Reviewing the complexities of bacterial biocide susceptibility and in vitro biocide adaptation methodologies

Abstract

Decreased bacterial susceptibility to biocides raises concerns due to their influences on antibiotic resistance. The lack of standardized breakpoints, established methods, and consistent terminology complicates this research. This review summarizes techniques for studying biocide resistance mechanisms, susceptibility testing, and in-vitro adaptation methods, highlighting their benefits and limitations. Here, the challenges in studying biocide susceptibility and the need for standardized approaches in biocide research are emphasized for commonly studied biocide classes.

Fig. 1. A summary of the various techniques used for in vitro biocide adaptation of bacteria.

In all panels, a right-angle triangle represents biocide concentration gradients, where the increasing biocide (blue) intensifies as its concentration increases. Bacterial colonies (panels B–E) are colored in pink for clarity. A The test tube biocide adaptation experiment core design is shown based on previously published methods listed in Table 2. Numbers above Day 1 and Day 2 subcultures represent gradually increasing arithmetic dilutions of a given biocide and red boxes show an example of a culture with the highest measured growth. B Cup plate dilution agar experimental design; black dotted arrows shown on the agar plate after the step 2 represents the zone of biocide growth inhibition. C The gradient agar biocide adaptation method is shown. D The spot gradient biocide agar technique at step 1, requires a ≥1 h time incubation for spotted biocides to adsorb into the agar^,,. E A continuous culture biocide adaptation technique is shown. Continuous culture designs can be machine-automated or operated manually with peristaltic pumps. The duration of the culture incubation period and when to sample adapted isolates are determined by the operator (ranges from days-years). The images shown in this diagram were generated using Inkscape v. 1.4 graphic design tools.