Disinfection susceptibilities of detached biofilm clusters compared to planktonic cells and biofilms in single species and dual species cultures

Abstract

Detachment of cells and clusters from biofilms is an important process in the dissemination of microorganisms in industrial, environmental, and clinical settings but the disinfection susceptibilities of these cell clusters have not been sufficiently characterized. With the help of fluorescent microscopy and image analysis, naturally detaching cells and clusters from single species and dual species biofilms of Burkholderia cepacia and Pseudomonas aeruginosa grown in biofilm tubing reactors were analyzed for cluster size distributions and compared to the cluster sizes in chemostat cultures. The commonly used oxidizing agents free chlorine, chlorine dioxide and dissolved ozone were used for disinfection experiments and susceptibilities of detached clusters, planktonic cells, and intact biofilms in single species and dual species cultures were determined. Additionally, disinfection rates were calculated for chlorine and chlorine dioxide disinfection for all sample types and species. In experiments with chlorine as the disinfectant, a correlation between cluster sizes and disinfection efficacy was observed for single species only. Samples with the higher percentage of large clusters were more tolerant than samples with fewer large clusters. Chemostat samples and detached clusters from dual species reactors contained lower numbers of large clusters but were equally or less susceptible than their single species counterparts. Biofilms required chlorine doses up to ten times higher than chemostat or detached biofilm cells for total inactivation. Chlorine dioxide disinfection was independent of cluster size so that chemostat cells and detached clusters were similar with respect to log reductions and disinfection rates. Dual species chemostat cells, detached clusters, and biofilms were more tolerant to chlorine dioxide than the single species samples. As with chlorine, biofilms required much higher chlorine dioxide doses for total inactivation. Ozone was very efficient against B. cepacia chemostat cells and detached clusters but failed to inactivate biofilm samples with the concentrations used in this study. In general, detached clusters were more similar to chemostat cells and very different from biofilms with respect to disinfection susceptibilities and disinfection rates suggesting that biofilm-specific physical and physiological protection mechanisms may be lost shortly after the detachment event or may be absent in small clusters.