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Ask microbiologists what the word ‘biofilm’ brings to mind, and many would speak about Pseudomonas aeruginosa, about microtitre plates or flowcells, about ‘mushrooms’ and ‘voids’, and about antibiotic resistance and device-related infections. They would perhaps speak of biofilm growth as development: distinct phases or behaviours such as attachment, spreading and dispersal. They would almost certainly mention quorum sensing or communication, and almost all would use the word ‘community’ or ‘population’. Yet, despite their well-founded enthusiasm and the numerous impressive examples they would cite, the populations to which they typically refer are in fact quite homogeneous; most of what is clearly recognized as biofilm research has been conducted in vitro using single bacterial pure cultures. However, working in relative obscurity and beginning several decades prior to popularization of the word biofilm, oral microbiologists established the paradigm for our understanding of development in real-world biofilm communities: dental plaque. Irene Dige and colleagues continue this tradition in the current issue of Microbiology by describing and quantifying the spatiotemporal population dynamics of Actinomyces naeslundii in early supragingival biofilms (Dige et al., 2009).
Historically, examination of biofilm growth at the level of single cells began in earnest when the elecron microscope became a common biological tool. Microbiologists and ‘cariologists’, especially those in Scandinavian dental schools, used this tool to study the most easily accessible biofilms of the human body: those that form on tooth surfaces. The biofilms were retrieved at sequential time points on small pieces of glass or bovine enamel that had been carried in the oral cavity of a volunteer: typically an eager(?) student. To isolate, count, and identify organisms after removal, the biofilm was scraped off, homogenized, and plated on various media. With the perfect hindsight that the oral cavity is home to some 800 phylotypes, and compounded by the relatively rudimentary speciation techniques at the time, this approach was laborious, duplicative, and often yielded datasets that were difficult to compare from laboratory to laboratory. However, one arrived at the conclusion that a lot of different bugs were present, and one could say that the great majority of those were streptococci, with other bacteria such as actinomyces also present. Carrier pieces were simultaneously examined with the electron microscope. Early on (≤4 h of carrier wear), the biofilm consisted of small aggregates of spherical cells (generally no more than three or four cells) with the occasional non-coccoid morphotype thrown in. As time progressed, the total cell count as well as the size of the cell aggregates increased rapidly, in parallel with species diversity determined by plating. However, the electron microscope provided little information on the spatial arrangement of different bacterial species within the biofilm; cocci are cocci and rods are rods. Were the aggregates clonal, at least early on? Could the later, much bigger aggregates be mixtures of streptococci with any number of other coccoid organisms such as veillonellae? What is the arrangement of species after the aggregates join to confluence? A comb with finer teeth was needed.
The advent of immunofluorescence microscopy and FISH answered some of these questions. Clonality was out and communities were in, at least for the cocci. Dige et al. (2009) confirm an observation from the early days: that a Gram-positive bacterium, thought to be an actinomyces based on plating results, was regularly found at the substratum. The authors use FISH, confocal microscopy and stereological enumeration to expand upon that initial observation. They show that A. naeslundii grows in a quasi-clonal manner, that its numbers increase slowly relative to those of streptococci, and that it occupies pockets within the biofilm that can always be followed to the substratum. Thus, A. naeslundii is not only an early colonizer of the tooth surface, but also demonstrates a patchy, mosaic-like distribution in older biofilms. These observations, together with the lack of single A. naeslundii cells at the top of older biofilms, suggest that the organism gets into the biofilm early through recognition of and adherence to streptococci, but then putters along on its own little patch of real estate. Part of this behaviour may be due to its ability to use the streptococcal fermentation product lactic acid as an energy source. Veillonellae share this ability and are also found intimately associated with streptococci (Palmer et al., 2006). Interestingly, Actinomyces oris, until recently known as A. naeslundii but now reclassified as a unique species (Henssge et al., 2009), does not grow in clumps but prefers to mix itself throughout the streptococcal biomass, at least when grown in saliva in vitro (Palmer et al., 2001). Could these different behaviours between closely related organisms indicate physiological traits which are detectable only when saliva is the nutrient source? Or only when appropriate partner organisms are present? Such questions of the physiological basis for spatial distribution in natural biofilms are being explored in other ecosystems (Boetius et al., 2000), and the paradigm system of dental plaque presents many such opportunities.