Induction of Escherichia coli Into a VBNC State by Continuous-Flow UVC and Subsequent Changes in Metabolic Activity at the Single-Cell Level

Abstract

A viable but non-culturable (VBNC) state of bacteria induced by disinfection in water treatment poses serious health risks because of possible resuscitation of VBNC cells during transportation. In this study, a setup using continuous-flow ultraviolet (UVC) irradiation ranging from 0 to 172.2 mJ cm-2 was designed to simulate real-world disinfection in both drinking water (SDW) and reclaimed water (SRW) treatment plants. A systematic investigation of UVC-induced VBNC bacteria, including occurrence, resuscitation, and time-dependent recovery of metabolic activity during post-incubation, was conducted. Different techniques including two new ones of “single cell culture” and D2O-labeled single-cell Raman spectroscopy were employed to gain comprehensive insights into VBNC cells. Heterotrophic plate counts (HPC) and 5-cyano-2,3-ditoyl tetrazolium chloride flow cytometry (CTC-FCM) assay demonstrated that exposure to continuous-flow UVC can induce E. coli into a VBNC state. Membranes integrity and 16S rRNA transcription level of VBNC bacteria were demonstrated to be unaffected by UVC exposure even at a high dose of 172.2 mJ cm-2. Resuscitation of VBNC bacteria was identified in a more accurate way based on “single cell culture.” Finally, time-dependent evolution of metabolic activity of UVC-treated cells during post-incubation was examined by D2O-labeled Raman spectroscopy at a high-resolution of single-cell level. C-D Raman bands resulting from incorporation of D2O-derived D into bacterial biomass were used as a sensitive and quantitative indicator of bacterial metabolic activity. A lower UVC dose, longer post-incubation time, and higher initial number of bacteria were demonstrated to result in a faster recovery of metabolic activity. Heterogeneous metabolic activity and subpopulation with higher metabolic activity were also revealed by single-cell Raman, even for UVC-treated cells losing cultivability. The comprehensive assessment of VBNC bacteria in UVC-disinfected drinking and reclaimed water points out treatment deficiencies of UVC and the necessity to develop more effective strategies to eliminate VBNC cells.

Developing a new class of engineered live bacterial therapeutics to treat human diseases

“…Traditionally, enumeration of LBPs has relied on agar plating techniques to determine Colony Forming Units (CFU) 10 , and while this methodology remains a staple for the field, it may not be the most appropriate metric for all live bacterial therapeutics. For example, cells in a viable but nonculturable (VBNC) state may be unable to divide and form colonies but may nonetheless retain sufficient metabolic activity to perform some engineered functions in situ 87 . In this case, assessment of viability using commercial live/dead stains to detect intact cell membranes may be more appropriate for enumerating cells in the drug product 88 .…”

Virulence Factors in Coagulase-Negative Staphylococci

“…These cells were identified by Xu et al, which showed that these bacterial cells could not grow on routine or selective media [168]. Moreover, VBNC cells were discriminated from dead cells, since they were similar to live cells, containing an intact membrane, an active mRNA transcription, metabolic activity, and respiration [169,170]. At least eighty-five bacterial species have been shown to enter a VBNC state, including foodborne and clinical pathogens [171].…”

Alterations in the Cell Wall of Rhodococcus biphenylivorans Under Norfloxacin Stress

“…Bacteria in the VBNC state still have active respiration. CTC can be reduced by electron transport chains to give off red fluorescence whose intensity reflects the number of viable cells ( Zhang et al, 2018 ). CTC solution was added to 500 μL of R. biphenylivorans TG9 at a final CTC concentration of 1 mM.…”