A Multi-Purpose Approach to the Mechanisms of Action of Two Biocides (Benzalkonium Chloride and Dibromonitrilopropionamide): Discussion of Pseudomonas fluorescens’ Viability and Death

Abstract

Biocides are widely used in water treatment for microbiological control. The rise of antimicrobial resistance and the need to assure properly managed water systems require a better understanding of the mechanisms of action of biocides and of their impact on cell’s viability as a function of dosage concentrations. The present work addresses these two aspects regarding the biocides benzalkonium chloride (BAC) and dibromonitrilopropionamide (DBNPA)—two biocides commonly found in the water treatment industry. For that, the following parameters were studied: culturability, membrane integrity, metabolic activity, cellular energy, and the structure and morphology of cells. Also, to assess cell’s death, a reliable positive control, consisting of cells killed by autoclave (dead cells), was introduced. The results confirmed that BAC is a lytic biocide and DBNPA a moderate electrophilic one. Furthermore, the comparison between cells exposed to the biocides’ minimum bactericidal concentrations (MBCs) and autoclaved cells revealed that other viability parameters should be taken into consideration as “death indicators.” The present work also shows that only for the concentrations above the MBC the viability indicators reached values statistically similar to the ones observed for the autoclaved cells (considered to be definitively dead). Finally, the importance of considering the biocide mechanism of action in the definition of the viability parameter to use in the viable but non-culturable (VBNC) determination is discussed.

Pseudomonas fluorescens Cells’ Recovery after Exposure to BAC and DBNPA Biocides

“…The definition of MBC is the lowest biocidal concentration able to kill 99.9% of the initial bacterial population [ 24 ]. Therefore, when cells were first exposed to the MBC and higher concentrations [ 23 ], no CFU/mL counts were observed. This means that, for both biocides, cells were able to significantly regrow when stress conditions (biocide effect) were removed, and nutritional requirements were met.…”

“…The work of Barros et al [ 23 ] discussed that BAC, due to its cationic nature, strongly interacts with bacterial cell membranes, inducing small membrane pores that allow PI entrance into the cells. The authors demonstrated that only 30 mg/L of BAC can lead to 100% of PI uptake, even though P. fluorescens remained culturable for concentrations below the MBC.…”

“…In a previous work [ 23 ], the authors studied in detail the mechanism of action of two widely used biocides: benzalkonium chloride (BAC) and dibromonitrilopropionamide (DBNPA), after exposing Pseudomonas fluorescens to different concentrations of such biocides. That work also discussed cells labelled as ‘dead’ based on (i) the assessment of standard indicatorsmethodologies (culturability, membrane integrity, metabolic activity, cellular energy, and transmission electron microscopy (TEM) micrographs); (ii) comparing the results from each indicator to a negative control of cells (without biocidal effect) and a positive control (from autoclaved cells).…”

“…To address these goals, the biocide was removed from the bacterial suspension, and P. fluorescens cells were supplied with a fresh nutrient medium and incubated overnight at optimal growth conditions (agitation and temperature). Then, the same parameters analysed by Barros et al [ 23 ] were evaluated, namely culturability, viability (PI uptake), metabolic activity (resazurin), cellular energy (ATP), and structural and morphological changes (TEM imaging).…”

A fluorescently labelled quaternary ammonium compound (NBD-DDA) to study mode-of-action and resistance mechanisms in bacteria

“…The main target of BAC is thought to be the bacterial cell envelope 2,7 . The general mode of action likely proceeds as follows: the cationic headgroup attaches to anionic sites on the cell surface, which possesses a net negative charge.…”