Actinomyces naeslundii in initial dental biofilm formation

Abstract

The combined use of confocal laser scanning microscopy (CLSM) and fluorescent in situ hybridization (FISH) offers new opportunities for analysis of the spatial relationships and temporal changes of specific members of the microbiota of intact dental biofilms. The purpose of this study was to analyse the patterns of colonization and population dynamics of Actinomyces naeslundii compared to streptococci and other bacteria during the initial 48 h of biofilm formation in the oral cavity. Biofilms developed on standardized glass slabs mounted in intra-oral appliances worn by ten individuals for 6, 12, 24 and 48 h. The biofilms were subsequently labelled with probes against A. naeslundii (ACT476), streptococci (STR405) or all bacteria (EUB338), and were analysed by CLSM. Labelled bacteria were quantified by stereological tools. The results showed a notable increase in the number of streptococci and A. naeslundii over time, with a tendency towards a slower growth rate for A. naeslundii compared with streptococci. A. naeslundii was located mainly in the inner part of the multilayered biofilm, indicating that it is one of the species that attaches directly to the acquired pellicle. The participation of A. naeslundii in the initial stages of dental biofilm formation may have important ecological consequences. INTRODUCTIONDental biofilm is an archetypal example of a complex biofilm (Costerton et al., 1999;Davies, 2003;DuPont, 1997). Biofilm formation on tooth surfaces follows the same basic rules as biofilm formation elsewhere in nature. Dental biofilms develop readily because of the optimal temperature, the rich nutrient supply in the oral cavity, and the hard non-shedding surface. They are easily accessible for experimentation using intra-oral devices (Auschill et al., 2004;Kilian et al., 1979;Nyvad & Fejerskov, 1987a;Palmer et al., 2003), and therefore dental biofilms can be used to demonstrate colonization phenomena and ecological principles of universal interest. Concurrent with the increasing recognition of the significance of biofilms in infectious diseases, the development of techniques such as confocal laser scanning microscopy (CLSM), fluorescence in situ hybridization (FISH) and immunofluorescence has enabled visualization of bacteria in their natural undisturbed environment. This offers a substantial improvement upon previous microbiological studies of bacteria grown in planktonic settings (Anwar et al., 1992;Davies, 2003).Previous studies of dental biofilm that took advantage of these methods mainly focused on streptococci Dige et al., 2007; Hannig et al., 2007;Palmer et al., 2003) because culture-based studies suggested that this group of bacteria is prominent during the initial stages of biofilm formation on teeth (Li et al., 2004;Nyvad & Kilian, 1987. However, other genera such as Actinomyces are also among the earliest colonizers of dental surfaces and may constitute up to 27 % of the pioneer bacteria (Kilian et al., 1979;Li et al., 2004;Nyvad & Kilian, 1987). Several culturebased studies indicated that Ac...

Biogeography of a human oral microbiome at the micron scale

“…Clean enamel, glass, or hydroxyapatite surfaces in the mouth are initially colonized by a mixed community in which Streptococcus and Actinomyces are prominent (32)(33)(34). Previous models of development and succession in plaque, after initial colonization, assign a central role to Fusobacterium spp.…”

“…It seems likely that Corynebacterium finds attachment sites on the preexisting biofilm consisting of Streptococcus and Actinomyces, which are among the early colonizers (33) and can be found near the base of hedgehog structures. EM examination of the microbial community colonizing removable enamel chips worn inside the mouth showed scattered filamentous cells oriented perpendicularly to the primarily coccus-covered surface at 24 hours and a mixed community of abundant filamentous organisms by 48 hours (39), suggesting that colonization with Corynebacterium may take place around the 24-hour stage in plaque development.…”

l -Arginine Modifies the Exopolysaccharide Matrix and Thwarts Streptococcus mutans Outgrowth within Mixed-Species Oral Biofilms

“…These three strains were selected because S. mutans is a well-established virulent cariogenic bacterium (32). S. gordonii, a pioneer colonizer of dental biofilm, is arginolytic and ADS positive (33), and A. naeslundii is also detected during the early stages of dental biofilm formation (34). Furthermore, S. mutans UA159 and S. gordonii DL1 have been used in multiple laboratories in several in vitro biofilm models, and they are the strain of choice for in vivo (rodent) models of dental caries (35)(36)(37)(38)(39).…”

Subgingival Biofilm Structure

“…While all these studies could only identify the biofilm bacteria based on morphology or cell wall structure (if electron microscopy was used) and differentiate between live and dead organisms using vitality stains, some more recent studies used fluorescent in situ hybridization (FISH) technologies to identify targeted biofilm bacteria at the species level [e.g. [18][19][20][21]. The application of phylogenetic group-or species-specific single cell identification techniques to undisturbed biofilms formed supragingivally in the oral cavity on retrievable surfaces is currently the state of the art, and promises to reveal important new information on supragingival plaque formation and architecture.…”

“…These bacteria are embedded in somewhat fibrous membrane- Electron microscopic views. Reproduced with permission of the American Academy of Periodontology [21,22]. c Stained with a FITC-labeled universal bacterial probe that stains the bacteria green.…”