duvivier B, Boutigny H, Roussel-Delvallez M, Chopin C. Colonization of dental plaque: a source of nosocomial infections in intensive care unit patients. Crit Care Med. 1998;26:301–8. 15. Garrouste-Orgeas M, Chevret S, Arlet G, Marie O, Rouveau M, Popoff N, et al. Oropharyngeal or gastric colonization and nosocomial pneumonia in adult intensive care unit patients. Am J Respir Crit Care Med. 1997;156:1647–55. 16. Russell SL, Boylan RJ, Kaslick RS, Scannapieco FA, Katz RV. Respiratory pathogen colonization of the dental
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
191
plaque of institutionalized elders. Spec Care Dentist. 1999; 19:128–34. Scannapieco FA, Stewart EM, Mylotte JM. Colonization of dental plaque by respiratory pathogens in medical intensive care patients. Crit Care Med. 1992;20:740–5. Leuckfeld I, Obregon-Whittle MV, Lund MB, Geiran O, Bjørtuft Ø, Olsen I. Severe chronic obstructive pulmonary disease: association with marginal bone loss in periodontitis. Respir Med. 2008;102:488–94. Scannapieco FA, Ho AW. Potential associations between chronic respiratory disease and periodontal disease: analysis of National Health and Nutrition Examination Survey III. J Periodontol. 2001;72:50–6. Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, et al. Bacterial diversity in human subgingival plaque. J Bacteriol. 2001;183:3770–83. Paster BJ, Olsen I, Aas JA, Dewhirst FE. The breadth of bacterial diversity in the human periodontal pocket and other oral sites. Periodontol 2000. 2006;42:80–7. National Heart, Lung and Blood Institute and World Health Organization. Global initiative for chronic obstructive lung disease: global strategy for diagnosis, management, and prevention of chronic pulmonary disease. 2007 update. Available from URL: http://www.goldcopd.com (accessed July 2008). Edwards U, Rogall T, Blocker H, Emde M, Bottger EC. Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res. 1989;17:7843–53. Cole JR, Chai B, Farris RJ, Wang Q, Kulam-SyedMohideen AS, McGarrell DM, et al. The ribosomal database project (RDP-II): introducing myRDP space and quality controlled public data. Nucleic Acids Res. 2007;35 (Database issue):D169–72. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4:406–25. Jukes TH, Cantor CR. Evolution of protein molecules. In: Munro HN, editor Mammalian protein metabolism, vol. 3. New York: Academic Press; 1969. p. 21–132. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. 2007;24:1596–9. Van de Peer Y, De Wachter R. TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci. 1994;10:569–70. Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defining the normal bacterial flora of the oral cavity. J Clin Microbiol. 2005;43:5721–32. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. Microbial complexes in subgingival plaque. J Clin Periodontol. 1998;25:134–44. Bahrani-Mougeot FK, Paster BJ, Coleman S, Barbuto S, Brennan MT, Noll J, et al. Molecular analysis of oral and respiratory bacterial species associated with ventilator-associated pneumonia. J Clin Microbiol. 2007;45: 1588–93. Fagon J-Y, Chastre J, Trouillet J-L, Domart Y, Dombret M-C, Bornet M, et al. Characterization of distal bronchial microflora during acute exacerbation of chronic bronchitis. Use of the protected specimen brush technique in 54 mechanically ventilated patients. Am Rev Respir Dis. 1990; 142:1004–8. Monsó E, Ruiz J, Rosell A, Manterola J, Morera J, Ausina V. Bacterial infection in chronic obstructive pulmonary disease. A study of stable and exacerbated outpatients using the
192
34.
35.
36.
37.
38.
39.
40.
I. Leuckfeld et al. protected specimen brush. Am J Respir Crit Care Med. 1995;152:1316–20. Pela R, Marchesani F, Agostinelli C, Staccioli D, Cecarini L, Bassotti C, et al. Airway microbial flora in COPD patients in stable clinical conditions and during exacerbations: a bronchoscopic investigation. Monaldi Arch Chest Dis. 1998; 53:262–7. Soler N, Torres A, Ewig S, Gonzalez J, Celis R, El-Ebiary M, et al. Bronchial microbial patterns in severe exacerbations of chronic obstructive pulmonary disease (COPD) requiring mechanical ventilation. Am J Respir Crit Care Med. 1998; 157:1498–505. Kamma JJ, Nakou M, Baehni PC. Clinical and microbiological characteristics of smokers with early onset periodontitis. J Periodontal Res. 1999;34:25–33. Shiloah J, Patters MR, Waring MB. The prevalence of pathogenic periodontal microflora in healthy young adult smokers. J Periodontol. 2000;71:562–7. Umeda M, Chen C, Bakker I, Contreras A, Morrison JL, Slots J. Risk indicators for harboring periodontal pathogens. J Periodontol. 1998;69:1111–18. Haffajee AD, Socransky SS. Relationship of cigarette smoking to the subgingival microbiota. J Clin Periodontol. 2001; 28:377–88. Lie MA, van der Weijden GA, Timmerman MF, Loos BG, van Steenbergen TJM, van der Velden U. Oral microbiota in smokers and non-smokers in natural and experimentally induced gingivitis. J Clin Periodontol. 1998;25:677–86.
41. Preber H, Bergstrom J, Linder LE. Occurrence of periopathogens in smoker and non-smoker patients. J Clin Periodontol. 1992;19:667–71. 42. Stoltenberg JL, Osborn JB, Pihlstrom BL, Herzberg MC, Aeppli DM, Wolff LF, et al. Association between cigarette smoking, bacterial pathogens, and periodontal status. J Periodontol. 1993;64:1225–30. 43. Monsó E, Rosell A, Bonet G, Manterola J, Cardona PJ, Ruiz J, et al. Risk factors for lower airway bacterial colonization in chronic bronchitis. Eur Respir J. 1999;13:338–42. 44. Qvarfordt I, Riise GC, Andersson BA, Larsson S. Lower airway bacterial colonization in asymptomatic smokers and smokers with chronic bronchitis and recurrent exacerbations. Respir Med. 2000;94:881–7. 45. Zalacain R, Sobradillo V, Amilibia J, Barrón J, Achótegui V, Pijoan JI, et al. Predisposing factors to bacterial colonization in chronic obstructive pulmonary disease. Eur Respir J. 1999;13:343–8. 46. Clarridge JE, Zhang Q. Genotypic diversity of clinical Actinomyces species: phenotype, source, and disease correlation among genospecies. J Clin Microbiol. 2002;40:3442–8. 47. Doré P, Robert R, Grollier G, Rouffineau J, Lanquetot H, Charrière J-M, et al. Incidence of anaerobes in ventilatorassociated pneumonia with the use of a protected specimen brush. Am J Respir Crit Care Med. 1996;153:1292–8. 48. Tunney MM, Field TR, Moriarty TF, Patrick S, Doering G, Muhlebach MS, et al. Detection of anaerobic bacteria in high numbers in sputum from patients with cystic fibrosis. Am J Respir Crit Care Med. 2008;177:995–1001.
