Contributions of extracellular polymeric substance to biofilm defense against antibiotic attack

Emily Kaitlin Davenport

This dissertation presents research on biofilm extracellular polymeric substances, or EPS, and its role in defense against antibiotic attack. EPS is a significant component of the biofilm and has many functions to protect and support its viability. The major components of EPS are polysaccharides, proteins, and DNA. Each component can serve different functions in protecting the biofilm against antibiotics. This work demonstrates that EPS protection can vary depending on the producing bacterial strain. It was observed that EPS from Gram-negative biofilms protects cell cultures from aminoglycosides, unlike EPS from Gram-positive strains. The magnitude of this protection was determined for nine antibiotics and five bacteria. Some EPS also displayed protection against β-lactams. This protection was dependent on the mechanism of protection from the cell. EPS containing β-lactamase enzymes showed the highest protection from β-lactams (protection not diminished at 3.2×minimum inhibitory concentration (MIC)), while EPS containing mostly binding proteins showed a lower protection (2.8×MIC). It was concluded that bacterial resistance to an antibiotic does not necessarily translate to protection by EPS. However, EPS without antibiotic-specific protective properties may still protect the viability of the biofilm by non-specific mechanisms. In 24h E. faecalis biofilms, incubation with 0.5×MIC ciprofloxacin resulted in increased biofilm accumulation (2.1-fold, P=0.004), significant aggregation (CLSM, P<0.02), and an increase in many extracellular proteins (LC/MS/MS). The increased abundance of proteins involved in reducing oxidative stress, DNA repair, and growth under nutrient-limited conditions gives an indication of the survival mechanisms at work. Biofilms incubated with both proteinase K and ciprofloxacin during growth did not exhibit these characteristics, and instead, resembled control biofilms. The increase of biomass and the aggregation of cells in treated biofilms make them more likely to be resistant to further antimicrobial attack than the control biofilm. It was observed that treated biofilms retained significantly more of their viability (up to 30%) after a subsequent 6-hour antibiotic treatment (P=0.005). This adverse response from biofilm cells was triggered by sub-inhibitory levels of antibiotic. The ability to predict adverse reaction and the magnitude of EPS protection against an antibiotic can provide guidance on the selection of antibiotics for biofilm treatment.

Contributions of extracellular polymeric substance to biofilm defense against antibiotic attack

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