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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.

Image cytometry is the process of converting image data to flow cytometry-style plots, and it usually requires computer-aided surface creation to extract out statistics for cells or structures. One way of dealing with structures stained with multiple markers in three-dimensional images, is carrying out multiple rounds of channel co-localization and image masking before surface creation, which is cumbersome and laborious. We propose the application of the hue-saturation-brightness color space to streamline this process, which produces complete surfaces, and allows the user to have a global view of the data before flexibly defining cell subsets. Spectral compensation can also be performed after surface creation to accurately resolve different signals. We demonstrate the utility of this workflow in static and dynamic imaging datasets of a needlestick injury on the mouse ear, and we believe this scalable and intuitive approach will improve the ease of performing histocytometry on biological samples.