(316c) Inoculum Concentration As an Independent Variable to Evaluate Biofilm Structure-Function Relationships

Material selection for implanted medical devices is a challenging problem with as many as 70 % of hospital-acquired infections attributed to devices. Ideally, materials would fully integrate into the host with minimal fibrous capsule formation or immune response. However, complete integration means the material will be continuously exposed to any microbes present in host fluids. “Non-fouling” materials that use a variety of mechanisms to resist microbial attachment over the lifetime of the device are promising, but depending on material and cellular properties, the surface coverage profile of any microbes that do adhere could vary greatly from sparse to high coverage. This initial surface density is expected to impact the structure of the biofilm as it forms, which could in turn modulate the biofilm functional properties such as susceptibility to antimicrobial treatments.

Our objective was to develop a simple model system to mimic variations in cell attachment density and evaluate how the initial density relates to biofilm structure and function. Streptococcus mutans (S. mutans) was selected as a representative biofilm-forming bacterium and is a commensal oral bacterium that adheres to tooth and dental materials where it contributes to caries formation. We varied the inoculum concentration of S. mutans over four orders of magnitude and then quantified the resultant biofilm structures at 48 h using biofilm characterization methods based on colorimetric reporters (crystal violet, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), coomassie blue) and fluorescent imaging (Syto9). Changes in the starting concentration of dispersed cells produced initial surface coverage profiles with distinct differences appearing as early as 6 h after inoculation, with higher inoculum concentrations leading to more surface coverage. These cell surface densities led to different biofilm structures, with higher inoculum concentrations yielding sheet-like biofilms with a homogenous appearance and lower concentrations resulting in isolated microcolonies with tower-like structures. These visible differences were maintained over a 72 h culture period. To evaluate the functional impact of the structural profiles, biofilms were subject to antibiotic challenge at various concentrations (erythromycin), and metabolic activity was measured using the MTT assay immediately after antibiotic exposure and after a 24 h recovery period with fresh medium. Based on the potential differences in antibiotic access to the S. mutans due to the different biofilm structures, our hypothesis was that there would be an inoculum concentration-dependent response to antibiotic challenge.

Most measures of the biofilms showed increased biofilm activity and component staining as the inoculum concentration increased. However, the antibiotic challenge did not reflect these changes and presented a more complicated picture. Inoculum concentration (and the corresponding biofilm structure) did not seem related to metabolic activity immediately after the challenge, where even 100 µg/mL erythromycin only somewhat reduced biofilm activity for all biofilms. Interestingly, providing a 24 h recovery period revealed a different response to the antibiotic challenge, with a much greater reduction in biofilm activity at the higher antibiotic concentrations and a potential dependence on inoculum concentration for some antibiotic levels. These results suggest that an immediate MTT assay may not fully represent the biofilm response to the erythromycin treatment.

The key finding from this work is that seeding concentration alone can result in large differences in initial cell attachment on a surface, which leads to differences in biofilm appearance and may affect response to antibiotics. Engineering biofilms via inoculum concentration is a promising approach to modulate biofilm structure and to develop model systems that mimic biofilms that might develop on low-attachment, “non-fouling” surfaces. Moreover, this model system could also be used for broader studies of biofilm structure-function relationships especially in the context of recovery from antibiotic treatment.

Acknowledgements: KP and HL are supported by the NIST National Research Council Postdoctoral Research Associateship Program.