The suitability of traditional microbial indicators (i.e., Escherichia coli and enterococci) has been challenged due to the lack of correlation with pathogens and evidence of possible regrowth in the natural environment. In this study, the relationships between alternative microbial indicators of potential human fecal contamination (Bacteroides thetaiotaomicron, Methanobrevibacter smithii, human polyomaviruses [HPyVs], and F؉ and somatic coliphages) and pathogens (Salmonella spp., Pseudomonas aeruginosa, rotavirus, astrovirus, norovirus GI, norovirus GII, and adenovirus) were compared with those of traditional microbial indicators, as well as environmental parameters (temperature, conductivity, salinity, pH, dissolved oxygen, total organic carbon, total suspended solids, turbidity, total nitrogen, and total phosphorus). Water samples were collected from surface waters of urban catchments in Singapore. Salmonella and P. aeruginosa had significant positive correlations with most of the microbial indicators, especially E. coli and enterococci. Norovirus GII showed moderately strong positive correlations with most of the microbial indicators, except for HPyVs and coliphages. In general, high geometric means and significant correlations between human-specific markers and pathogens suggest the possibility of sewage contamination in some areas. The simultaneous detection of human-specific markers (i.e., B. thetaiotaomicron, M. smithii, and HPyVs) with E. coli and enterococcus supports the likelihood of recent fecal contamination, since the human-specific markers are unable to regrow in natural surface waters. Multiple-linear-regression results further confirm that the inclusion of M. smithii and HPyVs, together with traditional indicators, would better predict the occurrence of pathogens. Further study is needed to determine the applicability of such models to different geographical locations and environmental conditions. Indicator bacteria, such as Escherichia coli and enterococci (ENT), have been used extensively in past decades to indicate the presence of human enteric pathogens because of the great diversity of pathogens, the large volumes required, and the difficulty in concentrating the typically low concentrations of pathogens from water samples. In Singapore, microbial water quality guidelines for recreational freshwater currently target enterococci (less than 200 counts per 100 ml for 95% of the time).Despite the wide use of fecal indicator bacteria in microbial water quality monitoring, their suitability to represent a level of health risk is still being questioned. One of the reasons is the lack of data correlating the presence and concentrations of the indicators with the presence of disease-associated pathogens. With improvements in microbiological test methods and bacterial species identification, there is growing evidence that fecal indicator bacteria, including E. coli and ENT, do not necessarily correlate well with some of the enteric pathogens, particularly viruses (1). There is also evidence that E. ...

Assessment of physiological states of individual bacterial cells can be useful in the monitoring of the biotechnological processes. Physiological heterogeneity of Escherichia coli population by respiration activity and membrane potential during growth and starvation in batch cultures was evaluated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and bis-(1,3-dibutylbarbituric acid) trimethine oxo (DiBAC₄)(3) fluorescent probes in combination with flow cytometry. The shares of CTC-reducing cells (CTC(+)-cells) and cells with positively charged outside cytoplasmic membrane which were not stained by DiBAC₄(3) (ΔΨ(+)-cells), were 90% and 95% in the exponential phase of batch culture, respectively. After short-term starvation for 10 h, the shares of CTC(+)-cells and ΔΨ(+)-cells in the samples taken from the exponential phase dropped to 78% and 72%, respectively. After long-term starvation for 40 days, the share of CTC(+)-cells dropped to 5%, whereas the share of ΔΨ(+)-cells was about 50%. The conclusions from this research are as follows: (a) the physiological heterogeneity of bacterial population increased after starvation; (b) the cell respiratory activity is more sensitive to starvation than the cell membrane potential; (c) a probe for the cell membrane potential DiBAC₄(3) is more suitable than a probe for the cell respiratory activity CTC in the detection of viable indicator bacteria in environment.

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It is considered that all growing cells, for exception of acidophilic bacteria, have negatively charged inside cytoplasmic membrane (Δψ⁻-cells). Here we show that growing populations of microbial cells contain a small portion of cells with positively charged inside cytoplasmic membrane (Δψ⁺-cells). These cells were detected after simultaneous application of the fluorescent probes for positive membrane potential (anionic dye DIBAC⁻) and membrane integrity (propidium iodide, PI). We found in exponentially growing cell populations of Escherichia coli and Saccharomyces cerevisiae that the content of live Δψ⁻-cells was 93.6 ± 1.8 % for bacteria and 90.4 ± 4.0 % for yeasts and the content of live Δψ⁺-cells was 0.9 ± 0.3 % for bacteria and 2.4 ± 0.7 % for yeasts. Hypothetically, existence of Δψ⁺-cells could be due to short-term, about 1 min for bacteria and 5 min for yeasts, change of membrane potential from negative to positive value during the cell cycle. This change has been shown by the reversions of K⁺, Na⁺, and Ca²⁺ ions fluxes across the cell membrane during synchronous yeast culture. The transformation of Δψ(⁻-cells to Δψ⁺-cells can be explained by slow influx of K⁺ ions into Δψ⁻-cell to the trigger level of K⁺ concentration ("compression of potassium spring"), which is forming "alternative" Δψ⁺-cell for a short period, following with fast efflux of K⁺ ions out of Δψ⁺-cell ("release of potassium spring") returning cell to normal Δψ⁻ state. We anticipate our results to be a starting point to reveal the biological role of cell dualism in form of Δψ⁻- and Δψ⁺- cells.