Table_1.DOCX

Abstract

Bacterial vitality after water disinfection treatment was investigated using Bioorthogonal Non-canonical Amino Acid Tagging (BONCAT) and flow cytometry. Protein synthesis activity and DNA integrity (BONCAT-SYBR Green) was monitored in E. coli monocultures and in natural marine samples after UV irradiation (from 25 to 200 mJ/cm2) and heat treatment (from 15 to 45 min at 55°C). UV irradiation of E. coli caused DNA degradation followed by the decrease in protein synthesis within a period of 24h. Heat treatment affected both DNA integrity and protein synthesis immediately, with an increased effect over time. Results from the BONCAT method were compared with results from well-known methods such as plate counts (focusing on reproduction) and LIVE/DEAD BacLight (focusing on membrane permeability). The methods differed somewhat with respect to vitality levels detected in bacteria after the treatments, but the results were complementary and revealed that cells maintained metabolic activity and membrane integrity despite loss of replication ability. Similarly, analysis of protein synthesis in marine bacteria with BONCAT displayed residual activity despite inability to grow/reproduce. Background controls (time zero blanks) prepared using different fixatives (formaldehyde, isopropanol, acetic acid) and several different bacterial strains revealed that the BONCAT protocol still resulted in labelled, i.e. apparently active, cells. The reason for this is unclear and needs further investigation to be understood. Our results show that BONCAT and flow cytometry can detect, enumerate and differentiate bacterial cells after physical water treatments such as UV irradiation and heating. The method is reliable to enumerate and explore vitality of single cells, and a great advantage with BONCAT is that all protein synthesized within cells are analysed, compared to assays targeting specific elements like enzyme activity.