Self-repair and resuscitation of viable injured bacteria in chlorinated drinking water: Achromobacter as an example

Chlorine disinfection for the treatment of drinking water can cause injury to the membrane and DNA of bacterial cells and may induce the surviving injured bacteria into a viable but non-culturable (VBNC) state. It is difficult to monitor viable injured bacteria by heterotrophic plate counting (HPC), and their presence is also easily miscalculated in flow cytometry intact cell counting (FCM-ICC). Viable injured bacteria have a potential risk of resuscitation in drinking water distribution systems (DWDSs) and pose a threat to public health when drinking from faucets. In this study, bacteria with injured membranes were isolated from chlorinated drinking water by FCM cell sorting. The culture rate of injured bacteria varied from 0.08% to 2.6% on agar plates and 0.39% to 6.5% in 96-well plates. As the dominant genus among the five identified genera, as well as an opportunistic pathogen with multiple antibiotic resistance, Achromobacter was selected and further studied. After treatment with chlorine at a concentration of 1.2 mg/L, Achromobacter entered into the intermediate injured state on the FCM plot, and the injury on the bacterial surface was observed by electron microscopy. However, the CTC respiratory activity assay showed that 75.0% of the bacteria were still physiologically active, and they entered into a VBNC state. The injured VBNC Achromobacter in sterile drinking water were resuscitated after approximately 25 h. The cellular repair behavior of injured bacteria was studied by Fourier transform infrared attenuated total reflectance (FTIR-ATR) and comet assays. It was found that DNA injury rather than membrane injury was repaired first. The expression of Ku and ligD increased significantly during the DNA repair period, indicating that non-homologous end-joining (NHEJ) played an important role in repairing DNA double-strand breaks. This study deepened the understanding of the effect of chlorine disinfection on bacterial viability in drinking water and will provide support for the improvement of the chlorine disinfection process for the treatment of drinking water.