Comparing the chlorine disinfection of detached biofilm clusters with those of sessile biofilms and planktonic cells in single- and dual-species cultures

Abstract

Although the detachment of cells from biofilms is of fundamental importance to the dissemination of organisms in both public health and clinical settings, the disinfection efficacies of commonly used biocides on detached biofilm particles have not been investigated. Therefore, the question arises whether cells in detached aggregates can be killed with disinfectant concentrations sufficient to inactivate planktonic cells. Burkholderia cepacia and Pseudomonas aeruginosa were grown in standardized laboratory reactors as single species and in coculture. Cluster size distributions in chemostats and biofilm reactor effluent were measured. Chlorine susceptibility was assessed for planktonic cultures, attached biofilm, and particles and cells detached from the biofilm. Disinfection tolerance generally increased with a higher percentage of larger cell clusters in the chemostat and detached biofilm. Samples with a lower percentage of large clusters were more easily disinfected. Thus, disinfection tolerance depended on the cluster size distribution rather than sample type for chemostat and detached biofilm. Intact biofilms were more tolerant to chlorine independent of species. Homogenization of samples led to significantly increased susceptibility in all biofilm samples as well as detached clusters for single-species B. cepacia, B. cepacia in coculture, and P. aeruginosa in coculture. The disinfection efficacy was also dependent on species composition; coculture was advantageous to the survival of both species when grown as a biofilm or as clusters detached from biofilm but, surprisingly, resulted in a lower disinfection tolerance when they were grown as a mixed planktonic culture.

Fig. 1.

Cell cluster size distributions of single and dual species of B. cepacia (FS-3) and P. aeruginosa (RB-8) from chemostat (CS) and tube reactor effluent (TRE) samples.

Fig. 2.

Log₁₀ reduction (LR) comparison among reactors per species after chlorine treatment. Each point represents the mean LR over multiple experiments. Error bars indicate one standard deviation of the LR (n ≥ 3). Significance at 5% is indicated by an asterisk. FS-3, B. cepacia; RB-8, P. aeruginosa.

Fig. 3.

Log₁₀ reduction (LR) comparison among the biofilm sample types after chlorine disinfection. Each point is the mean LR over multiple experiments. Error bars indicate one standard deviation of the LR (n ≥ 3). See Table 1 for all chlorine treatment doses. FS-3, B. cepacia; RB-8, P. aeruginosa.

Fig. 4.

Log₁₀ reduction (LR) comparison among FS-3 (B. cepacia), RB-8 (P. aeruginosa), dual FS-3 (dual-species B. cepacia), and dual RB-8 (dual-species P. aeruginosa) for each reactor (chemostat, tubing reactor effluent, and biofilm [BF]) across multiple representative doses (not all doses are shown). The top of each bar represents the mean LR. Error bars indicate one standard deviation of the LR (n ≥ 3).

Fig. 5.

k_initial values for all species in the chemostat (CS) and the tubing reactor effluent (TRE) and k_end values for all species. Error bars indicate one standard deviation. Note the difference in y axis values.