Bacterial biofilms are able to persist in drinking water distribution systems (DWDS) even if disinfectants such as monochloramine are used to inhibit bacterial colonization and biofilm formation. While studies have determined the monochloramine concentrations required to inhibit bacterial biofilms, not much is known about how bacterial biofilms develop resistance towards monochloramine. This study covers the development of resistance to monochloramine in both single species and mixed bacterial biofilms. Through culturability tests and flow cytometry, exposing bacterial biofilms to monochloramine disinfection using a sub-lethal concentration (1.5 mg/L Cl2, experimentally determined) was sufficient to cause an increase of the monochloramine's inhibitory concentrations by as much as two times than what is initially required to inhibit biofilm growth. Through persister cultures and 16S rRNA next generation sequencing (NGS) studies, mixed bacterial biofilms experienced to monochloramine exposure resulted in more bacterial genera becoming persistent and resistant towards monochloramine. Through this study, bacterial genera that were persistent towards monochloramine were suggested to share common traits including the ability (1) to readily enter a persister or viable but non-culturable (VBNC) state and (2) to form biofilms primarily comprising proteinaceous extra-polymeric substances (EPS). Both of these traits also suggested that selected bacterial genera tended to be more persistent to monochloramine and produce EPS. This study advances our understanding of bacterial biofilm resistance towards monochloramine and showed the importance of maintaining monochloramine concentrations in DWDS to prevent the development of bacterial resistance towards monochloramine. KEY POINTS: • Monochloramine-resistant biofilm was developed after sub-lethal disinfection. • Mixed-species culture experienced monochloramine showed more persistence to monochloramine. • Ability to enter a persister/VBNC state is a common trait of persistent bacteria genera.
Keywords: Biofilms; Inhibitory concentrations; Mixed bacterial community; Monochloramine.