Exploring the use of flow cytometry for understanding the efficacy of disinfection in chlorine contact tanks.

A pilot scale chlorine contact tank (CCT) with flexible baffling was installed at an operational water treatment plant (WTP), taking a direct feed from the outlet of the rapid gravity filters (RGF). For the first time, disinfection efficacy was established by direct microbial monitoring in a continuous reactor using flow cytometry (FCM). Disinfection variables of dose, time, and hydraulic efficiency (short circuiting and dispersion) were explored following characterisation of the reactor's residence time distributions (RTD) by tracer testing. FCM enabled distinction to be made between changes in disinfection reactor design where standard culture-based methods could not. The product of chlorine concentration (C) and residence time (t) correlated well with inactivation of microbes, organisms, with the highest cell reductions (N/N0) reaching <0.025 at Ctx¯ of 20 mg.min/L and above. The influence of reactor geometry on disinfection was best shown from the Ct10. This identified that the initial level of microbial inactivation was higher in unbaffled reactors for low Ct10 values, although the highest levels of inactivation of 0.015 could only be achieved in the baffled reactors, because these conditions enabled the highest Ct10 values to be achieved. Increased levels of disinfection were closely associated with increased formation of the trihalomethane disinfection by-products. The results highlight the importance of well-designed and operated CCT. The improved resolution afforded by FCM provides a tool that can dynamically quantify disinfection processes, enabling options for much better process control.

Thermal and chemical disinfection of water and biofilms: only a temporary effect in regard to the autochthonous bacteria.

Thermal and chemical disinfection of technical water systems not only aim at minimizing the level of undesired microorganisms, but also at preventing excessive biofouling, clogging and interference with diverse technical processes. Typically, treatment has to be repeated in certain time intervals, as the duration of the effect is limited. The transient effect of disinfection was demonstrated in this study applying different treatments to water and biofilms including heat, chlorination, a combination of hydrogen peroxide and peracetic acid and monochloramine. Despite the diverse treatments, the reduction in live bacteria was followed by regrowth in all cases, underlining the universal validity of this phenomenon. The study shows that autochthonous bacteria can reach the concentrations given prior to treatment. The reason is seen in the nutrient concentration that has not changed and that forms the basis for regrowth. Nutrients are released by disinfection from lysed cells or are still fixed in dead biomass that is subsequently scavenged by necrotrophic growth. Treatment cycles therefore only provide a transient reduction of water microbiology if nutrients are not removed. When aiming at greater sustainability of the effect, biocidal treatment has to be equally concerned about nutrient removal by subsequent cleaning procedures as about killing efficiency.

Microbiological examination of water and aerosols from four industrial evaporative cooling systems in regard to risk of Legionella emissions and methodological suggestions for surveillance.

The hygienic risk associated with evaporative cooling systems in Germany is currently only assessed by determining concentrations of Legionella spp. in the corresponding cooling waters. Relevant for the health risk is however the load of Legionella in emitted aerosols. In this work aerosol emissions from four industrial cooling systems (A - D) were analyzed. A microbiological air bioburden factor (MABF) is suggested to be useful to assess the overall microbiological load of emitted air and to judge the efficiency of droplet separation and overall microbiological retention. Whereas the MABF by itself only serves as a technical quality assurance (QA) parameter, the hygienic relevance has to be seen in combination with the assessment of Legionella either contained in the aerosol or in the cooling water. Plate counting of colonies was an appropriate method to quantify Legionella spp. in aerosols given the short time of flight at the chosen sampling locations and resulting low risk of desiccation. qPCR data on the other hand proved more reproducible than the culture approach to quantify Legionella spp. concentrations in cooling water-. The application of qPCR also allowed to assess the relative proportion of Legionella pneumophila within the total pool of Legionella which adds epidemiological relevance to risk assessment. A traffic light system was proposed to guide interpretation of qPCR data. The four industrial systems greatly differed in all measured parameters leading to different associated risks.

Lysis Performance of Bacteriophages with Different Plaque Sizes and Comparison of Lysis Kinetics After Simultaneous and Sequential Phage Addition.

Background: Although bacteriophages see a revival for specifically removing undesired bacteria, there is still much uncertainty about how to achieve the most rapid and long-lasting clearance. Materials and Methods: This study investigated the lysis kinetics of three distinct environmental coliphages, reproducibly forming different plaque sizes (big, medium, and small). Lysis performance by individual phages was compared with the one obtained after simultaneous or sequential addition of all three phages. Kinetics was monitored by density absorbance or by flow cytometry, with the latter having the advantage of providing higher sensitivity. Results: Plaque size happened to correlate with lysis kinetics in liquid suspensions, with phages producing big (phage B), medium (phage M), and small (phage S) plaques showing maximal bacterial clearance under the chosen conditions within ∼6, 12, and 18 h, respectively. Use of a phage cocktail (all three phages added simultaneously) resulted in slower initial lysis compared with the fastest lysing phage with the greatest plaque size alone, but it showed longer efficacy in suppression. When adding phages sequentially, overall lysis kinetics could be influenced by administering phages at different time points. The lowest bacterial concentration after 36 h was obtained when administering phages in the sequence S, M, and B although this combination initially took the longest to achieve bacterial clearance. Conclusions: Results support that timing and order of phage addition can modulate strength and duration of bacterial suppression and, thus, influence the overall success of phage treatment.

Comparing flow cytometry with culture-based methods for microbial monitoring and as a diagnostic tool for assessing drinking water treatment processes.

Flow cytometry (FCM) and the ability to measure both total and intact cell populations through DNA staining methodologies has rapidly gained attention and consideration across the water sector in the past decade. In this study, water quality monitoring was undertaken over three years across 213 drinking water treatment works (WTW) in the Scottish Water region (Total n = 39,340). Samples subject to routine regulatory microbial analysis using culture-based methods were also analysed using FCM. In addition to final treated water, the bacterial content in raw water was measured over a one-year period. Three WTW were studied in further detail using on-site inter-stage sampling and analysis with FCM. It was demonstrated that there was no clear link between FCM data and the coliform samples taken for regulatory monitoring. The disinfectant Ct value (Ct = mg·min/L) was the driving factor in determining final water cell viability and the proportion of intact cells (intact/total cells) and the frequency of coliform detections in the water leaving the WTW. However, the free chlorine residual, without consideration of treatment time, was shown to have little impact on coliform detections or cell counts. Amongst the three treatment trains monitored in detail, the membrane filtration WTW showed the greatest log removal and robustness in terms of final water intact cell counts. Flow cytometry was shown to provide insights into the bacteriological quality of water that adds significant value over and above that provided by traditional bacterial monitoring.

Effect of turbidity on water disinfection by chlorination with the emphasis on humic acids and chalk.

Chlorine is globally the most widely used chemical for water disinfection. Whereas disinfection efficiency is well known to depend on water pH and temperature, the effect of turbidity is less well studied. Although turbidity is measured online in most drinking water works and most countries where regulations exist have set limits of <1 NTU for water leaving the works, the composition of turbidity is typically unknown. Given the heterogeneous nature of substances contributing to turbidity, the aim of this work was to study the effect of selected compounds on chlorination efficacy. The effect of humic acids and chalk on the inactivation of the indicator bacteria Escherichia coli and Enterococcus faecalis was assessed at neutral pH at different turbidity levels using both plate counting and flow cytometry in combination with membrane integrity staining. For humic acids, a turbidity of 1 NTU (corresponding to 2 mg L-1) was identified as a critical threshold, which when exceeded was found to have a negative impact on chlorine disinfection. Chalk, on the other hand, had no measurable impact up to 5 NTU. The observation applied to both bacterial species with identical conclusions from the two diagnostic methods. Results corroborate that different turbidity causing substances affect chlorination efficiency to very different extents with chlorine demand by organic material probably being the most important determinant. In the case of turbidities >1 NTU, turbidity measurement benefits from the consideration of the organic content as mere NTU values do not allow predicting an impact on chlorination efficiency.

Optimization of viability qPCR for selective detection of membrane-intact Legionella pneumophila.

Although a number of viability qPCR assays have been reported to selectively detect signals from membrane-intact Legionella pneumophila, the efficient suppression of amplification of DNA from dead membrane-compromised bacteria remains an ongoing challenge. This research aimed at establishing a new oligonucleotide combination that allows for a better exclusion of dead Legionella pneumophila on basis of the mip gene. Propidium monoazide (PMA) was chosen as viability dye. An oligonucleotide combination for the amplification of a 633 bp sequence was established with 100% specificity for different Legionella pneumophila strains compared with 17 other Legionella species tested. Apart from increasing amplicon length, the study aimed at optimizing dye incubation time and temperature. An incubation temperature of 45 °C for 10 min was found optimal. Dye treatment of heat-killed bacteria in the presence of EDTA improved signal suppression, whereas deoxycholate also affected signals from live intact bacteria. Suppression of signals from heat-treated bacteria was found to be approx. twice as efficient compared to a commercial kit, although the detection sensitivity is superior when targeting short amplicons. With a limit of detection of 10 genome copies per PCR well and a 6-log signal reduction of bacteria killed at 80 °C, the assay appears useful for applications where pathogen numbers are not limiting and where the priority is on the distinction between intact and damaged Legionella pneumophila for the evaluation of hygienic risk and of disinfection efficiency.

Flow cytometry-based evaluation of the bacterial removal efficiency of a blackwater reuse treatment plant and the microbiological changes in the associated non-potable distribution network.

The study evaluated the changes in bacterial numbers across a full-scale membrane bioreactor (MBR) blackwater reuse system. Flow cytometry was used to quantify total and intact bacterial concentrations across the treatment train and during distribution of the recycled water. Membrane passage reduced bacterial numbers by up to 5-log units resulting in coliform-free permeate. A 2-log increase in bacterial cell concentration was subsequently observed after the granular activated carbon unit followed by a reduction in intact cells after chlorination, which corresponds to an overall intact bacteria removal of 3.4-log units. In the distribution network, the proportion of intact cells greatly depended on the free chlorine residual, with decreasing residual enabling regrowth. An initial target of 0.5 mg L-1 free chlorine ensured sufficient suppression of intact cells for up to 14 days (setting the time intervals for system flushes at times of low water usage). Bacterial regrowth was only observed when the free chlorine concentration was below 0.34 mg L-1. Such loss of residual chlorine mainly applied to distant points in the distribution network from the blackwater reuse treatment plant (BRTP). Flushing these network points for 5 min did not substantially reduce cell numbers. At points closer to the BRTP, on the other hand, flushing reduced cell numbers by up to 1.5-log units concomitant with a decreasing proportion of intact cells. Intact cell concentrations did not correlate with DOC, total nitrogen, or soluble reactive phosphate, but it was shown that dead biomass could be efficiently converted into new biomass within seven days.

The absence or presence of a lytic coliphage affects the response of Escherichia coli to heat, chlorine, or UV exposure.

Disinfection aims at maximal inactivation of target organisms and the sustainable suppression of their regrowth. Whereas many disinfection efforts achieve efficient inactivation when the effect is measured directly after treatment, there are questions about the sustainability of this effect. One aspect is that the treated bacteria might recover and regain the ability to grow. In an environmental context, another question is how amenable surviving bacteria are to predation by omnipresent bacteriophages. Provisional data suggested that bacteria when subjected to sublethal heat stress might develop a phage-resistant phenotype. The result made us wonder about the susceptibility to phage-mediated lysis for bacteria exposed to a gradient of chlorine and UV-LED disinfection strengths. Whereas bacteria exposed to low sublethal chlorine doses still underwent phage-mediated lysis, the critical chlorine Ct of 0.5 mg min/L eliminated this susceptibility and induced phage resistance in the cells that survived treatment. In the case of UV, even the smallest tested dose of 2.8 mJ/cm2 abolished phage lysis leading to direct regrowth. Results suggest that bacteria surviving disinfection might have higher environmental survival chances directly after treatment compared to non-treated cells. A reason could possibly lie in their compromised metabolism that is essential for phage replication.

Turbidity composition and the relationship with microbial attachment and UV inactivation efficacy.

Turbidity in water can be caused by a range of different turbidity causing materials (TCM). Here the characteristics and attachment of bacteria to TCMs was assessed and the resultant impact on UV disinfection determined. TCMs represent potential vehicles for bacterial penetration of water treatment barriers, contamination of potable supplies and impact on subsequent human health. The TCMs under investigation were representative of those that may be present in surface and ground waters, both from the source and formed in the treatment process. The TCMs were chalk, Fe (III) hydroxide precipitate, kaolin clay, manganese dioxide and humic acids, at different turbidity levels representative of source waters (0, 0.1, 0.2, 0.4, 1, 2, and 5 NTU). Escherichia coli and Enterococcus faecalis attachment followed the order of Fe(III)>chalk, with little to no attachment seen for MnO2, humic acids and clay. The attachment was postulated to be due to chalk and Fe(III) particles having a more neutral surface charge resulting in elevated aggregation with bacteria compared to other TCMs. The humic acids and Fe(III) were the TCMs which influenced inactivation of E. coli and E. faecalis due to decreasing UV transmittance (UVT) with increasing TCM concentration. The presence of the Fe(III) TCM at 0.2 NTU resulted in the poorest E. coli inactivation, with 2.5 log10 reduction at UV dose of 10mJcm-2 (kd of -0.23cm2mJ-1) compared to a 3.9 log10 reduction in the absence of TCMs. E. faecalis had a greater resistance to UV irradiation than E. coli for all TCMs. Effective disinfection of drinking water is a priority for ensuring high public health standards. Uniform regulations for turbidity levels for waters pre-disinfection by UV light set by regulators may not always be appropriate and efficacy is dependent on the type, as well as the amount, of turbidity present in the water.

Schrödinger's microbes: Tools for distinguishing the living from the dead in microbial ecosystems.

While often obvious for macroscopic organisms, determining whether a microbe is dead or alive is fraught with complications. Fields such as microbial ecology, environmental health, and medical microbiology each determine how best to assess which members of the microbial community are alive, according to their respective scientific and/or regulatory needs. Many of these fields have gone from studying communities on a bulk level to the fine-scale resolution of microbial populations within consortia. For example, advances in nucleic acid sequencing technologies and downstream bioinformatic analyses have allowed for high-resolution insight into microbial community composition and metabolic potential, yet we know very little about whether such community DNA sequences represent viable microorganisms. In this review, we describe a number of techniques, from microscopy- to molecular-based, that have been used to test for viability (live/dead determination) and/or activity in various contexts, including newer techniques that are compatible with or complementary to downstream nucleic acid sequencing. We describe the compatibility of these viability assessments with high-throughput quantification techniques, including flow cytometry and quantitative PCR (qPCR). Although bacterial viability-linked community characterizations are now feasible in many environments and thus are the focus of this critical review, further methods development is needed for complex environmental samples and to more fully capture the diversity of microbes (e.g., eukaryotic microbes and viruses) and metabolic states (e.g., spores) of microbes in natural environments.

The feasibility of improved live-dead distinction in qPCR-based microbial source tracking.

PCR-based microbial source tracking (MST) has become a useful tool to identify dominant sources of fecal pollution in water. The method has previously been successfully combined with viability PCR (using propidium monoazide) allowing the preferential detection of membrane-intact bacteria. This study aimed at further improving the selectivity for intact cells when targeting host-specific markers in Bacteroidales bacteria. One approach was to increase amplicon sizes that had been shown to be useful for other applications of viability PCR. For this purpose, two different amplicon sizes were compared when targeting either the genus of Bacteroidales or subgroups thereof specifically associated with human and ruminant fecal material. When applied to different environmental samples, the proposed proportion of intact cells could drop by up to 38% (for sewage treatment effluent from 64 to 26%) when targeting longer sequences. Furthermore co-incubation of the viability dye with dimethylsulfoxide (DMSO) was found to be beneficial, although this observation is currently still empirical. When examining signal decay of artificially contaminated unfiltered river water over six weeks, the PMA treatment effect was observed from the beginning, but the ratio of intact and damaged cells remained constant over time with signals disappearing at the same rate independent of PMA treatment. In this instance the contribution of other factors to overall signal decay seemed more important than loss of membrane integrity.

When are bacteria dead? A step towards interpreting flow cytometry profiles after chlorine disinfection and membrane integrity staining.

Flow cytometry is increasingly employed by drinking water providers. Its use with appropriate fluorescent stains allows the distinction between intact and membrane-damaged bacteria, which makes it ideally suited for assessment of disinfection efficiency. In contrast to plate counting, the technology allows the visualization of the gradual loss of membrane integrity. Although this sensitivity per se is very positive, it creates the problem of how this detailed viability information compares with binary plate counts where a colony is either formed or not. Guidelines are therefore needed to facilitate interpretation of flow cytometry results and to determine a degree of membrane damage where bacteria can be considered 'dead'. In this study we subjected Escherichia coli and environmental microorganisms in real water to increasing chlorine concentrations. Resulting flow cytometric patterns after membrane integrity staining were compared with culturability and in part with redox activity. For laboratory-grown bacteria, culturability was lost at lower disinfectant concentrations than membrane integrity making the latter a conservative viability parameter. No recovery from chlorine was observed for four days. For real water, loss of membrane integrity had to be much more substantial to completely suppress colony formation, probably due to the heterogenic composition of the natural microbial community with different members having different susceptibilities to the disinfectant.

Impact of Treated Sewage Effluent on the Microbiology of a Small Brook Using Flow Cytometry as a Diagnostic Tool.

Flow cytometry was applied to assess the microbiological impact of treated sewage effluent discharge into a small brook carrying surface runoff water. Increases in dissolved organic carbon and soluble reactive phosphorous were accompanied by increases in counts of intact bacteria by up to eightfold. Effluent ingress furthermore resulted in a pronounced shift of bacterial clusters. Whereas brook water upstream of the discharge point was characterised by a bacterial cluster with low nucleic acid (LNA) content, downstream water showed a shift to bacteria with high nucleic acid (HNA) content. Changes in the LNA/HNA ratio were largely maintained along the course of the brook. Results suggest that the LNA/HNA ratio can under certain conditions serve as an indicator of anthropogenic nutrient impact. Measuring impact on this low trophic level might be more sensitive and straightforward than measuring macroindicators. More evidence will however be required to assess the usefulness of LNA/HNA measurements to assess the ecological nutrient status of natural waters and the impact of nutrient pollution.

PCR-RFLP as a useful tool for diagnosis of invasive mycoses in a healthcare facility in the North of Brazil

Background The incidence of invasive mycoses is increasing worldwide. PCR-RFLP was applied to the identification of 10 reference strains and 90 cultures of agents of invasive mycoses. In addition, the new approach was applied to detect fungal agents in 120 biological samples (blood, cerebrospinal fluid and bone marrow). PCR-RFLP results were compared with the ones obtained with conventional methods (culture, microscopy, and biochemical testing). Results The assays carried out with the reference strains (Candida albicans, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida guilliermondii, Cryptococcus neoformans, Cryptococcus gattii and Histoplasma capsulatum), demonstrated that the RFLP profiles were correctly predicted by the in silico investigation and allowed unequivocal identification of all chosen reference strains. The PCR-RFLP also identified 90 cultures of agents of invasive mycoses correctly, 2.5 times faster than the conventional assays. Evaluating PCR-RFLP with biological samples it was observed that the PCR was found to be 100% accurate and the RFLP profiles allowed the identification of the etiological agents: C. neoformans (n = 3) and C. gattii (n = 1) in CSF samples, H. capsulatum (n = 1) in bone marrow and C. albicans (n = 2) in blood cultures. The detection and identification by PCR-RFLP were found to be between two to ten times faster than the conventional assays. Conclusion The results showed that PCR-RFLP is a valuable tool for the identification of invasive mycoses that can be implemented in hospital laboratories, allowing for a high number of clinical analyses per day.

Antimicrobial properties of magnesium chloride at low pH in the presence of anionic bases.

Magnesium is an element essential for life and is found ubiquitously in all organisms. The different cations play important roles as enzymatic co-factors, as signaling molecules, and in stabilizing cellular components. It is not surprising that magnesium salts in microbiological experiments are typically associated with positive effects. In this study with Listeria monocytogenes as a model organism, we focus however on the usefulness of magnesium (in form of MgCl2) as a stress enhancer. Whereas MgCl2 does not affect bacterial viability at near-neutral pHs, it was found to strongly compromise culturability and redox activity when cell suspensions were exposed to the salt at acidic pH. The principle was confirmed with a number of gram-negative and gram-positive species. The magnesium salt dramatically increased the acidity to a level that was antimicrobial in the presence of anionic bases such as phosphate, lactate, or acetate, but not TRIS. The antimicrobial activity of MgCl2 was much stronger than that of NaCl, KCl, or CaCl2. No effect was observed with MgSO4 or when cells were exposed to MgCl2 in phosphate buffer with a pH ≥ 5. Acid stress was reinforced by an additional, salt-specific effect of MgCl2 on microbial viability that needs further examination. Apart from its implications for surface disinfection, this observation might support the commonly stated therapeutic properties of MgCl2 for the treatment of skin diseases (with healthy skin being an acidic environment), and could contribute to understanding why salt from the Dead Sea, where Mg(2+) and Cl(-) are the most abundant cation/anion, has healing properties in a microbiological context.

Assessing microbiological water quality in drinking water distribution systems with disinfectant residual using flow cytometry.

Flow cytometry (FCM) as a diagnostic tool for enumeration and characterization of microorganisms is rapidly gaining popularity and is increasingly applied in the water industry. In this study we applied the method to obtain a better understanding of total and intact cell concentrations in three different drinking water distribution systems (one using chlorine and two using chloramines as secondary disinfectants). Chloramine tended to result in lower proportions of intact cells than chlorine over a wider residual range, in agreement with existing knowledge that chloramine suppresses regrowth more efficiently. For chlorinated systems, free chlorine concentrations above 0.5 mg L(-1) were found to be associated with relatively low proportions of intact cells, whereas lower disinfectant levels could result in substantially higher percentages of intact cells. The threshold for chlorinated systems is in good agreement with guidelines from the World Health Organization. The fact that the vast majority of samples failing the regulatory coliform standard also showed elevated proportions of intact cells suggests that this parameter might be useful for evaluating risk of failure. Another interesting parameter for judging the microbiological status of water, the biological regrowth potential, greatly varied among different finished waters providing potential help for investment decisions. For its measurement, a simple method was introduced that can easily be performed by water utilities with FCM capability.

Microbiological tap water profile of a medium-sized building and effect of water stagnation.

Whereas microbiological quality of drinking water in water distribution systems is routinely monitored for reasons of legal compliance, microbial numbers in tap water are grossly understudied. Motivated by gross differences in water from private households, we applied in this study flow cytometry as a rapid analytical method to quantify microbial concentrations in water sampled at diverse taps in a medium size research building receiving chlorinated water. Taps differed considerably in frequency of usage and were located in laboratories, bathrooms, and a coffee kitchen. Substantial differences were observed between taps with concentrations (per mL) in the range from 6.29 x 10(3) to 7.74 x 10(5) for total cells and from 1.66 x 10(3) to 4.31 x 10(5) for intact cells. The percentage of intact cells varied between 7% and 96%. Water from taps with very infrequent use showed the highest bacterial numbers and the highest proportions of intact cells. Stagnation tended to increase microbial numbers in water from those taps which were otherwise frequently used. Microbial numbers in other taps that were rarely opened were not affected by stagnation as their water is probably mostly stagnant. For cold water taps, microbial numbers and the percentage of intact cells tended to decline with flushing with the greatest decline for taps used least frequently whereas microbial concentrations in water from hot water taps tended to be somewhat more stable. We conclude that microbiological water quality is mainly determined by building-specific parameters. Tap water profiling can provide valuable insight into plumbing system hygiene and maintenance.

Opportunities and limitations of molecular methods for quantifying microbial compliance parameters in EU bathing waters.

The debate over the suitability of molecular biological methods for the enumeration of regulatory microbial parameters (e.g. Faecal Indicator Organisms [FIOs]) in bathing waters versus the use of traditional culture-based methods is of current interest to regulators and the science community. Culture-based methods require a 24-48hour turn-around time from receipt at the laboratory to reporting, whilst quantitative molecular tools provide a more rapid assay (approximately 2-3h). Traditional culturing methods are therefore often viewed as slow and 'out-dated', although they still deliver an internationally 'accepted' evidence-base. In contrast, molecular tools have the potential for rapid analysis and their operational utility and associated limitations and uncertainties should be assessed in light of their use for regulatory monitoring. Here we report on the recommendations from a series of international workshops, chaired by a UK Working Group (WG) comprised of scientists, regulators, policy makers and other stakeholders, which explored and interrogated both molecular (principally quantitative polymerase chain reaction [qPCR]) and culture-based tools for FIO monitoring under the European Bathing Water Directive. Through detailed analysis of policy implications, regulatory barriers, stakeholder engagement, and the needs of the end-user, the WG identified a series of key concerns that require critical appraisal before a potential shift from culture-based approaches to the employment of molecular biological methods for bathing water regulation could be justified.