Abstract
Bacterial vitality after water disinfection treatment was investigated using bio-orthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM). Protein synthesis activity and DNA integrity (BONCAT–SYBR Green) was monitored in Escherichia 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 24 h. 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 growth) 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 cell division. Similarly, analysis of protein synthesis in marine bacteria with BONCAT displayed residual activity despite inability to grow or reproduce. Background controls (time zero blanks) prepared using different fixatives (formaldehyde, isopropanol, and acetic acid) and several different bacterial strains revealed that the BONCAT protocol still resulted in labeled, i.e., apparently active, cells. The reason for this is unclear and needs further investigation to be understood. Our results show that BONCAT and FCM 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 proteins synthesized within cells are analyzed, compared to assays targeting specific elements such as enzyme activity.
Highlights
Determining bacterial physiological states is important for a wide range of applications
The bio-orthogonal non-canonical amino acid tagging (BONCAT) results were compared with other vitality analysis methods [i.e., LIVE/DEADTM BacLightTM (Invitrogen, Thermo Fisher Scientific, United States) and plate counts], and the experimental setup in Figure 1 gives an overview of the different steps
Results from the vitality assessment with BONCAT-flow cytometry (FCM) are shown in Figures 2, 3 and in Supplementary Table 1
Summary
Determining bacterial physiological states is important for a wide range of applications. Hereafter, these physiological states are described as viable cells when bacteria have the capacity to reproduce (i.e., divide) and vital when they present metabolic activity and cell integrity but are unable to reproduce. Processes, and functions necessary for bacterial growth and development. BONCAT-FCM After Water Disinfection are necessary to assess their role in aquatic ecosystems (Czechowska et al, 2008). The determination of effects of antimicrobial treatment is highly relevant to fight resistant bacterial pathogens (Léonard et al, 2016). Analysis of viable microbes in water is necessary to ensure safety and the absence of pathogens. Wastewater, drinking water, aquaculture, or ballast water onboard ships are all the examples where water treatment is required to prevent the spread of pathogens or non-indigenous species
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
