methanogenic archaea, sulfate-reducing bacteria, and acetog

21 Pages·2008·0.14 MB·English

JB Accepts, published online ahead of print on 7 March 2008
J. Bacteriol. doi:10.1128/JB.01861-07
Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Quantitative analysis of three hydrogenotrophic microbial groups -
methanogenic archaea, sulfate-reducing bacteria, and acetogenic bacteria -
within plaque biofilms associated with human periodontal disease
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M.E. Vianna1, S. Holtgraewe2, I. Seyfarth1, G. Conrads1, and H.P. Horz1
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1Division of Oral Microbiology and Immunology, Department of Operative and D
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Preventive Dentistry & Periodontology, and DepaPrtment of Medical Microbiology, w
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RWTH Aachen University Hospital, Germany lo
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2Department of Operative and Preventive Dentistry & Periodontology, RWTH Aachen ed

University Hospital, Germany fr
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Short title: Hydrogenotrophic microorganisms and periodontal disease .
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KAey words: periodontal disease, methanogens, sulfate-reducers, acetogens, .o
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interspecies hydrogen transfer o
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Number of words of the abstract: 87 u
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Total Number of words: 2,435 ,

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Number of tables: 1 b
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Number of figures: 6 e
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Number of cited references: 43
*Corresponding author:
Hans-Peter Horz,. Dr.rer.nat.
Division of Oral Microbiology and Immunology
RWTH Aachen University Hospital
Pauwelsstrasse 30
D-52057 Aachen
Phone [phone]
Fax [phone]
E.mail: [email protected]
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Abstract

Human subgingival plaque biofilms are highly complex microbial ecosystems that
may depend on H -metabolising processes. Here we investigated the ubiquity and
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proportions of methanogenic archaea, sulfate-reducers and acetogens in plaque D
samples from 102 periodontitis patients. As opposed to 65 healthy control subjects
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hydrogenotrophic groups were almost consistently detected in periodontal pockets
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with the proportions of methanogens and sulfate-reducers being significantly
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elevated in severe cases. In addition, antagonistPic interactions among the three w
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microbial groups indicated that they may function as alternative syntrophic partners
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of secondary fermenting periodontal pathogens. fr
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Periodontal disease is a polymicrobial anaerobic infection that, besides leading
possibly to loss of the involved teeth (if remain untreated), is also considered to
constitute a risk factor contributing to the development of life-threatening systemic D
diseases, such as endocarditis, atherosclerosis, and stroke as well as co-causative
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for preterm birth (6,7,34). The list of oral microorganisms involved in periodontal
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disease is large, consisting of several hundred species of which approximately 50%
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represent as yet uncultivable phylotypes (4,16,32,3P8). No single species has been w
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identified so far as the ultimate causative agent. Instead the disease is likely to be a
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result of the activity of different and varying microbial complexes (37). Recent fr
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implication of methanogenic archaea (methanogens) and dissimilatory sulfate- h
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reducing bacteCria (SRB) in periodontal pockets (18,23) prompted us to perform a /jb
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cross-sectional examination of the ubiquity of hydrogen (H )-utilizing organisms m
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(hydrogenotrophs) in subgingival plaque biofilms from periodontitis patients. Such /

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knowledge could provide basic information on the role of H -consumption for the J
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regulation of the periodontal biofilm ecosystem (i.e. “interspecies hydrogen transfer”
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(36) as a possible driving force to promote proliferation of fermenting pathogens). 2
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Because of their polyphyletic nature, hydrogenotrophs are not amenable as
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functional microbial entities with 16S rRNA gene-based approaches. We therefore u
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surveyed the major functional microbial guilds methanogens, SRB and acetogenic
bacteria (acetogens) by recovering and analyzing three unifying group-specific genes
(i.e. mcrA, dsrAB, and fhs) encoding for key enzymes involved in H -consumption,
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namely methyl-coenzyme M reductase, dissimilatory sulfite-reductase, and
formyltetrahydrofolate synthetase, respectively. Our analysis encompassed 102
plaque samples from patients at different stages of severity of chronic periodontal
disease (the most common form of periodontitis) along with 65 age-matched healthy
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control individuals. Patient inclusion (42 males, 60 females, mean age 50.7, SD
11.23, attending the Clinic of Operative and Preventive Dentistry and Periodontology,
RWTH Aachen) was performed in accordance with the Ethics Commitee of the
RWTH University Hospital, Aachen. Subgingival plaque samples were collected and D
pooled from the 4 deepest periodontal pockets of each patient with sterile paper
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points (ISO 45, Alfred Becht GmbH, Offenburg, Germany), after isolation and
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supragingival plaque removal. For the healthy subjects, plaque was collected and
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pooled from vestibular sulcus of first molars from Pall quadrants with sterile paper w
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points. Periodontitis sites with periodontal probing depth ≥ 6 mm (clinical attachment a
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loss: ≥ 4 mm) were defined as “severe” cases, and with periodontal probing depth < 6 fr
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mm (clinical attachment loss: < 4 mm) as “moderate” cases (31). Healthy sites were h
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defined as thCose with periodontal probing depths < 3 mm and no bleeding on /jb
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probing. Whole genomic community DNA was extracted as described previously m
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(14). Abundance of target microorganisms was determined by real-time quantitative /

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PCR (RTQ-PCR) using primer pairs with validated target specificity as published J
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earlier (15,21,27,30,43) and thermal profiles modified from the original protocols for
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adapting to a LightCycler-based amplification (Light Cycler 2.0) according to the 2
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recommendations of the manufacturer (Roche Molecular Biochemicals, Technical
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Note No. 2/99, Roche Applied Science, Penzberg, Germany, Table 1). Reactions u
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were performed using LightCycler FastStart DNA Masterplus SYBR Green I in a total
volume of 20 µl. Final reactions contained 100 nM of each primer, and 3 µl of
template DNA (approximately 75 ng). Quantification (i.e. determination of crossing
points and conversion to initial gene target molecule numbers based on calibration
standards) as well as melting curve analysis followed the protocol described in
Vianna et al., 2006 (42) except for preparations of DNA standards. For this purpose
DNA from Methanobrevibacter oralis DSM 7256T was amplified with primers “ME1”
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(11) and “LuR”, from Desulfovibrio piger DSM 749T with primers ”DSR1F” (43) and
“DSR4R” and from Eubacterium limosum DSM 20543T, with primers “FTHFS-F” and
“FTHFS-R”. Purified amplicons (Qiagen Purification Kit, Qiagen, Hilden, Germany)
were quantified with the PicoGreen dsDNA quantification kit (Molecular Probes, D
Leiden, Netherlands). Knowing the exact size of the amplicons (in base pairs) and
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using the average molecular weight of a single DNA base pair (i.e. 650) the target
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molecule numbers for each PCR product could be determined and appropriate
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dilutions series of the PCR products could be used aPs standards. The linear scope of w
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detection ranged from 102 to 108 target molecule numbers, with an amplification a
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efficiency of 1.695, error: 0.025 (mcrA); 1.755, error: 0.01 (dsrAB); and 1.830, error: fr
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0.007 (fhs). For quantifying sulfate-reducers a pre-amplification with the “external” h
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primer DSR1FC and the assay primer DSR4R was performed with the following /jb
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temperature profile: 96°C, 10 min; 16 cycles: 96°C, 10 s; 53°C, 10 s; 72°C, 180 s. m
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The efficiency of this reaction measured based on dilution series of genomic DNA /

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from D. piger 749T was found to be 1.57, error 0.06. Thus, dsrAB gene levels were Ja
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assumed to be enriched by the pre-amplification step by factor 1.5716 (= factor 1363). ar
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Total bacteria were quantified exactly as described previously (42). All samples were 2
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run in triplicate, with the mean value used for analysis. The coefficient of variation b
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among replicates was below 1%. u
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A representative set of RTQ-PCR products from randomly chosen patient
samples was directly sequenced as described previously (42) and phylogenetic
analysis performed using the ARB software package (26). The partial mcrA-, dsrAB-,
and fhs-gene sequences determined in this study have been deposited in the EMBL,
GenBank, and DDBJ nucleotide sequence databases under accession no EU294497
to EU294506. Differences in the microbial population size between disease states
and between patients grouped by presence/absence of hydrogenotrophic groups
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were assessed by the Mann–Whitney test, SPSS, 10.0.1, Chicago, Illinois. All errors
are reported as standard errors.
Prevelance, proportions and interactions of hydrogenotrophic groups
While methanogens and SRB were detected in samples of periodontitis D
patients only (43.1 and 41.2%, respectively) acetogens were found in 84.3% of
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patient samples but also in 64.4% of healthy control subjects (Fig. 1). Only 4.9% of
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patient samples did not contain any of three functional groups. Single presence
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occurred in 3.9% (methanogens), 2.9% (SRB) anPd 30.4% (acetogens) of cases, w
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respectively. Co-occurrence of acetogens with one or both groups was found with
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roughly equal frequency (between 15 and 19% of cases), while co-occurence of fr
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methanogens and SRB was a rarity (3.9%). Absolute abundance of total bacteria and h
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acetogens waCs significantly lower in healthy control patients compared to /jb
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periodontitis patients (P < 0.0001, Fig. 2). Within the periodontitis patients roughly m
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equal amounts of methanogens and acetogens were found but SRB were about one /

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order of magnitude lower (Fig. 2). In order to normalize for variations in microbial J
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biomass among samples absolute abundance data were referred to the total
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bacterial load. Methanogens made up the largest proportion with 0.26%, followed by 2
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acetogens (0.11%) and SRB (0.01%). The mean proportion of acetogens in the
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healthy control subjects was 1.67%. Significant antagonistic interactions among the u
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three hydrogenotrophic groups were such that the mean proportion of acetogens was
twice as high in SRB-negative samples (n=51) compared to SRB-positive samples
(n=42, P = 0.001) and 2.4 times higher in methanogen-negative samples (n=50) than
in methanogen-positive samples (n=44, P = 0.028). Likewise the mean proportion of
SRB was 2.7 times higher in methanogen-negative samples (n=22) compared to
methanogen-positive samples (n=44, P = 0.002), but co-occurrence with acetogens
did not alter the population size of SRB. Lastly, the mean proportion of methanogens
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was 3.3 times higher in SRB-negative samples (n=24) as opposed to SRB-positive
samples (n=42, P = 0.001) and even 9.0 times higher in samples devoid of
acetogens (n=8) compared to acetogen-positive samples (n=86, P = 0.001).
While the total bacterial load did not significantly differ between the moderate D
and the severe cases of periodontitis (Fig. 3A), mean proportions of the
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hydrogenotrophs were significantly different although with opposite amplitudes (Fig.
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3B-D). The mean proportion of acetogens was reduced by factor 3 in the severe
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cases compared to the moderate cases (P = P0.088). Conversely, the mean w
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proportion of methanogens was elevated by factor 4 (P = 0.038) and that of sulfate-
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reducers even by factor 10 (P = 0.032) in the severe cases compared to the fr
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moderate cases. h
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IdentityC of hydrogenotrophic groups /jb
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Phylogenetic analysis of sequenced PCR products confirmed the identity of m
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the target genes. All but one mcrA gene sequence were highly related to /

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Methanobrevibacter oralis with 100% identity at the protein level (Fig. 4) while the J
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remaining sequence was approximately 96% identical at the protein level. Most
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dsrAB gene sequences determined in this study formed a distinct clade related to 2
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Desulfovibrio piger and D. desulfuricans with a sequence identity of approximately
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93% at the protein level (Fig. 5). Sequence identities within this clade were beyond u
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98%. Its position within the overall tree-topology prompted us to test for a possible
close relationship to the recently reported periodontal isolate “Desulfovibrio strain
NY682” (DSM12803 (19)). A 1320 bp-stretch of the dsrAB sequence of this strain
was therefore determined and incorporated into the ARB database. In fact, the
dsrAB-clade determined in our study shared ancestry with strain NY682 to the
exclusion of D. piger and D. desulfuricans with a sequence identity of approximately
96% at the protein level. The fhs sequence types determined in our study, were
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highly related to each other with a protein-sequence identity of at least 95%. They
formed a distinct lineage with moderate relationship to the fhs-gene from an
uncultured microorganism previously identified in the gut system of ruminants (28)
with a sequence identity of approximately 80%, Fig. 6. D
Hydrogenotrophy and periodontal disease
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This epidemiologic survey provides evidence for the ubiquity of hydrogenotrophs in
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periodontal pockets with niche exclusion (single presence of one hydrogenotrophic
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group) in approximately 40% of patients. Co-occurrePnce of two (or all three groups) in w
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the remaining 60% indicates H -levels to be sufficiently high in periodontal plaque to
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allow partitioning of H2-consumption or alternative metabolic strategies of SRB and/or fro
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acetogens. This could include the use of electron donors others than H or h
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fermentation oCf short chain fatty acids with possible production of H2 (8,12). In the /jb
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latter case a mutual relationship with methanogens rather than competition could be m
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possible (3,13,22,41). The actual in situ-activity may depend on the availability of /

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nutrients and the redox potential of the terminal electron acceptors (5). This in turn J
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may vary from host to host and also within plaque biofilms of one patient during the
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cause of disease. Irrespective of such site-specific and temporary varying 2
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interactions and of the intersubject-variability of the subgingival microflora our data
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suggest that from a global point of view, antagonistic interactions, hence competition u
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among H -utilizers seem to prevail. Although SRB should out-compete methanogens
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for the substrate H if sulfate is not a limiting factor (10,25), a recent study provided
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evidence for the possibility of an opposite order of competitivity (i.e. methanogens
outcompete SRB) in the human colon (39). The close phylogenetic affinity of gut and
oral methanogens as well as their proportional dominance over SRB as determined
in our study could be a reflection of an analogous situation in subgingival plaque.
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The proportions of the hydrogenotrophs were in most cases below one percent
of the total microflora. This accords with a study from Kumar et al. (17), in which 93%
of 274 phylotypes identified in subgingival plaque and healthy sites had a proportion
below one percent and approximately 50% of phylotypes were even below 0.1%. D
Similar magnitudes have also been reported in another study which found that 99% of
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phylotypes in soil samples each make up less than 1% of the community (1). This
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means that complex microbial communities may not only be dominated by “rare”
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species but that the population size of any givePn species is not an appropriate w
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measure for assessing its ecological impact. This should in particular be true for
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organisms for which a high degree of functional redundancy does not exist and which fr
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are considered to parallel keystone species in certain environments (e.g. h
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hydrogenotropChs, (2). /jb
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The mcrA and dsrAB sequence types determined in our study are in line with m
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previous findings including culture-based studies. For instance, M. oralis and closely /

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related phylotypes are the dominant oral methanogens (9,23,42) yet strikingly, the J
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overall archaeal diversity in the human oral cavity seems to be restricted to few
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members of the genus Methanobrevibacter. In addition, most dsrAB- gene types 2
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determined in our study were closely related to the oral isolate “Desulfovibrio strain
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NY682” (19) which in turn matches D. fairfieldensis based on 16S rRNA treeing u
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analysis (19). D. fairfieldensis, a resident of the human gastrointestinal tract, has not
only frequently been isolated from periodontal pockets (24) but also from various
other sites of infection such as a pyogenic liver abscess (40), from blood (29,40) and
in association with choledocholithiasis (35), designating this species as a potential
human pathogen. Its ten-fold increase in severe periodontitis cases as observed in
the present study underscores this hypothesis. It should be noted, though that the
diversity of SRB in subgingival plaque is not restricted to Desulfovibrio species (19).
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In contrast to the former sequences it remains unclear to which organisms the
fhs sequence types correspond however, their position within the phylogenetic
radiation of known acetogenic organisms (Fig. 6) and the antagonistic interactions
with methanogens and SRB, as observed in this study, suggests that they reflect D
authentic acetogens. It could be speculated that these fhs-sequences correspond to
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Treponema sp. since Lepp et al (23) found larger proportions of Treponema
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populations at periodontal sites lacking methanogenic archaea, hypothesizing a
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possible competition for H between these two grPoups of organisms. However, as w
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opposed to Treponema colonizing the gut of termites (20,33) human-associated
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Treponema have not yet been demonstrated to be capable of (hydrogen-consuming) fr
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acetogenesis. Clearly, more work is needed here for assessing the identity and h
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function of aceCtogenic bacteria in human plaque biofilms. /jb
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Final consideration m
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Unlike acetogens, detectable levels of methanogens and sulfate-reducers were /

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only found at diseased sites with proportions of methanogens and sulfate-reducers J
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significantly elevated in the more severe cases. Although this finding demonstrates
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association with - rather than causation of – disease, the increased importance of 2
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both groups for the progression of periodontitis is evident and principally designates
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the genes mcrA and dsrAB as potential biomarkers for progressive periodontal u
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disease with eminent positive predictive values.
With respect to species diversity and species function, we are only at the
beginning of understanding the polymicrobial periodontal disease. An ultimate
etiological agent of the various forms of periodontitis may probably not exist. Instead,
the concerted activity of the microbial community as a whole may be one cause of the
disease. Attempts to identify fundamental principals driving the infectious process
regardless of site- or host-specific community structure might strongly advance our
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