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Bacteria Interfere with A. actinomycetemcomitans Colonization
1 Catholic University Leuven, Research Group for Microbial Adhesion, Department of Periodontology, Kapucijnenvoer 7, 3000 Leuven, Belgium; Correspondence: * corresponding author, Wim.Teughels{at}med.kuleuven.be
It is known that beneficial bacteria can suppress the emergence of pathogenic bacteria, particularly in the gastrointestinal tract. This study examined the potential for a similar suppression of Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans colonization of epithelial cells, due to its potential relevance in periodontal diseases. Seven presumed beneficial bacteria were examined for their ability to interfere, exclude, or displace A. actinomycetemcomitans from epithelial cells in vitro. Streptococcus sanguinis, Streptococcus mitis, and Streptococcus salivarius showed prominent inhibitory effects on either A. actinomycetemcomitans recovery or colonization. These results confirmed the hypothesis that bacterial interactions interfere with A. actinomycetemcomitans colonization of epithelial cells in vitro, and demonstrated the potential beneficial effects of S. mitis, S. salivarius, and S. sanguinis.
Key Words: Aggregatibacter actinomycetemcomitans • probiotic • adherence • epithelial cells • polymicrobial
Colonization of host tissues by pathogens is an important step in the development of infectious diseases (Reed and Williams, 1978). The periodontal microbiota consists of more than 500 different bacterial species (Kumar et al., 2005). The emergence of selected pathogens [e.g., Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans (recently re-classified as Aggregatibacter actinomycetemcomitans) (Norskov-Lauritsen and Kilian, 2006), and Tannerella forsythensis] can lead to the development of periodontitis, an infectious disease of the tooth-supporting tissues (Rudney et al., 2005). It is believed that non-pathogenic, or beneficial, bacteria are important for maintaining a healthy subgingival ecosystem (Roberts and Darveau, 2002). In periodontal microbiology, bacteria are considered beneficial when their numbers are high in periodontal health and low in diseased situations. Despite our rapidly increasing knowledge of periodontopathogen–host cell interactions, the role of beneficial bacteria in preventing the emergence of pathogenic species remains obscure. Beneficial bacteria can protect mucosal surfaces from colonization of pathogens (Brook, 1999). Mechanisms such as competition for, and exclusion of, adhesion receptors, displacement, competition for nutrients, and production of antimicrobials or surfactants may be important to the protective effect of beneficial bacteria (Wilson, 2005). It is important to identify beneficial bacteria and characterize the mechanisms underlying their protective effects, to improve our understanding of the ecology of periodontal disease and, ultimately, to develop new treatment strategies. This in vitro study examined presumed beneficial oral bacteria for their ability to interfere with epithelial colonization by the periodontopathogen A. actinomycetemcomitans.
Growth Conditions for Bacteria and Cells Test bacteria, selected for their presumed beneficial effects (Table 1  ), and A. actinomycetemcomitans strain ATCC 43718 were grown as previously described (Van Hoogmoed et al., 2005). Selective media for cultivation of A. actinomycetemcomitans were prepared by the addition of 10 mg/L vancomycin (Sigma, St. Louis, MO, USA), and used as indicated in the text. HeLa cell monolayers were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) at 37°C in 5% CO2, and passaged, when near confluent, to 24-well tissue culture plates as described previously (Teughels et al., 2005). HeLa cells were used because A. actinomycetemcomitans epithelial colonization has been predominantly characterized on KB cells, now considered to be HeLa cells (Ogura et al., 1993). Additionally, A. actinomycetemcomitans gene expression during invasion of HeLa cells mimics what occurs in vivo (Richardson et al., 2005).
Adhesion Assays with Viable Test Bacteria Bacterial cultures were washed 3 times with phosphate-buffered saline (PBS) and re-suspended in DMEM at a concentration of 2 x 108 colony-forming units (CFU)/mL. For competition assays, epithelial cells were incubated with A. actinomycetemcomitans and a test strain together. For exclusion assays, cells were incubated first with a test strain, washed 3 times with PBS, and then incubated with A. actinomycetemcomitans. For displacement assays, cells were incubated first with A. actinomycetemcomitans, washed 3 times with PBS, and then incubated with a test strain. Single-species inoculations performed in parallel were used as controls. In all cases, the ratio of A. actinomycetemcomitans to the test strain was 1:1 (Table 1  ). All incubations were conducted for 2 hrs at 37°C in 5% CO2. The colonization of epithelial cells was determined by a standard bacterial adherence assay (Teughels et al., 2005). After epithelial cell lysis, serial dilutions were plated on blood-agar and incubated for bacterial cultivation. The distinct colonial morphology of A. actinomycetemcomitans in comparison with that of the test bacteria allowed for differentiation between the species. We calculated the total number of CFU/well to determine the cell-associated bacterial load.
Dose-response Effect
Effect of Environmental Conditioning on Bacterial Recovery and Adhesion Competition assays as described above were performed with the conditioned media but without the addition of viable S. mitis, S. sanguinis, and S. salivarius.
Statistical Analysis
Adhesion Assays with Viable Test Bacteria The effect of the test bacteria on the epithelial colonization of A. actinomycetemcomitans was addressed in competition, exclusion, and displacement assays and compared with mono-infection controls (Fig. 1 ). All test species had effects on A. actinomycetemcomitans colonization (Fig. 1A). A. actinomycetemcomitans colonization was increased in the presence of A. naeslundii and decreased in the presence of S. salivarius and S. sanguinis, regardless of the inoculation protocol. The other species showed inoculation-protocol-dependent effects. S. mitis competitively interfered with and displaced A. actinomycetemcomitans. Modest but significant displacement properties were also evident for S. cristatus. In contrast, in displacement assays with F. nucleatum and exclusion assays with H. parainfluenzae, the recovery of A. actinomycetemcomitans was increased.
Epithelial colonization of the test bacteria was also influenced by the inoculation protocol (Fig. 1B ). H. parainfluenzae, S. salivarius, and S. sanguinis showed increased colonization in displacement assays with A. actinomycetemcomitans, whereas the colonization of F. nucleatum was decreased. S. salivarius, S. mitis, and S. cristatus showed an increased colonization in competition assays, whereas the colonization of F. nucleatum was decreased. In exclusion assays, F. nucleatum, H. parainfluenzae, and S. salivarius colonization was increased in contrast to S. mitis. Additional studies, described below, focused on interactions of beneficial bacteria that inhibit A. actinomycetemcomitans colonization.
Dose-response Effect
Effects of Bacterial Interactions in Post-incubation Cultivation The 3 streptococcal species examined induced minor, if any, growth inhibition of A. actinomycetemcomitans in the agar-plate assay, whereas the positive control did inhibit growth (APPENDIX). These results exclude the possibility that the results in Fig. 1A were induced during the post-adhesion-experiment culturing period.
Effect of Environmental Conditioning on Bacterial Recovery
Bacterial-conditioned medium, but not cellular-and-bacterial-conditioned medium, from S. sanguinis and S. mitis increased A. actinomycetemcomitans recovery, whereas S. salivarius-derived conditioned media had no effect. However, lowering the pH of the DMEM to 5.5, 5.0, or 4.6 also resulted in a relatively increased A. actinomycetemcomitans recovery, by 16% ± 18, 35% ± 18 (P < 0.05), and 46% ± 16 (P < 0.05), respectively. Therefore, the effect of each conditioned medium was compared with that of its pH-matched control (DMEM at pH of the conditioned medium), or after the pH of the conditioned medium was increased to the pH of DMEM. S. sanguinis-derived bacterial-conditioned medium increased A. actinomycetemcomitans recovery, whereas it was decreased by cellular-and-bacterial-conditioned medium. Adjustment of the cellular-and-bacterial-conditioned medium pH resulted in an increased recovery. S. mitis-derived bacterial-conditioned medium lowered A. actinomycetemcomitans recovery independent of the pH. S. mitis-derived cellular-and-bacterial-conditioned medium resulted in a similar decrease, but the effect disappeared at pH 7.6. None of the S. salivarius-derived conditioned media showed an effect on A. actinomycetemcomitans recovery.
Effect of Medium Conditioning on Adhesion Significant differences in A. actinomycetemcomitans colonization were found, depending on the origin of the conditioned medium. Bacterial-conditioned medium and cellular-and-bacterial-conditioned medium derived from S. sanguinis induced more pronounced increases than the pH-induced increase (P < 0.05). This effect was stronger at pH 7.6 (P < 0.05). The effect of bacterial-conditioned medium from S. mitis was similar to that of S. sanguinis bacterial-conditioned medium. In contrast, after correction for pH, S. mitis cellular-and-bacterial-conditioned medium decreased A. actinomycetemcomitans colonization at a low pH. At pH 7.6, S. mitis cellular-and-bacterial-conditioned medium increased colonization. Bacterial-conditioned medium and cellular-and-bacterial-conditioned medium from S. salivarius increased A. actinomycetemcomitans colonization. However, after correction for pH, a reduction in A. actinomycetemcomitans colonization by bacterial-conditioned medium and cellular-and-bacterial-conditioned medium was evident at low pH.
It is believed that certain indigenous bacterial species and their products are beneficial for a healthy periodontium (Roberts and Darveau, 2002). For example, in the gastro-intestinal tract, lactobacilli prevent pathogen colonization and infection (Roach and Tannock, 1979). Beneficial species of the indigenous oral microbiota and their role in epithelial colonization of oral pathogens are largely unexplored. This study is the first to describe in vitro interference with epithelial colonization of an oral pathogen. Similar to lactobacilli and enteropathogens (Todoriki et al., 2001), both species- and strain-specific effects on A. actinomycetemcomitans colonization were evident. S. mitis, S. salivarius, and S. sanguinis showed prominent inhibitory properties, whereas A. naeslundii and H. parainfluenzae facilitated A. actinomycetemcomitans colonization. The colonization of the test strains was also affected by the pathogen, indicating that these are two-way interactions, influencing the colonization of both pathogen and test strain. A similar two-way interaction was reported for bifidobacteria and enteropathogens (Candela et al., 2005). This was thought to originate from rapid oxygen consumption by the pathogens, which favors the growth of anaerobic bifidobacteria. The biological basis of our findings remains to be investigated. The complexity inherent in interference studies (e.g., differences in cells and bacterial strains) makes comparisons with different systems difficult (Osset et al., 2001). However, it is interesting that the 3 species with the most prominent colonization-inhibiting properties in this study are effective as effector strains in replacement therapy for ENT infections (Falck et al., 1999; Roos et al., 2001) and caries development (Tanzer et al., 1985). Bacteriocins or bacteriocin-like inhibitory substances, acidification, competition for nutrients, hydrogen peroxide, and lactic acid production are known mechanisms of bacterial interference (Talarico and Dobrogosz, 1989; Sreenivasan et al., 1993; Leriche and Carpentier, 2000). The incidence of bacteriocin-like inhibitory-substance production is relatively high in the Streptococcus genus (Jack and Tagg, 1992), and they produce hydrogen peroxide (Garcia-Mendoza et al., 1993). However, minor, if any, interference with A. actinomycetemcomitans growth was observed on the agar plates in our investigation. Therefore, we conclude that the diminished recovery of A. actinomycetemcomitans is not an artifact of post-incubation growth inhibition. However, agar composition can influence bacteriocin-like inhibitory-substance sensitivity, as shown for mutans streptococci (Balakrishnan et al., 2001). Additionally, our findings do not rule out the possible effects of bacteriocin-like inhibitory substances or hydrogen peroxide production in vivo or in liquid media. The streptococci exerted species-specific interactions with the A. actinomycetemcomitans growth rate in DMEM during the adhesion experiment. Similar species-specificity has been documented for lactobacilli on Gram-negative bacteria (Vignolo et al., 1993). In our experiments, S. salivarius-conditioned media did not interfere, whereas S. mitis-conditioned medium lowered the A. actinomycetemcomitans growth rate. Inhibition of growth rate without a bactericidal activity was previously described for Lactobacillus casei rhamnosus against several human pathogens (Forestier et al., 2001). In contrast, the difference between bacterial-conditioned medium and cellular-and-bacterial-conditioned medium seems to indicate that S. sanguinis can trigger epithelial cells to secrete pH-sensitive factors that lower the A. actinomycetemcomitans growth rate. These conditioned media also changed A. actinomycetemcomitans colonization of epithelial cells, depending on pH and origin. The impact of the conditioned media on colonization outweighed their effect on growth rate. Similarly, a pH effect inhibits Salmonella typhimurium adhesion to Caco-2 monolayers (Lehto and Salminen, 1997). These experiments indicate that S. sanguinis facilitates A. actinomycetemcomitans adhesion by lowering the pH and conditioning DMEM. Similarly, S. mitis induces pH lowering and conditions DMEM so that A. actinomycetemcomitans colonization is enhanced. However, S. mitis triggers epithelial cells to condition DMEM so that it inhibits A. actinomycetemcomitans colonization, especially at a low pH. In contrast, S. salivarius did not trigger the epithelial cells to condition the medium, but conditioned it by itself. Although conditioning of the environment is an attractive hypothesis for the observed interactions, it is important to point out that the effects induced by the conditioned media were considerably less than the effects evident in the dose-response assay. Additionally, they do not explain the effect of S. sanguinis in the competition assays. Probably, steric hindrance, as described for uropathogens by Chan and co-workers (1984), had a minor effect on pathogen colonization, as evidenced by the absence of competition and exclusion with S. cristatus. Therefore, epithelial cell membrane changes induced by a direct bacterial interaction, or the necessity of A. actinomycetemcomitans presence for substantial environmental conditioning, are alternative hypotheses that remain to be explored. Additionally, a direct competition for the same epithelial cell receptor is possible. In conclusion, our findings indicate that selected bacterial interactions interfere with A. actinomycetemcomitans colonization of epithelial cells. S. mitis, S. salivarius, and S. sanguinis have protective properties that interfere with A. actinomycetemcomitans colonization. The nature of these interactions warrants further exploration, and environmental conditioning by epithelial cells or beneficial bacteria appears to contribute to these effects. Our in vitro results highlight the importance of the indigenous microbiota in oral ecology, and suggest that replacement therapy may offer a new therapeutic approach for the prevention of plaque-related periodontal diseases.
This study was supported by NIDCR grant DE015360 (MQ). The authors thank Prof. P. Fives-Taylor of the University of Vermont for critical reading of the manuscript, and Prof. J. Tanzer of the University of Connecticut and Prof. H. Van Der Mei of the University of Groningen for sending bacterial strains. This paper is based on a thesis submitted to the Catholic University of Leuven in partial fulfillment of the requirements for the PhD degree of W. Teughels.
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org. Received for publication September 7, 2006. Revision received February 23, 2007. Accepted for publication March 11, 2007.
Journal of Dental Research, Vol. 86, No. 7,
611-617 (2007) This article has been cited by other articles:
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