Coliphages as an indicator of faecal pollution in water. Its relationship with indicator and pathogenic microorganisms

The study was designed to test the proposal that Escherichia coli specific bacteriophages might serve as universal faecal pollution indicators in water. A highly specific, sensitive and rapid technique for the detection and quantification of these virus particles was developed. The numerical relationship between E. coli and its parasitic phages was investigated in three different aqueous ecosystems such as sea water in the vicinity of sewage outfalls, river water contaminated by domestic and industrial sewage discharges, and estuarine waters, and found to be very close. In addition, the results obtained indicate that the coliphages are good indicators of the presence of the pathogenic microorganisms studied. In nearly all the water samples tested, the results suggest that coliphages are better indicators of faecal pollution than the classical indicator systems currently employed.     

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.     

Pathogenic or potentially pathogenic bacteria as contaminants of fresh water from different sources in Araraquara, Brazil

The microbiological quality of various fresh waters in the Araraquara region, state of São Paulo, Brazil was investigated. Ninety-nine water samples were taken from rivers, reservoirs, artesian and non-artesian wells, springs and tap waters, and these waters were analysed using: plate counts of heterotrophic microorganisms (per 1 ml); Most Probable Number (MPN) of fecal coliforms and E. coli (per 100 ml); tests for presence of Salmonella, Shigella, Yersinia, the E. coli pathogens of classes EPEC, ETEC and EIEC and Mycobacterium, Shigella, Yersinia and enteroinvasive E. coli (EIEC) were not isolated. The other types of microorganisms were isolated in varying proportions. We conclude that the waters investigated represent a potential microbiological health risk.     

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.     

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.     

Microbial indicators and pathogens: removal, relationships and predictive capabilities in water reclamation facilities

Four water reclamation facilities in north-eastern Spain were monitored over 2 years to determine the occurrence and concentrations of a set of microbial indicators (total coliforms, Escherichia coli, enterococci, spores of sulphite reducing clostridia, somatic coliphages, F-specific RNA phages, phages infecting Bacteroides fragilis strain RYC2056 and phages infecting Bacteroides tethaiotaomicron strain GA-17), and two selected pathogens (cytopathogenic enteroviruses and viable Cryptosporidium oocysts). The indicator (survival) and index (presence) functions of the various indicators tested were evaluated through the wastewater treatments. The inactivation pattern of all groups of bacteriophages tested was closer to the inactivation of enteroviruses than to the inactivation of the conventional bacterial indicators tested. The inactivation of sulfite reducing clostridia spores and bacteriophages more closely approximates the reduction of viable Cryptosporidium than do the conventional bacterial indicators. We observed neither index functions nor a predictive relationship between any of microbial indicators and viable Cryptosporidium oocysts. In contrast, several regression models (r > 0.6) and discriminant functions (67-88% well classified samples) based mostly on numbers of bacteriophages were able to predict both the presence and concentrations of enteroviruses. A combination of both bacterial and bacteriophage indicators seem to be the best choice for ensuring the microbial quality of reclaimed water.     

Photodynamic inactivation of E. coli by immobilized or coated dyes on insoluble supports

The photodynamic inactivation of E. coli by methylene blue, rose bengal and eosin covalently immobilized on polystyrene beads and that of methylene blue coated on granular activated carbon, silica gel and XAD-2 (polystyrene resin) was studied. For the study light-exposed and aerated tap-water contaminated with E. coli subjected to dye sensitized photo-oxidation was used. After 0, 10, 20, 30, 40 and 60 min treatment time, samples were diluted and plated on agar to determine the number of Colony Forming Units (CFU). The study gave the following results: all the light exposed and aerated immobilized dyes (methylene blue, rose bengal and eosin) had an elevated photodynamic action on E. coli. After only 30 min treatment time there was 97.5% inactivation of E. coli by methylene blue, 92.2% by rose bengal and 81.6% by eosin. Methylene blue proved to be the most active. Methylene blue coated on activated carbon, silica gel and XAD-2 also had a high photodynamic action. Activated carbon was found to be the best support; since after 30 min contact it had adsorbed 2 mg g⁻¹ of dye and inactivated 94.4% of E. coli. Comparing the differences in the regression lines of the various methods employed (parallel test) it was found that the effect of each dye was significantly high (P = 0.001) whereas there was no significant difference between methylene blue immobilized and coated on active carbon. From these results it seems that sensitized photo-oxidation using methylene blue coated on activated carbon can be used as an alternative for disinfecting waters for potable purposes, but its effectiveness against other micro-organism present in the water must also be tested.     

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.     

Tracer study and profiling of a tertiary lagoon in the United Kingdom: II

Monitoring data over a year showed that the performance of a tertiary lagoon system in the United Kingdom was very variable. Distinct seasonal patterns were seen for all the determinands. Tracer studies and profile sampling of one of the lagoons were carried out in order to investigate the factors affecting performance. The tracer studies, using sodium fluoride, indicated that the hydraulic regime was dispersed plug flow. The mean retention time was found to be 26 h, but the peak in tracer concentration occurred after 12 h, showing significant short-circuiting. During the profile sampling, the pH, temperature, DO and conductivity were measured down through the lagoon at 16 positions and four depths. E. coli, enterococci and total coliform numbers were measured in samples taken from the surface and the base of the lagoon. The results showed temperature decreased down through the lagoon, but the other parameters increased with depth, indicating that the short-circuiting was caused by the cooler influent sinking to the base of the lagoon.     

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.     

Dose—response of Escherichia coli in ozone demand-free phosphate buffer

The intention of this investigation was to determine the mean response of E. coli at predetermined ozone dose levels. By using replicated, randomized, independent batch experiments, precise estimates of the response could be obtained. Particular care was taken in preparing the bacteria and glassware to eliminate effects caused by extraneous ozone demand. A total of 57 batch experiments were conducted at bench-scale using ozone doses of 4.4, 11.45, 200 and 800 μg l⁻¹ at contact times of 30, 60 and 120 s.

The logarithm of the E. coli survival ratio was plotted as a function of applied ozone dose, utilized ozone and contact time. Contact time was found to have some effect for intermediate doses of ozone, but the effects were small relative to those associated with the utilized ozone dose. It was also found that the disinfection reaction was not first-order with respect to the surviving bacteria concentration and two distinct stages were observed; an initial rapid inactivation stage followed by a slower inactivation stage.

Regression analysis of the E. coli response on the logarithm of the dose parameter is typical of dose-response models for water and wastewater disinfection. However, this model form was inadequate when fitted to the data of this study as a result of a significant tailing effect in the log-log dose-response plots. It was postulated that the observed dose-response could be explained by chemical kinetic theory and that the dose-response was affected by the disinfection reaction rates which were dependent upon the surviving number of bacteria and the residual ozone concentration.

For practical application, it was found that an adequate dose-response model for 0–99.99% reduction of E. coli could be developed using the logarithm of utilized ozone as the dose function.     

Isolation and characterization of Bacteroides host strain HB-73 used to detect sewage specific phages in Hawaii

Previous studies have shown that Escherichia coli and enterococci are unreliable indicators of fecal contamination in Hawaii because of their ability to multiply in environmental soils. In this study, the method of detecting Bacteroides phages as specific markers of sewage contamination in Hawaii’s recreational waters was evaluated because these sewage specific phages cannot multiply under environmental conditions. Bacteroides hosts (GB-124, GA-17), were recovered from sewage samples in Europe and were reported to be effective in detecting phages from sewage samples obtained in certain geographical areas. However, GB-124 and GA-17 hosts were ineffective in detecting phages from sewage samples obtained in Hawaii. Bacteroides host HB-73 was isolated from a sewage sample in Hawaii, confirmed as a Bacteroides sp. and shown to recover phages from multiple sources of sewage produced in Hawaii at high concentrations (5.2-7.3 × 10⁵ PFU/100 mL). These Bacteroides phages were considered as potential markers of sewage because they also survived for three days in fresh stream water and two days in marine water. Water samples from Hawaii’s coastal swimming beaches and harbors, which were known to be contaminated with discharges from streams, were shown to contain moderate (20-187 CFU/100 mL) to elevated (173-816 CFU/100 mL) concentrations of enterococci. These same samples contained undetectable levels (<10 PFU/100 mL) of F+ coliphage and Bacteroides phages and provided evidence to suggest that these enterococci may not necessarily be associated with the presence of raw sewage. These results support previous conclusions that discharges from streams are the major sources of enterococci in coastal waters of Hawaii and the most likely source of these enterococci is from environmental soil rather than from sewage.     

The performance of tertiary lagoons in the United Kingdom: I

Two tertiary lagoons at Holmwood Sewage Treatment Works in the United Kingdom were sampled over a 1 year period. Each lagoon is approximately 39×28 m, receives domestic wastewater only, and serves approximately 2000 people. The aim of the work is to gain a greater understanding of the performance of the lagoon with respect to a number of chemical, biological and physical parameters. Samples were analysed for E. coli, enterococci, total coliforms, BOD, COD, suspended solids, ammoniacal nitrogen, nitrite, nitrate, total oxidised nitrogen and soluble phosphate. Occasional samples were also analysed for Cryptosporidium, Giardia and enteroviruses. Cusum charts were used to analyse the cumulative variations that were seen for the parameters being investigated, and had the advantage of emphasising their distinct seasonal trends. Although good percentage removals were seen, there was a lack of consistency in performance for all parameters over the year. No ammonia or phosphorus removal was found, although limited nitrification does appear to occur. None of the bacterial criteria for the WHO guidelines on wastewater reuse or the EC directive on bathing water were reached.     

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.     

噬菌体作为水中病毒指示物的研究进展

噬菌体作为潜在的水中病毒指示生物，可用于污水再生利用过程中的病毒学安全评价、阐明病毒灭活机理以及改进病毒检测方法等领域的研究。介绍了常用的指示噬菌体——SC噬菌体、F—RNA噬菌体和Bacteroides fragilis噬菌体在环境中的分布、存活和去除特性及其作为水质评价和病毒指示生物的研究进展。     

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.     

Filtration methods for recovery of Bacillus anthracis spores spiked into source and finished water

Spores of Bacillus anthracis Sterne strain were recovered from 100 ml and 1 L volumes of tap and source waters using filtration through a 0.45 um filter, followed by overnight culture on agar plates. In a set of experiments comparing sheep red blood cell (SRBC) plates with a chromogenic agar formulation designed by R & F Laboratories, with a spiking dose of 47 plate-enumerated spores in 100 ml tap water, the mean spore recoveries were 34.0 and 30.8 spores, respectively. When a spiking dose of 100 fluorescence activated cell sorter(FACS)-enumerated spores was used in 100 ml potable water, the average recovery with SRBC plates was 48 spores. Detection efforts with spiking doses of 35 and 10 spores in 1 L tap water were successful, but recovery efforts from spiked 1 L volumes of source water were problematic due to the concomitant growth of normal spore-forming flora. Recoveries were also attempted on 10 L volumes of tap water. For a spiking dose of 100 spores, mean recovery from six replicates was 11 spores (±6.8, range 2–20), and for a spiking dose of 10 spores, mean recovery from six replicates was 2.3 spores (±3.5, range 0–9). Efforts were also made to “direct detect” spores via polymerase chain reaction (PCR) on washes from filters. When spiking 534 spores in 100 ml, 9/9 replicates of spiked tap water, 6/6 source water replicates, and 0/3 unspiked controls were positive by lef PCR. When 534 spores were spiked into 1 L tap water, the lef PCR was unsuccessful; however, using the nested vrrA PCR resulted in 4/9 spiked samples, and 0/3 unspiked controls, testing positive. Our results indicate that an inexpensive and user-friendly method, utilizing filtration apparatus commonly present in many water quality testing labs, can readily be adapted for use in detecting this potential threat agent.     

Influence of inter-annual variations in climatic factors on fecal coliform levels in Mississippi Sound

Information on the effects of inter-annual variations in climatic factors on fecal coliform levels in coastal waters is scarce. We used 11 years (1990–2001) of rainfall, water temperature, Pearl River stage and salinity data to assess the effects of the 1991–1992 and 1997–1998 El Niño events on fecal coliform levels in Mississippi Sound. El Niño-Southern Oscillation had major effects on Pearl River stage and water quality in the Sound. The geometric mean fecal coliform number differed among years (P=0.0001), being highest during 1991–1992 El Niño years (14.22 MPN per 100 ml) and lowest during 1999–2000 La Niña years (<1.8 MPN per 100 ml). Mean salinity varied among years (P=0.0001) from 9 ppt (1991–1992) to 21 ppt (1999–2000). Mean water temperature was lowest in 1997–1998 (14.5 °C) and highest in 1998–2000 (19.4 °C). Pearl River stage differed among years (P=0.0001), ranging from 1.96 m (1999–2000) to 3.57 m (1997–1998). Inverse relationships were observed between fecal coliform levels and salinity (r²=0.74; P=0.001) and water temperature (r²=0.69; P=0.001), whereas positive relationships were obtained with total rainfall (r²=0.52; P=0.013) and Pearl River stage (r²=0.90; P=0.0001). These relationships are useful for evaluating the potential effects of climate change on water quality and classification of shellfish growing waters in order to protect humans from consuming contaminated shellfish in shallow river-influenced estuaries.     

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.