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Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis are Components of a Polymicrobial Intracellular Flora within Human Buccal Cells
1 Department of Oral Science, School of Dentistry, and Correspondence: * corresponding author, jrudney{at}tc.umn.edu
Previously, we used in situ hybridization and confocal microscopy to detect the periodontal pathogens Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis within buccal epithelial cells taken directly from the mouth. This study tested the hypothesis that the intracellular flora of buccal cells is polymicrobial. Mixtures containing a red fluorescent universal probe paired with green fluorescent versions of either A. actinomycetemcomitans-, P. gingivalis-, or T. forsythensis-specific probes were hybridized with buccal cells collected from each of 38 healthy humans. We verified co-localization of probe pairs within cells by generating three-dimensional reconstructions. Intracellular bacteria were detected in every subject. Each cell that was labeled with a species-specific probe also contained bacteria recognized only by the universal probe. Bacteria labeled with specific probes often occupied smaller regions within larger masses of bacteria. Those findings suggest that future studies of invasion by oral bacteria may need to include microbial consortia.
Key Words: polymicrobial • bacterial invasion • Actinobacillus actinomycetemcomitans • Porphyromonas gingivalis • Tannerella forsythensis
The species Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis (formerly Bacteroides forsythus) are all present at multiple oral sites. Numerical dominance of one or more of those species in the gingival crevice is strongly associated with periodontal disease, but they also are present at lower numbers at sites including subgingival biofilm, supragingival biofilm, the tongue, buccal mucosa, and saliva. It has been suggested that those sites may provide reservoirs for re-colonization of the gingival crevice after periodontal treatment (Quirynen et al., 2001; Mager et al., 2003). All the above species are fastidious anaerobes when grown in culture. Yet they are able to maintain themselves under the aerobic conditions associated with saliva and buccal mucosa. The buccal mucosa presents those species with the additional challenge of remaining in place as epithelial cells are shed. All of them have been shown to be capable of invading oral epithelial cells in tissue culture (Lamont et al., 1995; Madianos et al., 1996; Meyer et al., 1996; Han et al., 2000). In previous studies, we addressed the question of whether buccal cell invasion might provide a protected environment in vivo. Fluorescence in situ hybridization (FISH) and confocal microscopy (LSCM) were used to detect, first, intracellular A. actinomycetemcomitans and P. gingivalis and, later, T. forsythensis, within buccal epithelial cells (BEC) taken directly from the mouths of human subjects (Rudney et al., 2001; unpublished data). In those studies, a FISH probe directed toward a 16S rRNA sequence present in all bacterial species consistently detected larger masses of intracellular bacteria than could be accounted for by 16S rRNA probes specific to our 3 target species (Rudney et al., 2001). One possible explanation for that observation could be that the intracellular flora of BEC is polymicrobial. In this study, we used double-labeling with universal and species-specific FISH probes to test that hypothesis.
Human Subjects Informed consent was obtained according to a protocol approved by the University of Minnesota Institutional Review Board, after the nature and possible consequences of the studies were explained to a convenience sample of 38 adults (20 males and 18 females). BEC were collected from each person with sterile cytological brushes, according to our published protocol (Rudney et al., 2001).
Bacterial Loads We have recently published detailed endpoint qPCR protocols for enumeration of A. actinomycetemcomitans, P. gingivalis, and T. forsythensis (Rudney et al., 2003). The same methods were used here. Briefly, Masterpure kits (Epicentre, Madison, WI, USA) were used for DNA extraction from bacterial standards and BEC. Total DNA was then determined with Picogreen kits (Molecular Probes, Eugene, OR, USA). Our qPCR approach is based on the AmplifluorTM system (Chemicon Inc., Temecula, CA, USA). A manufacturer-designed sequence (Z-tail) was added to the 5' end of one of each pair of species-specific primers. The Z-tail also constitutes the 3' end of a universal primer (UniPrimerTM), incorporating a quenched fluorescein. During the earliest stages of amplification, the specific tailed primer incorporates the Z-sequence into the PCR product. The complement to the Z-tail can then anneal to the UniPrimerTM. When the complementary strand is extended, the fluorescein is forced away from the quencher molecule. We determined the number of A. actinomycetemcomitans, P. gingivalis, and T. forsythensis cells in each BEC sample by semi-log regression against standard curves prepared as dilutions of DNA extracts from known quantities of each target species (grown in culture).
FISH A mixture of red and green versions of the complement to EUB338 was used as the negative control. The probe sequence for T. forsythensis was 5' TTC ACC GCG GAC TTA ACA 3'. It was derived from a published PCR primer previously found to be specific for T. forsythensis 16S rDNA (Meurman et al., 1997). All other probe sequences were as previously described (Rudney et al., 2001). We confirmed the specificities of each universal/specific probe pair by hybridization to cultures of target species mixed with cultures of other oral species (Appendix Fig. 1).
Confocal Microscopy
Bacterial loads We estimated the number of BEC per sample by dividing total sample DNA by 6 pg, an estimate of DNA yield per mammalian cell (Qiagen, 2004). The mean value ± SD was 7,959,239 ± 3,194,068. Mean bacterial loads per sample were expressed as logs, because they were strongly skewed to the right. The loads were highest for T. forsythensis (5.87 ± 2.62), lowest for P. gingivalis (2.41 ± 2.37), and intermediate for A. actinomycetemcomitans (3.90 ± 2.30). We divided bacterial loads (determined by qPCR) by the number of BEC to estimate the average numbers of bacteria per BEC. Those values also were highly skewed. As logs, they were –0.97 ± 0.62 for T. forsythensis, –4.40 ± 0.42 for P. gingivalis, and –2.95 ± 0.40 for A. actinomycetemcomitans.
FISH/LSCM
This could be seen in an example of a single z-section, with the red and green channels before and after superimposition (Figs. 1A  –1C). The specific probe in this case recognized A. actinomycetemcomitans. The yellow bacteria labeled by both the A. actinomycetemcomitans and universal probes were present as part of a larger mass of bacteria recognized only by the red universal probe.
In a three-dimensional reconstruction of the same field, the angle of view is different, due to rotation and zooming (Fig. 1D  ). The cell in the foreground contained small masses of intracellular bacteria. Some appeared to project out of the surface, while others were fully intracellular. Small masses labeled for A. actinomycetemcomitans were clustered on the right side of the cell. The smallest appeared to be mono-specific, but bacteria recognized only by the universal probe co-occurred in the larger ones. The cell in the background contained a much larger mass of bacteria. In that mass, A. actinomycetemcomitans clearly co-existed with other species.
An example of a cell where the species-specific probe was directed toward P. gingivalis showed extensive bacterial invasion, but only a relatively small proportion of the intracellular masses was labeled by the P. gingivalis probe (Fig. 2A
A typical cell probed for T. forsythensis could be described as "super-invaded", since it was almost completely filled with bacteria (Fig. 2B ). Large masses of T. forsythensis could be seen in this particular cell, but T. forsythensis still did not appear to be dominant relative to the bacteria recognized only by the universal probe. The prevalence of super-invaded cells appeared to vary among subjects, but most samples did contain them. One drawback of three-dimensional reconstructions made for printing is that the extensive image processing required leads to loss of resolution of individual bacterial cells. Single bacteria can more easily be seen in Confocal Assistant movies for these Figs., which are available online (Appendix Figs. 3–5).
Bacterial loads for P. gingivalis and T. forsythensis in BEC samples were lower than those we had previously found for disease-related subgingival biofilm, while A. actinomycetemcomitans loads appeared to be higher (Rudney et al., 2003). Analysis of our earlier qPCR data likewise suggests that buccal epithelia in healthy subjects may carry heavier loads of those species than supragingival biofilm (Rudney et al., 2003). That pattern of site distribution is in general agreement with results from a recent study (Mager et al., 2003) in which the authors reported a mean proportion of T. forsythensis from buccal surfaces (1.2%) which was approximately 4 times higher than that obtained for supragingival biofilm (0.3%), and higher than that seen for any other oral surface except subgingival biofilm (1.7%). Likewise, a culture-based study of the oral distribution of A. actinomycetemcomitans in a large number of periodontally healthy subjects also indicated that loads were higher on buccal mucosa than in subgingival or supragingival biofilm (Muller et al., 1996). Overall, there is strong evidence to suggest that the shedding buccal epithelial surface is capable of supporting surprisingly large bacterial populations. Moreover, those populations may include relatively greater numbers of some pathogenic species than are found on supragingival tooth surfaces. This seems consistent with the hypothesis that buccal cells may provide a reservoir for subgingival recolonization. In our BEC samples, "super-invaded" cells co-occurred with sparsely invaded cells and uninvaded cells. A similar pattern has been described for an in vivo mouse model in which bladder epithelium was invaded by uropathogenic E. coli (Mulvey et al., 2001). Those authors suggested that invasion provided a means for infection to persist, despite the frequent shedding of bladder epithelial cells. Their model was mono-specific, and the bladder contains only shedding surfaces. In the mouth, shedding mucosal surfaces co-occur with non-shedding tooth surfaces that harbor a complex diverse biofilm (Paster et al., 2001; Mager et al., 2003). Our study is the first to show that bacterial masses inside oral mucosal cells share the polymicrobial nature of tooth-surface biofilm. We do not yet know whether intracellular bacterial masses will prove to be as diverse as tooth-surface biofilm, although we are actively investigating that question. Several scenarios can be proposed. Invasiveness might be limited to a subset of oral species that use it as a virulence factor. Alternatively, a wide range of oral bacteria which principally live in biofilm might be capable of invasion as a means of persisting when they happen to encounter a shedding surface. Since species interaction appears to be widespread in oral biofilm (Cook et al., 1998; Fong et al., 2001; Palmer et al., 2001, 2003; McNab et al., 2003), another alternative could be that non-invasive species gain entrance to cells by forming consortia with invasive species. Previous studies of invasion have focused on putative periodontal pathogens such as A. actinomycetemcomitans, P. gingivalis, and T. forsythensis (Lamont et al., 1995; Madianos et al., 1996; Meyer et al., 1996; Han et al., 2000). That has tended to bring the virulence factor explanation to the forefront. However, our findings suggest that those 3 species are not dominant members of the buccal intracellular flora. That pattern is consistent with published observations of mucosal surfaces by DNA "checkerboard" analysis (Mager et al., 2003). We are looking at patients with active periodontitis, and it will be interesting to see whether pathogenic species are more prevalent inside buccal cells from those subjects. However, our present results seem more suggestive of invasion by either a broad range of species, or by consortia in which the non-invasive partners grow faster than those responsible for gaining entry to the cell. Invasive bacteria generally gain entry by co-opting and re-directing host cell mechanisms such as endocytosis (Lamont et al., 1995; Meyer et al., 1996, 1999; Sandros et al., 1996; Progulske-Fox et al., 1999; Han et al., 2000). Investigators have explored the molecular basis for such interactions by looking at mono-specific infections of host cells in tissue culture. As yet, however, there is no information on how such species-specific interactions will be influenced by the presence of multiple species. Such studies are likely to present challenging technical problems, but our results suggest that it will be important to make the attempt.
This study was supported by USPHS grant DE 14214 from the National Institute of Dental and Craniofacial Research, Bethesda, MD 20892, USA.
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org. Received for publication September 12, 2003. Revision received October 1, 2004. Accepted for publication October 13, 2004.
Journal of Dental Research, Vol. 84, No. 1,
59-63 (2005) This article has been cited by other articles:
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