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J Dent Res 84(12):1165-1171, 2005
© 2005 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Streptococci Dominate the Diverse Flora within Buccal Cells

J.D. Rudney*, R. Chen, and G. Zhang

Department of Diagnostic and Biological Sciences, School of Dentistry, University of Minnesota, 17-252 Moos Tower, 515 Delaware St. SE, Minneapolis, MN 55455, USA;

* corresponding author, jrudney{at}tc.umn.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previously, we reported that intracellular Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis were present within buccal epithelial cells from human subjects, as lesser components of a polymicrobial flora. In this study, we further characterized that intracellular flora by using the same double-labeling techniques to identify Fusobacterium nucleatum, Prevotella intermedia, oral Campylobacter species, Eikenella corrodens, Treponema denticola, Gemella haemolysans, Granulicatella adiacens, and total streptococci within buccal epithelial cells. All those species were found within buccal cells. In every case, species recognized by green-labeled species-specific probes were accompanied by other bacteria recognized only by a red-labeled universal probe. Streptococci appeared to be a major component of the polymicrobial intracellular flora, being present at a level from one to two logs greater than the next most common species (G. adiacens). This is similar to what is observed in oral biofilms, where diverse species interact in complex communities that often are dominated by streptococci.

KEY WORDS: Streptococcus • Fusobacterium nucleatum • Prevotella intermedia • Campylobacter • Eikenella corrodens • Treponema denticola • Gemella haemolysans • Granulicatella adiacens • buccal cells • bacterial invasion


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A variety of oral microbes associated with periodontal disease can invade cells in tissue culture. However, few studies have been done to determine whether host cell invasion actually occurs in the mouth. Our group previously addressed this question by using fluorescence in situ hybridization and laser-scanning confocal microscopy to detect bacteria within buccal epithelial cells from human subjects (Rudney et al., 2001). We focused on buccal cells because they were readily available, and because of the possibility that invaded buccal cells might provide a protected reservoir for re-colonization of the gingival crevice after periodontal treatment.

Our results indicated that buccal cells could contain intracellular Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis. Moreover, double-labeling studies with green species-specific rRNA probes and a red universal rRNA probe showed that those target periodontal pathogens were always accompanied within the same buccal cell by larger quantities of unidentified bacteria. This was the first demonstration that intracellular colonization of oral cells could be polymicrobial (Rudney et al., 2005).

Bacteria recognized only by the universal probe displayed morphotypes ranging from long filaments to cocci, with multiple types sometimes occurring within the same buccal cell. Cocci appeared to be the most prevalent. This heterogeneity of form is similar to what is observed in oral biofilms, where diverse species interact in complex communities that often are dominated by streptococci (Zee et al., 2000). We do not yet know whether true bacterial communities exist inside buccal cells. To begin investigating this question, we first wished to determine whether buccal cells contain additional species that have been implicated in periodontal disease. Second, we wanted to test the hypothesis that the intracellular cocci prevalent within buccal cells were streptococci.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Target Species
Fusobacterium nucleatum and Prevotella intermedia are putative periodontal pathogens that can invade oral epithelial cells in tissue culture (Dorn et al., 1998; Han et al., 2000). They were therefore logical choices for this investigation. We also included Campylobacter rectus, Eikenella corrodens, and Treponema denticola. Those species also are associated with periodontal disease (Ashimoto et al., 1996), and thus were of interest from the standpoint of our reservoir hypothesis. We could find no previous studies on the invasiveness of C. rectus, although Campylobacter curvus, another oral species, was not invasive in tissue culture. The same study found that E. corrodens likewise was non-invasive (Han et al., 2000). T. denticola has not been reported to enter host cells directly, but may invade tissues by a paracellular route (Lux et al., 2001).

Streptococcus mitis has been reported to be the dominant streptococcal species on buccal mucosa (Mager et al., 2003). However, because oral streptococci are very closely related, it has been difficult to design 16S rRNA probes that fully discriminate among species (Paster et al., 1998). We therefore chose to use a probe that would recognize all members of the genus Streptococcus. In a recent rRNA cloning study, Gemella haemolysans and Granulicatella adiacens also appeared to be common species on buccal mucosa (Aas et al., 2005). Since those species are morphologically similar to streptococci, they also were included in this study.

Collection of Buccal Epithelial Cells
Informed consent was obtained from subjects after the study protocol had been evaluated by the University of Minnesota Institutional Review Board. Buccal cells were collected from each person with sterile cytological brushes, according to our published method (Rudney et al., 2001). Technical limitations on equipment used during the hybridization protocol made it difficult to run more than 5 different species-specific probes at the same time. This study was therefore carried out in two phases. During the first phase, buccal cells were collected from 36 adult (age range, 20–60 yrs) subjects self-reporting as being in good periodontal and general health. Those samples were probed for oral Campylobacter, F. nucleatum, P. intermedia, and streptococci. Some of those subjects were not available for recall during the second phase, so additional persons were recruited to make up a second group of 37. Buccal cells from the second group were probed for E. corrodens, T. denticola, G. haemolysans, G. adiacens, and streptococci.

Bacterial Loads
A portion of each sample was frozen at –80°C, and set aside so that bacterial loads for each target species could be determined. As we have previously reported, it is difficult to estimate bacterial numbers by the direct counting of confocal images (Rudney et al., 2005). Our alternative has been to use quantitative polymerase chain-reaction (qPCR) assays. We have recently published endpoint qPCR protocols for the enumeration of total streptococci (Rudney et al., 2003b), A. actinomycetemcomitans, P. gingivalis, and T. forsythensis (Rudney et al., 2003a). Our qPCR approach is based on the AmplifluorTM system (Chemicon Inc., Temecula, CA, USA). The qPCR for total streptococci was used here exactly as we previously described it. The same principles were used to develop qPCR assays for the other target species. Briefly, we examined 16S rRNA gene sequences for those species to define species-specific forward and reverse primers that were within 100 to 250 base pairs apart (Table 1Go). When two specific primers could not be found within that distance, one specific and one universal primer were used instead. Primer specificity was initially evaluated with the use of Ribosomal Database Project Release 8.1 and BLAST software (Table 1Go; this Table includes updates on probe and primer specificity that have since been obtained from Ribosomal Database Project Release 9.28) (Maidak et al., 2001; Cole et al., 2005). Specificity then was confirmed by PCR against a panel containing the type strains for target species, plus A. actinomycetemcomitans, P. gingivalis, and T. forsythensis. We established quantitative standards by estimating the average ng DNA per cell of each target species (Table 1Go), according to the method we previously described (Rudney et al., 2003a). Masterpure kits (Epicentre, Madison, WI, USA) were used for DNA extraction from bacterial standards (and also buccal cells). Total DNA was then determined with Picogreen kits (Molecular Probes, Eugene, OR, 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.


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  		Table 1. Standard Strains, qPCR Primers, qPCR Parameters Varying between Assaysa, and in situ Hybridization Probes
 
We optimized the qPCR protocol for each target species by varying the location of the Z-tail (forward or reverse), the ratio of tailed primer to untailed primer, the annealing temperature, and the number of cycles. Optimal values for those parameters are reported in Table 1Go. All other qPCR reagents, reaction mixtures, and thermal cycling parameters were the same as those we described previously (Rudney et al., 2003a,b). We determined the number of bacterial cells in each buccal cell sample by semi-log regression against standard curves prepared as dilutions of DNA extracts from known quantities of each target species grown in culture. Results were cross-checked by agarose gel electrophoresis of the qPCR products. Only those samples that showed a band of the correct size were used for subsequent estimation of bacterial loads.

Fluorescent in situ Hybridization
The remainder of each buccal cell sample was fixed in 3.7% formaldehyde and processed for in situ hybridization. Species-specific probes for bacterial ribosomes were adapted from the species-specific PCR primers we had designed, or else from previously published probes (Table 1Go). Probes were obtained from Oligos Etc. (Wilsonville, OR, USA) as 5' conjugates of Alexa Fluor® dyes from Molecular Probes (Eugene, OR, USA). The protocol was identical to the one which we described in the online Appendix to our previous paper (Rudney et al., 2005). Briefly, 8 probe mixtures were prepared. Each mixture contained a red fluorescent (Alexa Fluor 594®) universal probe (EUB338), paired with a green fluorescent (Alexa Fluor 488®) conjugate of one of the specific probes for each target species. A mixture of red and green conjugates of the complement to EUB338 was used as the negative control. We confirmed the specificities of each probe pair by hybridization to cultures of target species mixed with cultures of other oral species, as described in the online Appendix to our previous paper (Rudney et al., 2005).

Confocal Microscopy
We used confocal microscopy to determine whether labeled bacteria were intracellular. This was done exactly as described in our previous paper (Rudney et al., 2005). In some cases, Z-stacks collected sequentially in the green and red channels (to minimize fluorescence crosstalk) were merged in Volocity software (Improvision, Lexington, MA, USA), which we used to generate three-dimensional reconstructions of invaded buccal cells. We then used Photoshop (Adobe Systems, San Jose, CA, USA) to adjust the color balance between the green and red channels, to optimize the visibility of bacteria against buccal cell background autofluorescence (which typically was strongest in the green channel).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Bacterial Loads
We estimated the number of buccal cells per sample by dividing total sample DNA by 6 pg, an estimate of DNA yield per mammalian cell (Qiagen, 2004). The average number of buccal cells was three-fold higher in the first set of subjects. We believe this to have been due to a change in the design of the cytological brushes between collection of the first and second sets. To facilitate comparison between sets, we standardized bacterial loads as the average number of bacteria per buccal cell. Those data were expressed as logs, because they were highly skewed. In each sample set, the estimated number of streptococci per buccal cell was highest, being one to two logs greater than G. adiacens and G. haemolysans (Set 2), which in turn were about 0.35 logs higher than oral Campylobacter species and F. nucleatum (Set 1), which were approximately one log higher than T. denticola (Set 2), P. intermedia (Set 1), and E. corrodens (Set 2). A similar relative ranking was obtained when we considered only the presence or absence of a PCR product (Table 2Go).


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  		Table 2. Results for qPCR Assays and Fluorescent in situ Hybridization/Confocal Microscopy
 
Fluorescent in situ Hybridization/Confocal Microscopy
The universal probe showed that every buccal cell sample contained invaded cells. The negative control was negative in every subject. Confocal microscopy was more sensitive than qPCR, with larger numbers of subjects being positive for each species (Table 2Go). Every buccal cell sample also contained uninvaded cells. For that reason, the qPCR-based estimates of the average numbers of bacteria per buccal cell appeared to underestimate bacterial numbers within invaded cells. For each person, semi-quantitative estimates of bacterial numbers were made for the buccal cell in each stored z-section image that appeared to contain the largest amount of intracellular bacteria labeled with a particular species-specific probe (Table 2Go). The modal value for intracellular streptococci was greater than 100 bacteria/cell in both sets of samples. The modal value for all the other species was 1–10 bacteria/cell, with a range from zero to approximately 100. Every stored z-section that was labeled with those species-specific probes also contained bacteria recognized only by the universal probe, with a modal value of greater than 100. Approximately 80% of buccal cells containing intracellular streptococci also contained intracellular bacteria recognized only by the universal probe.

Some buccal cells appeared to be dominated by intracellular and extracellular bacteria (Fig. 1Go). Cocci were the most common form in heavily invaded buccal cells. Double-labeling with the Streptococcus-specific and universal probes confirmed that the majority of those cocci were streptococci (Figs. 1A, 1BGo). Heavily invaded buccal cells also contained species recognized only by the universal probe. The other target species were present as lesser elements of a Streptococcus-dominated flora within heavily invaded buccal cells (Figs. 1C, 1DGo). Under those circumstances, bacteria of different species appeared to be in direct contact with each other.



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  		Figure 1. Three-dimensional reconstructions of buccal cells invaded by large numbers of cocci. (A) A cluster of buccal cells that contained cells invaded to various degrees by bacteria labeled by both the green Streptococcus-specific and red universal probes (shown in yellow), as well as bacteria recognized only by the universal probe (shown in red). The cell denoted by the arrow had been extensively invaded by streptococci. (B) Z-plane slices of three-dimensional reconstructions were generated to confirm that bacteria seen were intracellular. This slice of the reconstruction shown in panel A contained intracellular streptococci, as denoted by the arrows. (C) A buccal cell dominated by presumed streptococci that were labeled by the universal probe (red). This sample also was treated with the F. nucleatum-specific probe, and the buccal cell shown contained several yellow F. nucleatum cells (arrows) in close association with cocci. (D) A z-plane slice of the buccal cell shown in panel C, indicating that intracellular F. nucleatum were in association with intracellular cocci.

 
Samples from all subjects also contained buccal cells that showed relatively lower levels of invasion by target species (Fig. 2Go). In such cells, individual bacteria or bacteria in small groups were dispersed upon the surface and within cells. Those buccal cells could be considered to contain a polymicrobial flora, in the sense that bacteria recognized by each specific probe and bacteria recognized by the universal probe co-existed within them. In some cases, there was direct contact between different species. However, single-species clusters also were present.



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  		Figure 2. Three-dimensional reconstructions of buccal cells that were more sparsely invaded by bacteria. (A) Buccal cells invaded by distinct clusters of P. intermedia (yellow, denoted by arrows), as well as other bacteria recognized only by the universal probe (red). (B) A Z-plane slice of the reconstruction shown in panel A, which contained intracellular P. intermedia (arrows) that were not directly contiguous with other species. (C) Buccal cells invaded by G. haemolysans (yellow, denoted by arrows), as well as other bacteria recognized only by the universal probe (red). Some G. haemolysans were in direct contact with other species, and some were not. (D) A z-plane slice of the reconstruction shown in C. The arrow denotes intracellular G. haemolysans in contact with another species.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Thus far, we have detected 11 bacterial taxa within buccal cells. This indicates that the mucosal intracellular flora is diverse, and the full extent of that diversity remains to be determined. Our previous studies focused on species considered to be the most important periodontal pathogens, because we are interested in mucosal surfaces as a reservoir site for re-colonization of the gingival crevice. In this study, we looked for additional putative periodontal pathogens. F. nucleatum and P. intermedia are invasive in tissue culture, whereas E. corrodens was negative for invasion in the same study (Han et al., 2000). T. denticola can pass through layers of cultured epithelial cells, but has not been observed to enter cells directly (Lux et al., 2001). This is the first study to show that all of those species can invade epithelial cells in the mouth. To our knowledge, this is the first report that oral Campylobacter species are capable of invading human cells. However, invasion by Campylobacter jejuni and other enteropathogenic Campylobacter species has been well-documented (Carvalho et al., 2001). Overall, these findings suggest that the buccal mucosa has the potential to act as a reservoir for all of the named species that are currently recognized as periodontal pathogens. A recent study has identified novel uncultured species and phylotypes that also may be associated with periodontitis (Kumar et al., 2003). It will be important to determine whether those taxa also are members of the buccal intracellular flora.

G. haemolysans and G. adiacens appear to be commensal in the mouth (Kumar et al., 2003). A recent rRNA cloning study indicated that they were relatively common in samples from buccal mucosa (Aas et al., 2005). Our results are the first to show that those species can be invasive. All of the species we studied were typically present as minor components of an intracellular flora that was dominated by streptococci. Streptococcus mitis and Streptococcus oralis are the most prevalent species detected in buccal samples (Mager et al., 2003). A recent study indicated that neither of those species invades in tissue culture (Nobbs, 2002). However, only a single strain of each was used. S. mitis and S. oralis are very closely related to Streptococcus pneumoniae, which can be invasive (Brock et al., 2002). Horizontal gene transfer is believed to occur between those species (Whatmore et al., 2000), so it is possible that the intracellular streptococci we saw were S. mitis or S. oralis strains that had acquired the ability to invade.

Dental biofilm also is diverse, with a high prevalence of streptococci. A wide range of oral bacteria that principally live in biofilm might be capable of invading buccal cells as a means of persisting when they happen to encounter this shedding surface. Since species interaction appears to be widespread in oral biofilms, another alternative could be that non-invasive species gain entrance to cells by forming consortia with invasive species. We recently have shown that F. nucleatum greatly enhances invasion by Streptococcus cristatus in a tissue culture model (Edwards et al., 2005). It is possible that similar collaborations may occur in the mouth. This mode of entry also might explain our detection of species such as E. corrodens, which do not appear to invade in tissue culture.

Heavily invaded buccal cells could contain a more "mature" intracellular community, while more sparsely invaded cells might represent earlier stages of colonization. Alternatively, levels of invasion might differ because buccal cells differ in their susceptibility to bacterial invasion. In either case, it will be important to learn more about the way that epithelial cells respond to simultaneous invasion by multiple bacterial species. That is a current topic of investigation in our laboratory.


   ACKNOWLEDGMENTS
 
This study was supported by USPHS grant DE 14214 from the National Institute of Dental and Craniofacial Research. We thank Yaping Pan for early work on the qPCR primers. The University of Minnesota Biomedical Image Processing Laboratory (Jerry Sedgewick) and the University of Minnesota Supercomputing Institute (Seema Jaisinghani) provided imaging support.

Received May 12, 2005; Last revision July 26, 2005; Accepted August 5, 2005


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE (2005). Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 43 (in press).

Ashimoto A, Chen C, Bakker I, Slots J (1996). Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions. Oral Microbiol Immunol 11:266–273.[Medline]

Brock SC, McGraw PA, Wright PF, Crowe JE Jr (2002). The human polymeric immunoglobulin receptor facilitates invasion of epithelial cells by Streptococcus pneumoniae in a strain-specific and cell type-specific manner. Infect Immun 70:5091–5095.[Abstract/Free Full Text]

Carvalho AC, Ruiz-Palacios GM, Ramos-Cervantes P, Cervantes LE, Jiang X, Pickering LK (2001). Molecular characterization of invasive and noninvasive Campylobacter jejuni and Campylobacter coli isolates. J Clin Microbiol 39:1353–1359.[Abstract/Free Full Text]

Cole JR, Chai B, Farris RJ, Wang Q, Kulam SA, McGarrell DM, et al. (2005). The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis. Nucleic Acids Res 33:D294–D296.[Abstract/Free Full Text]

Dorn BR, Leung KL, Progulske-Fox A (1998). Invasion of human oral epithelial cells by Prevotella intermedia. Infect Immun 66:6054–6057.[Abstract/Free Full Text]

Edwards AM, Grossman TJ, Rudney JD (2005). Fusobacterium nucleatum transports non-invasive Streptococcus cristatus into human epithelial cells. Infect Immun (in press).

Han YW, Shi W, Huang GT, Kinder Haake S, Park NH, Kuramitsu H, et al. (2000). Interactions between periodontal bacteria and human oral epithelial cells: Fusobacterium nucleatum adheres to and invades epithelial cells. Infect Immun 68:3140–3146.[Abstract/Free Full Text]

Kumar PS, Griffen AL, Barton JA, Paster BJ, Moeschberger ML, Leys EJ (2003). New bacterial species associated with chronic periodontitis. J Dent Res 82:338–344.[Abstract/Free Full Text]

Lux R, Miller JN, Park NH, Shi W (2001). Motility and chemotaxis in tissue penetration of oral epithelial cell layers by Treponema denticola. Infect Immun 69:6276–6283.[Abstract/Free Full Text]

Mager DL, Ximenez-Fyvie LA, Haffajee AD, Socransky SS (2003). Distribution of selected bacterial species on intraoral surfaces. J Clin Periodontol 30:644–654.[ISI][Medline]

Maidak BL, Cole JR, Lilburn TG, Parker CT Jr, Saxman PR, Farris RJ, et al. (2001). The RDP-II (Ribosomal Database Project). Nucleic Acids Res 29:173–174.[Abstract/Free Full Text]

Nobbs AH (2002). Interactions of oral streptococci with epithelial cells (dissertation). Bristol, UK: Univ. of Bristol.

Paster BJ, Bartoszyk IM, Dewhirst FE (1998). Identification of oral streptococci using PCR-based, reverse-capture, checkerboard hybridization. Methods Cell Sci 20:223–231.

Qiagen, Inc. (2004). DNeasy® tissue handbook. Valencia, CA, USA: Qiagen, Inc.

Rudney JD, Chen R, Sedgewick GJ (2001). Intracellular Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in buccal epithelial cells collected from human subjects. Infect Immun 69:2700–2707.[Abstract/Free Full Text]

Rudney JD, Chen R, Pan Y (2003a). Endpoint quantitative PCR assays for Bacteroides forsythus, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans. J Periodontal Res 38:465–470.[ISI][Medline]

Rudney JD, Pan Y, Chen R (2003b). Streptococcal diversity in oral biofilms with respect to salivary function. Arch Oral Biol 48:475–493.[Medline]

Rudney JD, Chen R, Sedgewick GJ (2005). Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis are components of a polymicrobial intracellular flora within human buccal cells. J Dent Res 84:59–63.[Abstract/Free Full Text]

Whatmore AM, Efstratiou A, Pickerill AP, Broughton K, Woodard G, Sturgeon D, et al. (2000). Genetic relationships between clinical isolates of Streptococcus pneumoniae, Streptococcus oralis, and Streptococcus mitis: characterization of "atypical" pneumococci and organisms allied to S. mitis harboring S. pneumoniae virulence factor-encoding genes. Infect Immun 68:1374–1382.[Abstract/Free Full Text]

Zee KY, Samaranayake LP, Attström R (2000). An in vivo replica study of microbial colonization in "rapid" and "slow" dental plaque formers. APMIS 108:113–121.[Medline]





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