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Experimental Metabolism Studies of Oral Biofilm Communities

Paul Majors and Jeff McLean, Principal Investigator

Time-resolved µ-MRI images and <sup>1</sup>H NMR spectra for a <em>Streptococcus mutans</em> (S. mutans) biofilm
Time-resolved µ-MRI images and 1H NMR spectra for a Streptococcus mutans (S. mutans) biofilm growing on a substrate of brain heart infusion (BHI) medium enhanced with sucrose on a glass surface. By supplementing the BHI medium with sucrose, S. mutans grew vigorously on the glass. This growth was accompanied by a significant increase in carbohydrate resonances, including a distinct spectral signature for anomeric glucans that correlated qualitatively with the increasing biomass.
u-MRI and depth-resolved <sup>1</sup>H NMR for a <em>Streptococcus mutans</em> biofilm growing on a substrate of brain heart infusion (BHI) medium.
µ-MRI and depth-resolved 1H NMR for a Streptococcus mutans biofilm growing on a substrate of brain heart infusion (BHI) medium enhanced with glucose on a hydroxyapatite (HA) surface. HA surfaces are compatible for nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) studies. Click for a larger version.

Most bacteria in nature are attached to solid surfaces and to one another in a protective film of excreted polymers. These so called biofilms are complicated bacterial communities containing scores to hundreds of different bacterial species that are cooperating and/or competing for resources. Biofilm bacteria are physiologically and functionally distinct from free-floating bacteria - these differences play an important role in many environmental and health-related issues. Biofilms can display beneficial or detrimental properties. Beneficial biofilms include symbiotic plant root nodules and wastewater treatment bacteria. Detrimental biofilms include metal-pipe-corroding films and antibiotic-resistant medical infections. Unfortunately, most of what is known about bacterial function has been obtained from free-floating, single-species bacterial studies, and does not accurately represent biofilms. Thus, experimental methods are needed for the functional analysis of mixed-culture biofilms.

In a recent project, PNNL researchers developed novel nuclear magnetic resonance (NMR) and optical microscopy techniques to map metabolism in live, in-situ bacterial films [1,2]. Specifically, integrated optical and NMR microscopy was adapted to provide time- and depth-resolved (approximately 20 micrometer resolution) metabolic information for biofilms grown on small planar microscope slides. The model bacterial system under study was a mono-species biofilm of Shewanella oneidensis strain MR-1, a dissimilatory metal-reducing bacteria that has the potential for remediation of contaminated surface and ground water. These novel methods are equally applicable to other environmental or medical biofilms.

The "Oral Biofilms Project" team is combining NMR and optical microscopy with stable isotope probing (SIP) methodologies as a first step toward characterizing metabolic function in natural, mixed-community biofilms. NMR is being employed to characterize metabolism in pure and mixed-species biofilms comprised of Streptococcus mutans alone or with other bacterial species commonly associated with dental caries. Concurrently, stable isotope probing (SIP) will be used to identify the bacterial types that actively produce organic acids from the common sugars such as glucose.

Previous researchers have identified model dental biofilms containing up to ten species as viable and safe with which to work. These model communities thus provide a logical system for developing techniques to study community function. (Analogous environmental model communities are not available.) Dental-caries disease and associated tooth decay are correlated with a decrease in pH and increase in the proportion of certain species such as Streptococcus mutans in dental plaque (biofilm). The specific contributions of S. mutans and other species to the disease state are largely unknown. Furthermore, little information is available regarding the identities, concentrations, rates of production, and residence times of organic acids that are responsible for decreasing the pH in cariogenic biofilms.

The successful application of these combined approaches to address oral biofilm behavior will pave the way for addressing questions regarding metabolism of other, increasingly complicated microbial communities.

References

1. Majors PD, JS McLean, GE Pinchuk, JK Fredrickson, YA Gorby, KR Minard, and RA Wind. (2005). "NMR methods for in situ biofilm metabolism studies." Journal of Microbiological Methods. 62:337-344.

2. Majors PD, JS McLean, JK Fredrickson, and RA Wind. (2005). "NMR methods for in-situ biofilm metabolism studies: spatial and temporal resolved measurements." Water Science & Technology. 52(7):7-12.

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