Occurrence and levels of indicator bacteriophages in bathing waters throughout Europe

Somatic coliphages, F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, Escherichia coli and enterococci were counted in bathing waters in the late spring and summer. We tested fresh and marine bathing waters from North, South, East and West Europe expected to contain between 100 and 500 E. coli per 100 ml, although wider ranges were sometimes found. Bacteriophages were counted after concentration, since a preliminary study proved that this step was necessary to obtain positive counts. During monitoring, a first-line quality control with reference materials for bacteria and bacteriophages was performed by all the laboratories participating in the study. The same microbes were also counted in raw sewage samples from various areas in Europe, where the bacterial indicators and the three groups of bacteriophages were detected in roughly the same numbers. All groups of bacteriophages were detected in both fresh and marine bathing waters throughout Europe. Reliable and complete results from 147 samples showed that for log-transformed values, E. coli and bacteriophages were slightly correlated. However, the slope of the regression line changed according to E. coli concentration and the correlation diminished when this concentration was close to zero per 100 ml. The ratios between E. coli and phages in bathing waters differed significantly from those in sewage. The relative amounts of bacteriophages, mainly somatic coliphages and phages infecting Bact. fragilis RYC2056, increased in bathing waters with low E. coli concentration, especially in seawater samples containing <100 E. coli per 100 ml. The relationship of bacteriophages with respect to enterococci paralleled that of bacteriophages with respect to E. coli. Somatic coliphages and bacteriophages infecting Bact. fragilis are useful to predict the presence of some pathogens with the same origin as present bacterial indicators but with higher survival rates.     

Concentrations of coprostanol that correspond to existing bacterial indicator guideline limits

Coprostanol is a faecal sterol that has been proposed as an alternative measure of faecal pollution. While the technique has been used successfully to trace sewage-derived organic matter in a range of environments, it has not been embraced for use as a water quality indicator. This is mostly because of a lack of epidemiological evidence relating coprostanol abundance to any health risk. However, there is a valuable reason why the concentration of coprostanol should be related as quantitatively as possible to the abundance of bacterial indicators currently used to measure faecal pollution. The measurement of coprostanol (and concurrently other faecal sterols) offers many diagnostic and quantitative advantages over traditional techniques for detecting human sewage pollution versus faecal contamination from animal sources. Knowing the amount of coprostanol expected given a certain amount of human sewage pollution would provide a measure against which water managers could quantitatively assess faecal pollution as a whole and relate that assessment to variables with which they are more familiar. This study determines the relationships between coprostanol concentrations and indicator bacterial counts and synthesises the results from several environments to propose coprostanol concentrations broadly equivalent to existing bacterial standards. Our data suggest that 60 and 400 ng L⁻¹ of coprostanol correspond to currently defined primary and secondary contact limits for bacteria measured as thermotolerant coliforms (commonly referred to as faecal coliforms) or enterococci.     

Efficiency of removal of coliforms, faecal coliforms and coliphages in the tubli sewage treatment plant, bahrain

Total coliforms, faecal coliforms and coliphages in the raw sewage were detected in high numbers during the months averaging a maximum temperature of 20°C. The secondary and tertiary effluents show a 99–100% removal of the three indicators of faecal pollution. A direct correlation between the faecal coliforms and coliphages suggests that coliphages alone may be used as an indicator for the presence of pathogenic bacteria in contaminated waters.     

Occurrence and densities of fungi from northern Greek coastal bathing waters and their relation with faecal pollution indicators

The objective of this study was to determine the frequency and densities of yeasts and filamentous fungi in coastal water samples as well as their correlation with the indicator bacteria of faecal pollution. The prevalence of fungi was investigated in parallel with the standard pollution indicator microorganisms in 197 marine water samples from six northern Greek prefectures during the bathing season May–October 1999. Filamentous fungi were isolated from all the examined samples and yeasts from 29 (14.7%) of them; among the positive samples, their mean counts were 90.9 and 38.4 cfu/100 ml, respectively. A total of 23 genera of filamentous fungi and four genera of yeasts were identified. Prevailing genera of filamentous fungi were Penicillium, Aspergillus and Alternaria spp., whereas Candida spp. was the most frequently isolated yeast. Counts of yeasts were significantly (p<0.01) correlated with those of total and faecal coliforms, whereas no correlation was found between filamentous fungi and the indicator bacteria of faecal pollution. Significantly higher counts of total and faecal coliforms (p<0.05), and enterococci (p<0.001), were found during the months with the higher water temperatures and bather numbers. In the six prefectures, significant differences were observed in the counts of filamentous fungi and yeasts as well as in the counts of all the faecal pollution indicators. The results of this study indicate that coastal water can be a path for contamination of swimmers with yeasts and filamentous fungi and that the pollution indicator microorganisms cannot always predict their presence.     

A simple test for differentiation between E. coli and A. aerogenes

An important problem in water quality control is the ability to differentiate between E. coli (faecal) and A. aerogenes (non-faecal) coliforms. To perform this differentiation, a simple test, based on a modification of the selective mFC medium and optimal incubation temperature (37°C) is proposed. Under these specific conditions E. coli bacteria develop into dark blue colonies, whereas A. aerogenes into pink ones. The intermediate coliform bacteria appear greenish-blue or pink, depending on their relation (closeness) to E. coli or A. aerogenes.     

Fates of bacteriophages and bacterial indicators in the Moselle river (France)

It has been suggested that bacteriophages can provide useful information about the pathogenic microorganisms, particularly enteric viruses, present in water. This information is complementary to that obtained from bacterial indicators of faecal contamination, which would be of great value for evaluating the risks associated with the use of certain types of water. Before bacteriophages can be used as indicators of faecal contamination, we need to confirm that bacteriophages give a different response to that given by the well-known bacteria indicators and to determine what happens to bacteriophages in river water. Indeed, drinking water is often produced from river water, either by natural filtration through the soil or after undergoing various treatments. We collected 96 river water samples from six different sites between February and November 2000. The samples were analysed for three faecal indicator bacteria (thermotolerant coliforms, enterococci and spores of sulphite-reducing anaerobes) and three types of bacteriophages (somatic coliphages, F-specific phages and Bacteroides fragilis phages). The densities of thermotolerant coliforms and enterococci depended mainly on physical factors such as flow rate and water temperature. High temperature and low flow rate led to a decrease in the density of these microorganisms, especially in the absence of a major input of faecal pollution. Conversely, the densities of somatic coliphages, F-specific phages and spores of sulphite-reducing anaerobes remained constant regardless of the flow rate and temperature. The density of Bacteroides fragilis phages was too low for unambiguous determination of their fate in river water.     

Chemometric approach to validating faecal sterols as source tracer for faecal contamination in water

Faecal sterols detection is a promising method for identifying sources of faecal pollution. In this study, faecal contamination in water samples from point source (sewage treatment plants, chicken farms, quail farms and horse stables) was extracted using the solid phase extraction (SPE) technique. Faecal sterols (coprostanol, cholesterol, stigmasterol, β-sitosterol and stigmastanol) were selected as parameters to differentiate the source of faecal pollution. The results indicated that coprostanol, cholesterol and β-sitosterol were the most significant parameters that can be used as source tracers for faecal contamination. Chemometric techniques, such as cluster analysis, principal component analysis and discriminant analysis were applied to the data set on faecal contamination in water from various pollution sources in order to validate the faecal sterols’ profiles. Cluster analysis generated three clusters: coprostanol was in cluster 1, cholesterol and β-sitosterol formed cluster 2, while cluster 3 contained stigmasterol and stigmastanol. Discriminant analysis suggested that coprostanol, cholesterol and β-sitosterol were the most significant parameters to discriminate between the faecal pollution source. The use of chemometric techniques provides useful and promising indicators in tracing the source of faecal contamination.     

基于主成分分析的松花江流域黑龙江段水质评价

对松花江流域黑龙江控制区段13个监测断面不同水期的水质指标进行主成分分析,以鉴别主要的污染源,进而提出有针对性的水污染控制对策。结果表明,BOD5、COD、TP、TN为主要污染指标,合计贡献率为49. 529%,首要污染源为生活污水与农业废水。第二大污染指标主要为石油类、阴离子表面活性剂、氰化物,主要来源于工业废水。总污染最严重的主要是阿什河口内平水期与龙安桥平水期。龙安桥平水期等对应的河流区段受生活污水及农业废水污染较为严重,苗家平水期等对应的河流区段受工业污染较为严重。     

Rates of experimental microbiological contamination of fish exposed to polluted water

Fish inhabiting fecally polluted bodies of water are often used for human consumption. Such fish can be contaminated by enteric human pathogens and may pose a potential risk to public health. Controlled experiments with 132 fish of 100 g average weight were conducted to evaluate the rate of contamination of various tissues of fish (tilapia hybrids). The fish were exposed to E. coli introduced into the ambient water at concentrations of up to 10⁶ cfu/ml. Additional experiments were conducted with diluted wastewater containing Aeromonas, enterococci, fecal coliform and F+ coliphages. In another experiment poliovirus I was also added. The highest bacterial concentrations were recovered from the digestive tract (DT), some 5–24 h following exposure, with DT levels essentially similar to those in the inoculated water. In the E. coli experiments, geometric mean levels of about 10² cfu/cm² were recovered from the skin, 26 cfu/g in the spleen and 10² cfu/g in the liver. Most of the muscle samples were not contaminated. Greater contamination was not found under conditions of stress such as high organic load, a water temperature of 37°C or low levels of dissolved oxygen.     

Comparative survival and injury of Candida albicans and bacterial indicator organisms in streams receiving acid mine drainage

Microbiological studies were conducted to assess the survival and injury characteristics of Candida albicans and indicator bacteria in streams impacted by acid mine water (AMW) and organic wastes. Persistence of pure cultures of C. albicans in three AMW-polluted streams was studied in situ using environmental membrane diffusion chambers. Survival of the fungus (at least 3 days in AMW) indicated prolonged tolerance to acid conditions. In comparison, Escherichia coli was killed within several hours of acid stress. Persistence studies also demonstrated that C. albicans was less sensitive to seasonal water temperature fluctuations than E. coli or Streptococcus faecium. In addition to its prolonged survival, C. albicans incurred minimal sublethal injury whereas identical conditions of exposure resulted in significant injury to traditional indicator bacteria. The failure of standard microbiological procedures to detect AMW-damaged bacteria compromises the accuracy of public health safety determinations in these waters. On the other hand, the ability of C. albicans to survive in AMW streams with marked resistance to injury suggests its potential as a favorable alternative sanitary indicator organism for such environments.     

North and south american studies on the potential of coliphage as a water quality indicator

Studies were undertaken to assess the potential of coliphages to be used universally as water quality indicators and more specifically as health hazard indicators. Data were obtained from three water bodies, a northern Canadian River, inshore water samples from Lake Ontario and from marine beaches in Brazil. Data from this two continent, three water body study indicated (a) that within location fecal coliform and coliphages are positively correlated, (b) coliphage values can be predicted by using fecal coliform MPN, fecal streptococci MF and E. coli MF data and (c) a water quality guideline of 20 coliphage/100 ml for recreational fresh waters is proposed.     

Survival of infectious Poliovirus-1 in river water compared to the persistence of somatic coliphages, thermotolerant coliforms and Poliovirus-1 genome

The microbiological quality of water is currently assessed by search for fecal bacteria indicators. There is, however, a body of knowledge demonstrating that bacterial indicators are less resistant to environmental factors than human pathogenic viruses and therefore underestimate the viral risk. As river water is often used as a resource for drinking water production, it is particularly important to obtain a valid estimation of the health hazard, including specific viral risk. This work was conducted to compare the survival of infectious Poliovirus-1 used as a pathogenic virus model to the persistence of, on the one hand, thermotolerant coliforms commonly used as indicators and on the other hand, to somatic coliphages and Poliovirus-1 genome considered as potential indicators. We studied the behavior of infectious Poliovirus-1 and the three (potential) indicators of viral contamination in river water at three different temperatures (4 degrees C,18 degrees C and 25 degrees C). This experiment was performed twice with river water sampled at two different periods, once in winter and once in summer. Our results showed that the survival of thermotolerant coliforms can be 1.5-fold lower than infectious Poliovirus-1. In contrast, under all our experimental conditions, somatic coliphages and Poliovirus-1 genome persisted longer than infectious Poliovirus-1, surviving, respectively, 2-6-fold and about 2-fold longer than infectious Poliovirus-1. According to our results exclusively based on survival capacity, somatic coliphages and viral genome, unlike thermotolerant coliforms appear to be better indicators of viral contamination in river water. Moreover, the disappearance of viral genome is well-correlated to that one of infectious virus irrespective of the conditions tested.     

Evaluating the importance of faecal sources in human-impacted waters

Quantitative microbial risk assessment (QMRA) was used to evaluate the relative contribution of faecal indicators and pathogens when a mixture of human sources impacts a recreational waterbody. The waterbody was assumed to be impacted with a mixture of secondary-treated disinfected municipal wastewater and untreated (or poorly treated) sewage, using Norovirus as the reference pathogen and enterococci as the reference faecal indicator. The contribution made by each source to the total waterbody volume, indicator density, pathogen density, and illness risk was estimated for a number of scenarios that accounted for pathogen and indicator inactivation based on the age of the effluent (source-to-receptor), possible sedimentation of microorganisms, and the addition of a non-pathogenic source of faecal indicators (such as old sediments or an animal population with low occurrence of human-infectious pathogens). The waterbody indicator density was held constant at 35 CFU 100 mL⁻¹ enterococci to compare results across scenarios. For the combinations evaluated, either the untreated sewage or the non-pathogenic source of faecal indicators dominated the recreational waterbody enterococci density assuming a culture method. In contrast, indicator density assayed by qPCR, pathogen density, and bather gastrointestinal illness risks were largely dominated by secondary disinfected municipal wastewater, with untreated sewage being increasingly less important as the faecal indicator load increased from a non-pathogenic source. The results support the use of a calibrated qPCR total enterococci indicator, compared to a culture-based assay, to index infectious human enteric viruses released in treated human wastewater, and illustrate that the source contributing the majority of risk in a mixture may be overlooked when only assessing faecal indicators by a culture-based method.     

Fate of indicator microorganisms, Giardia and Cryptosporidium in subsurface flow constructed wetlands

Limited information is available on the ability of subsurface flow wetlands to remove enteric pathogens. Two multi-species wetlands, one receiving secondary sewage effluent and the other potable (disinfected) groundwater were studied from February 1995 to August 1996, at the Pima County Constructed Ecosystems Research Facility in Tucson, Arizona. Each wetland had a retention time of approximately 4 days. The objectives of this study were (1) to evaluate the ability of multi-species subsurface wetlands to physically remove Giardia cysts; Cryptosporidium oocysts, total and fecal coliforms, and coliphages; and (2) to determine the likely impact of local wildlife on the occurrence of these indicators and pathogens. In the wetland receiving secondary sewage effluent, total coliforms were reduced by an average of 98.8% and fecal coliforms by 98.2%. Coliphage were reduced by an average of 95.2%. Both Giardia cysts and Cryptosporidium oocysts were reduced by an average of 87.8 and 64.2%, respectively. In the wetland receiving disinfected groundwater, an average of 1.3 x 10(2) total coliforms/100 mL and 22.3 fecal coliforms/100 mL were most likely contributed by both flora and fauna. No parasites or coliphages were detected.     

Faecal contamination of greywater and associated microbial risks

The faecal contamination of greywater in a local treatment system at Viby?sen, north of Stockholm, Sweden was quantified using faecal indicator bacteria and chemical biomarkers. Bacterial indicator densities overestimated the faecal load by 100-1000-fold when compared to chemical biomarkers. Based on measured levels of coprostanol, the faecal load was estimated to be 0.04 g person(-1) day(-1). Prevalence of pathogens in the population and the faecal load were used to form the basis of a screening-level quantitative microbial risk assessment (QMRA) that was undertaken for rotavirus, Salmonella typhimurium, Campylobacter jejuni, Giardia lamblia and Cryptosporidium parvum. The different exposure scenarios simulated--direct contact, irrigation of sport fields and groundwater recharge--gave unacceptably high rotavirus risks (0.04 < Pinf < 0.60) despite a low faecal load. The poor reduction of somatic coliphages, which were used as a virus model, in the treatment was one main reason and additional treatment of the greywater is suggested. Somatic coliphages can under extreme circumstances replicate in the wastewater treatment system and thereby underestimate the virus reduction. An alternative QMRA method based on faecal enterococci densities estimated similar risks as for rotavirus. Growth conditions for Salmonella in greywater sediments were also investigated and risk modelling based on replication in the system increased the probability of infection from Salmonella 1000-fold, but it was still lower than the risk of a rotavirus infection.     

The influence of physical and chemical factors on the transport of E. coli through biological filters for wastewater purification

Transport studies of Escherichia coli were performed in laboratory columns over a period of 15 months. The effects of the filter media properties, effective grain size, specific surface area, pH and cation exchange capacity were examined for loading rates of 25 mm and 50 mm/day applied as 8 doses per day. Distilled water and two solutions of ionic strength 0.00725 and 0.097 M were applied to the columns. Physical factors were found to be the most important for the removal of E. coli. Reduced grain size, hydraulic loading rate and increased specific surface area of the grains significantly reduced transport of E. coli. Chemical factors such as pH, cation exchange capacity and wastewater ionic strength showed less significant effects. The results indicate that the chemical factors in biological wastewater filters have a minor influence on the removal of E. coli after a stabilizing period of three months. Minimum hydraulic retention time (time required for 10% breakthrough of a conservative tracer) was found to be the most relevant parameter for predicting bacterial removal in unsaturated filter systems. Correlation between observed data and a first order removal model, based on minimum retention time, was 0.70.     

Inactivation of f2 virus with ferrate (VI)

Disinfection of f2 virus with iron(VI) ferrate in buffered, distilled water and secondary treated sewage effluent has been studied. The relative resistance of f2 virus and Escherichia coli to iron(VI) ferrate has also been examined in buffered, distilled water. Potassium ferrate was found to rapidly inactivate f2 virus at low concentrations at pH = 6, 7 and 8 in pure water systems, and also in secondary effluent. The disinfection reactions observed did not follow first order reaction kinetics. f2 Virus appeared to be equally, or less resistant to potassium ferrate than were most bacteria, including E. coli, in buffered, distilled water at the pH values tested, and also in secondary effluent. Iron(VI) ferrate appears to be a good viricidal agent in raw water and wastewater.     

Coliform counts of polluted waters: a comparison of media and methods

Samples of sewage and polluted river water were used to compare various media for the enumeration of coliform organisms, including Escherichia coli, by the membrane filtration, pour-plate and MPN techniques. The pour-plate method with Lactose teepol agar was found to be at least as good as the other techniques, to which it is to be preferred as it is more economical, both in terms of time and materials.     

Airborne enteric coliphages and bacteria in sewage treatment plants

The concentrations of airborne culturable microorganisms were determined in wastewater and sludge treatment processes of seven sewage treatment plants. Two types of coliphages, Salmonella and total viable bacteria were sampled by the BioSampler and the numbers of faecal coliforms and enterococci were obtained from the Andersen 6-stage impactor. The BioSampler recovered higher numbers of airborne coliphage viruses than has been measured with other liquid samplers in previous studies, suggesting that this sampler has improved efficiency for sampling airborne coliphages. Airborne coliphages were detected in many stages of the wastewater or sludge treatment process. The highest microbiological air contaminations were found in pre-treatment and aerated grit separation stages of the operation. This was attributed to aerosolisation of microorganisms by mechanical handling or forced aeration. Aeration and settling processes located outdoors caused low microbial concentrations, but the brush aerator released more microorganisms into the air. Our results emphasize the necessity for controlling the exposure of sewage workers to airborne microorganisms, especially in process areas that involve mechanical agitation or forced aeration of wastewater.     

Influence of sampling depth on Escherichia coli concentrations in beach monitoring

While the US Environmental Protection Agency's (EPA) Beaches Environmental Assessment and Coastal Health (BEACH) Act requires coastal and Great Lakes’ states to implement plans for monitoring bacterial contamination of recreational beach water, exactly how this monitoring should occur has not been regulated. This study examined differences in concentration of Escherichia coli in water collected from different depths and from different horizontal locations across the beach. E. coli concentrations were significantly different (p<0.05), when water from different depths was compared. Sampling water at depths of 30, 60, and 120 cm resulted in significantly lower E. coli concentrations as depth increased. Had the State of Wisconsin chosen to collect beach water monitoring samples at a shallower or deeper depth, numbers of beach closures and the potential risk to public health would have changed substantially. These data imply that a revised and standardized protocol for monitoring beach water should be adopted by all states of a monitoring region to better compare microbial contamination of beaches and protect public health.