A biofilm model developed to investigate survival and disinfection of Mycobacterium mucogenicum in potable water

Abstract

Water in healthcare environments can be a source for healthcare-associated infections (HAI). However, information on the exposure risk to opportunistic pathogens in potable water distribution systems (PWDS) is lacking. Laboratory studies characterizing the interaction of opportunistic pathogens with biofilms are needed to understand their role in water systems within healthcare facilities. A stable, repeatable, PWDS multi-species biofilm model comprising Sphingomonas paucimobilis, Methylobacterium sp., Delftia acidovorans, and Mycobacterium mucogenicum was developed in the CDC Biofilm Reactor (CBR), reaching 6 log(10) CFU cm(-2) within 6 days. The model was used to investigate the interaction of the opportunistic pathogen M. mucogenicum with the other species, and to determine the efficacy of monochloramine (NH(2)Cl) as a disinfectant against 2-week-old biofilms. Addition of 1 or 2 mg l(-1) NH(2)Cl resulted in the same or an increased log density of viable M. mucogenicum in the biofilm while inactivating some of the Proteobacteria. Although M. mucogenicum preferentially resided in the biofilm, NH(2)Cl exposure caused release of viable M. mucogenicum from the biofilm into the water. Additional studies with this model should determine if sodium hypochlorite has a comparative effect and if other nontuberculous mycobacteria (NTM) respond to NH(2)Cl similarly.

Figure 1.

(a) PWDS biofilm model growth during a 2-week incubation. Total log₁₀ CFU cm⁻² (all four species) is displayed on the line plot. The percentage composition of the four species in biofilms from each sample day is represented by the bar chart (log density of each species divided by the log density of all four species, multiplied by 100). (b) Log₁₀ CFU ml⁻¹ of unattached bacteria in CBR liquid during incubation for 2 weeks; the three Gram-negative species are shown by the black line. Unattached M. mucogenicum was present in CBR erratically, as demonstrated by the grey line. The percentage composition of each species within water samples on each sample day are presented in the bar chart.

Figure 2.

Image of 14-day old multi-species base model PWDS biofilm formed in drinking water on PVC substratum. The image is a compiled z-stack obtained through a 63x objective. Biofilm was visualized after staining with Sybr Green I. Micrometers are labeled along the X- and Y-axis.

Figure 3.

(a) Culturable biofilm after batch NH₂Cl disinfection for 5 h, and (b) 24 h, (c) detached bacteria after batch NH₂Cl disinfection for 5 h, and (d) 24 h. Bars are percentage composition: solid grey (top), Methylobacterium sp.; diagonal stripes, M. mucogenicum; white bar, S. paucimobilis; and solid black bar, D. acidovorans. Line plots, solid square is total log density of culturable biofilm bacteria, and solid circle represents the log density of M. mucogenicum.

Figure 4.

(a) Culturable biofilm during continuous flow of 1 mg l⁻¹ NH₂Cl disinfection for 28 h and (b) culturable detached bacteria from the same experiments. Bars represent the percentage composition of each species, calculated from log densities: solid grey (top), Methylobacterium sp., diagonal stripes, M. mucogenicum; white bar, S. paucimobilis, and solid black bar, D. acidovorans. Line plots, solid square is total log density of culturable biofilm bacteria, and solid circle represents the log density of M. mucogenicum.