Antibacterial effects of peracetic acid disinfection assessed by culturability, enzyme activity and flow cytometry

Abstract

Aquaculture water disinfection with around 1 mg/L peracetic acid (PAA) was proved in various studies to effectively impair microbial proliferation and reduce pathogen-caused fish mortality. In the present study, we monitored the antibacterial effect of biweekly water disinfection with 1 mg/L PAA at a local flow-through fish farm for 8 weeks. Results from replicate water samples showed inconsistency of antibacterial efficacy assessed with colony forming units (CFU) of total culturable bacteria and a commercial enzyme activity assay. Single PAA disinfection caused up to 90% reduction of CFU in water. In contrast, the microbial hydrolase activity was hardly affected or even enhanced. Both methods have limitations and can't truly represent viable bacteria. We further monitored the temporal response of two Yersinia ruckeri isolates in suspensions to single PAA disinfection via high-throughput flow cytometry. We found out that the bacterial cell membrane damage appeared not instantly but with a lag phase of at least 2 h post disinfection at 4 °C. The abundance of cell membrane damage, considered as cumulative bacterial mortality, kept rising and maximized at 48 h post disinfection. The survived cells were more likely to enter the viable but non-culturable (VBNC) state at higher PAA concentrations. One isolate was more prone to enter the VBNC state than the other. Neither Y. ruckeri isolate showed measurable reaction with the enzyme substrate of the commercial microbial hydrolase activity assay. Our findings refresh knowledge about the dynamics of bacterial cell mortality post PAA disinfection (probably also similar non-thermal disinfection methods). Aquaculture practice should be aware of the delayed disinfection outcomes and pay attention to the dominance of VBNC state.