The occurrence of viable but non-culturable (VBNC) bacteria in drinking water may result in significant underestimation of viable cell counts detected by culture-based method, thus raising microbiological safety concern. Chlorine disinfection has been widely used in drinking water treatment to ensure microbiological safety. However, the effect of residual chlorine on inducing bacteria in biofilms into a VBNC state remains unclear. We determined cell numbers of Pseudomonas fluorescence in different physiological states (culturable, viable, dead) by heterotrophic plate count method and flow cytometer in a flow cell system under 0, 0.1, 0.5, 1.0 mg/L chlorine treatment. Numbers of culturable cells were 4.66 ± 0.47 Log10, 2.82 ± 0.76 Log10, 2.30 ± 1.23 Log10 (CFU/112.5 mm3) in each chlorine treatment group. However, viable cell numbers remained at 6.32 ± 0.05 Log10, 6.11 ± 0.24 Log10, 5.08 ± 0.81 Log10 (cells/112.5 mm3). Significant difference between numbers of viable and culturable cells demonstrated chlorine could induce bacteria in biofilms into a VBNC state. In this study, flow cells combination with Optical Coherence Tomography (OCT) were applied to construct an Automated experimental Platform for replicate Biofilm cultivation and structural Monitoring (APBM) system. The OCT imaging results demonstrated that changes of biofilm structure under chlorine treatment were closely related to their inherent characteristics. Biofilms with low thickness and high roughness coefficient or porosity were easier to be removed from the substratum. Biofilm with high rigid properties were more resistant to chlorine treatment. Even though >95 % bacteria in biofilms entered a VBNC state, the biofilm physical structure was still remained. This study revealed the possibility of bacteria to enter a VBNC state in drinking water biofilms and changes of biofilm structure with different characteristics under chlorine treatment, which provide reference for biofilms control in drinking water distribution systems.

To assess fecal pollution in coastal waters, current monitoring is reliant on culture-based enumeration of bacterial indicators, which does not account for the presence of viable but non-culturable or sediment-associated micro-organisms, preventing effective quantitative microbial risk assessment (QMRA). Seasonal variability in viable but non-culturable or sediment-associated bacteria challenge the use of fecal indicator organisms (FIOs) for water monitoring. We evaluated seasonal changes in FIOs and human enteric pathogen abundance in water and sediments from the Ribble and Conwy estuaries in the UK. Sediments possessed greater bacterial abundance than the overlying water column, however, key pathogenic species (Shigella spp., Campylobacter jejuni, Salmonella spp., hepatitis A virus, hepatitis E virus and norovirus GI and GII) were not detected in sediments. Salmonella was detected in low levels in the Conwy water in spring/summer and norovirus GII was detected in the Ribble water in winter. The abundance of E. coli and Enterococcus spp. quantified by culture-based methods, rarely matched the abundance of these species when measured by qPCR. The discrepancy between these methods was greatest in winter at both estuaries, due to low CFU\'s, coupled with higher gene copies (GC). Temperature accounted for 60% the variability in bacterial abundance in water in autumn, whilst in winter salinity explained 15% of the variance. Relationships between bacterial indicators/pathogens and physicochemical variables were inconsistent in sediments, no single indicator adequately described occurrence of all bacterial indicators/pathogens. However, important variables included grain size, porosity, clay content and concentrations of Zn, K, and Al. Sediments with greater organic matter content and lower porosity harbored a greater proportion of non-culturable bacteria (including dead cells and extracellular DNA) in winter. Here, we show the link between physicochemical variables and season which govern culturability of human enteric pathogens and FIOs. Therefore, knowledge of these factors is critical for accurate microbial risk assessment. Future water quality management strategies could be improved through monitoring sediment-associated bacteria and non-culturable bacteria. This could facilitate source apportionment of human enteric pathogens and FIOs and direct remedial action to improve water quality.

The long term survival of fecal indicator organisms (FIOs) and human pathogenic microorganisms in sediments is important from a water quality, human health and ecological perspective. Typically, both bacteria and viruses strongly associate with particulate matter present in freshwater, estuarine and marine environments. This association tends to be stronger in finer textured sediments and is strongly influenced by the type and quantity of clay minerals and organic matter present. Binding to particle surfaces promotes the persistence of bacteria in the environment by offering physical and chemical protection from biotic and abiotic stresses. How bacterial and viral viability and pathogenicity is influenced by surface attachment requires further study. Typically, long-term association with surfaces including sediments induces bacteria to enter a viable-but-non-culturable (VBNC) state. Inherent methodological challenges of quantifying VBNC bacteria may lead to the frequent under-reporting of their abundance in sediments. The implications of this in a quantitative risk assessment context remain unclear. Similarly, sediments can harbor significant amounts of enteric viruses, however, the factors regulating their persistence remains poorly understood. Quantification of viruses in sediment remains problematic due to our poor ability to recover intact viral particles from sediment surfaces (typically <10%), our inability to distinguish between infective and damaged (non-infective) viral particles, aggregation of viral particles, and inhibition during qPCR. This suggests that the true viral titre in sediments may be being vastly underestimated. In turn, this is limiting our ability to understand the fate and transport of viruses in sediments. Model systems (e.g., human cell culture) are also lacking for some key viruses, preventing our ability to evaluate the infectivity of viruses recovered from sediments (e.g., norovirus). The release of particle-bound bacteria and viruses into the water column during sediment resuspension also represents a risk to water quality. In conclusion, our poor process level understanding of viral/bacterial-sediment interactions combined with methodological challenges is limiting the accurate source apportionment and quantitative microbial risk assessment for pathogenic organisms associated with sediments in aquatic environments.

Salmonellosis, one of the most common food and water-borne diseases, has a major global health and economic impact. Salmonella cells present high infection rates, persistence over inauspicious conditions and the potential to preserve virulence in dormant states when cells are viable but non-culturable (VBNC). These facts are challenging for current detection methods. Culture methods lack the capacity to detect VBNC cells, while biomolecular methods (e.g. DNA- or protein-based) hardly distinguish between dead innocuous cells and their viable lethal counterparts. This work presents and validates a novel bacteriophage (phage)-based microbial detection tool to detect and assess Salmonella viability. Salmonella Enteritidis cells in a VBNC physiological state were evaluated by cell culture, flow-cytometry and epifluorescence microscopy, and further assayed with a biosensor platform. Free PVP-SE1 phages in solution showed the ability to recognize VBNC cells, with no lysis induction, in contrast to the minor recognition of heat-killed cells. This ability was confirmed for immobilized phages on gold surfaces, where the phage detection signal follows the same trend of the concentration of viable plus VBNC cells in the sample. The phage probe was then tested in a magnetoresistive biosensor platform allowing the quantitative detection and discrimination of viable and VBNC cells from dead cells, with high sensitivity. Signals arising from 3 to 4 cells per sensor were recorded. In comparison to a polyclonal antibody that does not distinguish viable from dead cells, the phage selectivity in cell recognition minimizes false-negative and false-positive results often associated with most detection methods.