Bacterial Plating is a Suitable Method for Determining the Effect of Alginate Lyase on Pseudomonas aeruginosa PAO1

Many research efforts have attempted to elucidate the mechanisms by which biofilms confer resistance to antibiotic treatment; this is particularly relevant to opportunistic pathogens, such as Pseudomonas aeruginosa. In previous studies, treatment of P. aeruginosa PAO1 biofilms with alginate lyase was correlated with an increase in cell division based on colony counts observed from plate counting. Given that the alginate cleaving activity of alginate lyase could contribute to artificially increased colony counts, which may be more accurately attributed to better dispersal of biofilm cells, the reliability of using bacterial plating to assay alginate lyase-treated biofilms was investigated. Physically disrupted alginate lyase-treated cells were concurrently treated with a reversible cell division inhibitor, sodium azide, for four hours. Plate counts were taken at regular intervals to monitor changes in cell counts. Alginate lyase-treated cells were observed to enter exponential phase growth sooner than untreated cells, resulting in higher initial plate counts. However, no difference in plate counts was observed between cells treated with alginate lyase and sodium azide, compared to sodium azide alone, thus ruling out enhanced dispersion of biofilms as the reason for increased counts. These results suggest that plate counting is a sufficient method for discerning increased cell division of P. aeruginosa PAO1 biofilms as induced by alginate lyase. Additionally, liberation of cells from the biofilm matrix by this enzyme points to increased growth rate likely due to their release from the biofilm, and subsequently, increased susceptibility to killing by bacteriostatic antibiotics compared to physical disruption.