The Use of Laboratory and Agent-based Models to Evaluate the Role of Natural Transformation in Biofilms in the Formation and Spread of Antibiotic Resistant Bacteria in Water Systems.

Abstract

Biofilms are aggregates of bacterial cells. Frequency of gene transfer in biofilms may be higher than in corresponding planktonic counterparts due to increased competence. Biofilms, antibiotics and antibiotic resistance determinants all occur in water systems. This dissertation assesses the role of natural transformation in biofilms in the formation and dissemination of antibiotic resistant bacteria in water networks using laboratory and agent-based models. We initially demonstrated detectable transformation frequencies in Acinetobacter baylyi strain AC811 biofilms exposed to varying genomic and donor DNA encoding antibiotic resistance in a once-through flow system replicating environmental conditions in water system pipes. Another set of experiments compared transformation frequencies of AC811 biofilm and planktonic cells and demonstrated that transformation frequencies of planktonic cells were approximately 10-fold higher than frequencies in biofilm cells. qPCR was used to quantify comP gene expression in AC811. Comparison of comP gene expression trends in biofilm and planktonic cells suggests that the observed frequency differences are due to a variation in competence state between biofilm and free-floating cells. These results suggest that the assumption of increased competence of biofilm cells as compared to planktonic cells may not be generalizable across all bacterial species. Development of an agent-based model allowed us to study additional factors that may affect transformation frequency and in a setting that allows visualization of the biofilm structure. We developed an extension to the iDynoMiCs agent-based model and used this extended model to assess the effect of resistance gene burden value on the persistence of resistant bacteria in a biofilm exposed to donor DNA and varying antimicrobial concentrations. Several trends are apparent in simulations results. Bacteria harboring no cost and low cost fitness genes will persist in the absence of selective pressure and increasing antimicrobial concentration in the influent promotes increased resistance expansion within the single-species biofilm. Results suggest that influent antimicrobial concentration can substantially affect the type and frequency of resistance genes circulating in the environment. This model can be a tool to test hypotheses that are difficult to conduct in the laboratory setting and can be used to drive future laboratory studies.