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Evaluation of coliphage detection as a rapid indicator of water quality.

A rapid coliphage analysis technique for enumerating coliphages in natural waters has been evaluated by water quality laboratories located throughout the United States. Correlations were established between coliphages and coliforms in natural water systems. These correlations were highly significant. This relationship can thus be used to determine the number of fecal or total coliforms present in natural water samples based on an enumeration of coliphages. With this method, coliphages in natural water systems (containing greater than or equal to six coliphages per 100 ml) can be enumerated within 6 h.

Coliphages

Coliphages as indicators of enteroviruses.

Coliphages were monitored in conjunction with indicator bacteria and enteroviruses in a drinking-water plant modified to reduce trihalomethane production. Coliphages could be detected in the source water by direct inoculation, and sufficient coliphages were detected in enterovirus concentrates to permit following the coliphage levels through different water treatment processes. The recovery efficiency by different filter types ranged from 1 to 53%. Statistical analysis of the data indicated that enterovirus isolates were better correlated with coliphages than with total coliforms, fecal coliforms, fecal streptococci, or standard plate count organisms. Coliphages were not detected in finished water.

Coliphages

Comparison of selective media for assay of coliphages in sewage effluent and lake water.

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.

Chloroform

Differential depuration of poliovirus, Escherichia coli, and a coliphage by the common mussel, Mytilus edulis.

The elimination of sewage effluent-associated poliovirus, Escherichia coli, and a 22-nm icosahedral coliphage by the common mussel, Mytilus edulis, was studied. Both laboratory-and commercial-scale recirculating, UV depuration systems were used in this study. In the laboratory system, the logarithms of the poliovirus, E. coli, and coliphage levels were reduced by 1.86, 2.9, and 2.16, respectively, within 52 h of depuration. The relative patterns and rates of elimination of the three organisms suggest that they are eliminated from mussels by different mechanisms during depuration under suitable conditions. Poliovirus was not included in experiments undertaken in the commercial-scale depuration system. The differences in the relative rates and patterns of elimination were maintained for E. coli and coliphage in this system, with the logarithm of the E. coli levels being reduced by 3.18 and the logarithm of the coliphage levels being reduced by 0.87. The results from both depuration systems suggest that E. coli is an inappropriate indicator of the efficiency of virus elimination during depuration. The coliphage used appears to be a more representative indicator. Depuration under stressful conditions appeared to have a negligible affect on poliovirus and coliphage elimination rates from mussels. However, the rate and pattern of E. coli elimination were dramatically affected by these conditions. Therefore, monitoring E. coli counts might prove useful in ensuring that mussels are functioning well during depuration.

Animals

Comparative inactivation of poliovirus type 3 and MS2 coliphage in demand-free phosphate buffer by using ozone.

MS2 coliphage (ATCC 15597-B1) has been proposed by the U.S. Environmental Protection Agency as a surrogate for enteric viruses to determine the engineering requirements of chemical disinfection systems on the basis of previous experience with chlorine. The objective of this study was to determine whether MS2 coliphage was a suitable indicator for the inactivation of enteric viruses when ozone disinfection systems were used. Bench-scale experiments were conducted in 2-liter-batch shrinking reactors containing ozone demand-free 0.05 M phosphate buffer (pH 6.9) at 22 degrees C. Ozone was added as a side stream from a concentrated stock solution. It was found that an ozone residual of less than 40 micrograms/liter at the end of 20 s inactivated greater than 99.99% of MS2 coliphage in the demand-free buffer. When MS2 was compared directly with poliovirus type 3 in paired experiments, 1.6 log units more inactivation was observed with MS2 coliphage than with poliovirus type 3. It was concluded that the use of MS2 coliphage as a surrogate organism for studies of enteric virus with ozone disinfection systems overestimated the inactivation of enteric viruses. It is recommended that the regulatory agencies evaluate their recommendations for using MS2 coliphage as an indicator of enteric viruses.

Buffers

Genotyping male-specific RNA coliphages by hybridization with oligonucleotide probes.

F-specific (F+) RNA coliphages are prevalent in sewage and other fecal wastes of humans and animals. There are four antigenically distinct serogroups of F+ RNA coliphages, and those predominating in humans (groups II and III) differ from those predominating in animals (groups I and IV). Hence, it may be possible to distinguish between human and animal wastes by serotyping F+ RNA coliphage isolates. Because serotyping is laborious and requires scarce antiserum reagents, we investigated genotyping using synthetic oligonucleotide probes as an alternative approach to distinguishing the four groups of F+ RNA coliphages. Oligoprobes I, II, III, IV, A, and B were selected to detect group I, II, III, IV, I plus II, and III plus IV phages, respectively. Methods for phage transfer from zones of lysis on a host cell lawn to candidate membrane filters and fixation of genomic nucleic acid on the membranes were optimized. The oligoprobes, which were end labeled with digoxigenin, were applied in DNA-RNA hybridization, and hybrids were observed by colorimetric, immunoenzymatic detection. Of 203 isolates of F+ RNA coliphages from environmental samples of water, wastes, and shellfish, 99.5 and 96.6% could be classified into each group by serotyping and genotyping, respectively. Probes A and B correctly identified 100% of the isolates. On the basis of these results, this method for genotyping F+ RNA coliphages appears to be practical and reliable for typing isolates in field samples.

Animals

Fate of human enteric viruses, coliphages, and Clostridium perfringens during drinking-water treatment.

The elimination of human enteric viruses, coliphages, and Clostridium perfringens was studied during a conventional complete drinking-water treatment process. The respective concentrations (geometric mean) of these microorganisms in 100-L samples of river water were, respectively, as follows: viruses, 79 mpniu (most probable number of infectious units) per 100 L, coliphages, 6565 pfu (plaque-forming units) per 100 L. and clostridia, 11,349 cfu (colony-forming units) per 100 L. After predisinfection, flocculation with alum, and settling, human enteric viruses were not detected in any of the 100-L samples (less than 4 mpniu/100 L), but coliphages were detected in 7 of 14 samples and clostridia in 15 of 16 samples. In filtered water samples, human enteric viruses were detected in 2 of 31 samples, coliphages in 10 of 33, and clostridia in 17 of 33. Finished water was free of human enteric viruses (0/162 samples), but coliphages were detected in one sample (1.5 pfu/100 L) and clostridia in three, at 1.0, 4.1, and 7.0 cfu/100 L. It thus appears that coliphages and clostridia, which are present in larger numbers than viruses in river water and which may have similar resistance to drinking-water treatments, may be useful for estimating the level of treatment attained when large volumes of water (1000 L or greater) are sampled.

Clostridium perfringens

[Coliphages, viral indicators of contamination].

This paper illustrates the importance of coliphages, which are specific bacteriophages for Escherichia coli strains, as viral indicators, and suggests the usefulness of a routine coliphage search in the detection of viral contamination. The samples studied consisted of 57 surface water, (46 of sea water and 11 of river water), 2 of mud taken from biological treatment plants and 3 samples of sludge. The results revealed that coliphages can be considered valid indicators of viral contamination as they show a good response to the general standards established for indicators. In particular: they are found in sludge (both untreated and treated) and in surface water (both river and sea) in higher concentrations than those of enterovirus; they show greater resistance than bacterial indicators to disinfection as, in different experimental conditions, the drop in the level does not exceed orders of magnitude they are relatively simple to identify, and are easily quantifiable by means of the MPN method, with the result that they can be used for the management of water resources. As regards the possible use of coliphages for the control of the necessary qualitative requisites of sea water for bathing, the relation with fecal coli enables us to make an assessment of coliphages corresponding to 100 fecal coli/100 ml. This levels is equivalent to 0.95 so that a suitable guide line for water for bathing purposes could for example be established at 10 coliphages/1. Although coliphages may be considered good indicators of viral contamination this clearly does not mean that they can replace classic bacterial indicators but rather, that they can provide useful additional information, particularly in those cases where a rapid drop in bacterial content may conceal high risk health and hygiene conditions.

Bacteria

[Inactivation of bacteria and coliphages in surface water highly polluted by secondary effluent and purified by flocculation and filtration by means of UV irradiation at a pilot plant scale].

The water of a channel in Berlin which is highly polluted by municipal sewage effluent is treated at the phosphate elimination plant (PEP) Tegel by flocculation and filtration in order to reduce eutrophication in the following Lake Tegel. The elimination of bacteria and coliphages in the effluent of the PEP was investigated in a scale pilot UV irradiation reactor installed at the outlet of the PEP Tegel. The influence of technical parameters such as flow rate and the arrangement of 23 UV lamps in the reactor on the inactivation was tested. The UV irradiation dose was calculated 119 mJ/cm2 and 49 mJ/cm2 at a flow rate of 50 m3/h and 120 m3/h, respectively and for an irradiation zone of 97.5 cm. The colony count of bacteria and concentrations of coliform organisms, E. coli, and feacal streptococci as well as the plaque forming units of coliphages in the influent of the UV reactor were reduced 2-3 lg units by an irradiation dose of 119 mJ/cm2. These elimination was found being only one lg unit at a UV irradiation dose of 49 mJ/cm2. The concentration of E. faecalis and Coliphages f2 seeded into the influent of the UV reactor decreased after UV irradiation by 119 mJ/cm2 by 2-4 lg units and 1-2 power of magnitude, respectively. A UV dose of 49 mJ/cm2 caused only a 90% elimination of E. faecalis and a 75% inactivation of Coliphages f2. Due to heterogenous distribution and the different retention period of the inflowing water in the irradiation zone, the inactivation of E. faecalis and Coliphages f2 was unequal. Both test organisms decreased in the middle of the reactor up to 2 lg units more than at the sides of the reactor. The hygienic-microbiological quality of a secondary effluent from sewage treatment plants can be improved by a combination of flocculation-filtration and UV irradiation due to their additive elimination effect. However, this UV reactor, which was tested under field conditions can only ensure the inactivation of bacteria and coliphages in the pretreated effluent, if more homogenous distribution of the inflowing water can be achieved. Further, the water must be irradiated by a higher UV dose.

Coliphages

Identification of the major adherence ligand of Klebsiella pneumoniae in the receptor for coliphage T7 and alteration of Klebsiella adherence properties by lysogenic conversion.

We have studied the adherence of both laboratory and wild-type Klebsiella pneumoniae strains, isolated from sputum, urine, and stool samples, to human buccal and intestinal and urinary tract epithelial cells. Of 32 unencapsulated strains, 30 adhered to all epithelial cells tested. Four K. pneumoniae strains lysogenic for AP3, a phage which causes conversion to resistance of coliphages T3, T7, and phi I, were all unable to adhere to epithelial cells. One of these strains was cured from phage infection and became capable of adhering, Spontaneous mutants resistant to coliphage T7, as well as K. pneumoniae K59-sensitive cells preadsorbed with inactivated T7 particles, did not adhere to epithelial cells. All strains capable of adhering were able to adsorb coliphage T7 and T3, whereas all nonadhesive strains were not. AP3-like prophages were induced from 7 of 12 nonadhesive Klebsiella strains. A laboratory strain which was able to adhere was lysogenized with 2 of these phages. In both cases, the strain lost its ability to adsorb coliphages T3, T7, and phi I and to adhere to human epithelial cells. All K. pneumoniae adhesive strains agglutinated yeast cells, whereas the nonadhesive strains did not. Competition studies have shown that D-mannose and concanavalin A prevented adherence to human epithelial cells, yeast agglutination, and adsorption of coliphage T7 to K. pneumoniae cells. It is concluded that in K. pneumoniae adherence to epithelial cells is mediated by the receptor for coliphages T7 (and T3), which in turn recognizes D-mannose in the receptors it binds.

Cell Adhesion

Concentration of coliphages from large volumes of water and wastewater.

Membrane filter adsorption-elution technology has been extensively used for the concentration and detection of animal viruses from large volumes of water. This study describes the development of positively charged microporous filters (Zeta Plus) for the concentration of coliphages from large volumes of water and wastewater. Four different coliphages were studied: MS-2, phi X174, T2, and T4. Positively charged microporous filters were found to efficiently adsorb these coliphages from tap water, sewage, and lake water at neutral pH. Adsorbed viruses were eluted with a 1:1 mixture of 8% beef extract and 1 M sodium chloride at pH 9. Using this method, coliphages could be concentrated from 17-liter volumes of tap water with recoveries ranging from 34 to 100%. Coliphages occurring naturally in raw and secondarily treated sewage were recovered with average efficiencies of 56.5 and 55.0%, respectively. This method should be useful in isolation of rare phages, the ecology of phages in natural waters, and the evaluation of water quality.

Coliphages

Incidence of coliphage in potable water supplies.

Samples of drinking water from different sources in greater Cairo, Egypt, and bottled drinking water were tested for total coliform, fecal coliform, and coliphage populations. Of the 147 samples tested, 4 samples were positive for both total coliforms and coliphage, 65 samples were negative for total coliforms, fecal coliforms, and coliphage, and 78 samples were positive for coliphage and negative for total coliforms and fecal coliforms. The incidence of coliphage in these potable water supplies reflects the probability of human pathogenic virus survival in these waters also.

Coliphages

Rates of inactivation of waterborne coliphages by monochloramine.

A sophisticated water quality monitoring program was established to evaluate virus removal through Denver's 1-million-gal (ca. 4-million-liter)/day Direct Potable Reuse Demonstration Plant. As a comparison point for the reuse demonstration plant, Denver's main water treatment facility was also monitored for coliphage organisms. Through the routine monitoring of the main plant, it was discovered that coliphage organisms were escaping the water treatment processes. Monochloramine residuals and contact times (CT values) required to achieve 99% inactivation were determined for coliphage organisms entering and leaving this conventional water treatment plant. The coliphage tested in the effluent waters had higher CT values on the average than those of the influent waters. CT values established for some of these coliphages suggest that monochloramine alone is not capable of removing 2 orders of magnitude of these specific organisms in a typical water treatment facility. Electron micrographs revealed one distinct type of phage capable of escaping the water treatment processes and three distinct types of phages in all.

Chloramines

Coliphages as ecological indicators of enteroviruses in various water systems.

The occurrence of coliphages and enteroviruses in a variety of water systems in Czechoslovakia was monitored for two years. Two host strains of Escherichia coli bacteria were used to test 1161 water samples for the presence of bacteriophages. These strains were polyvalent hosts for a broad spectrum of morphologically distinct coliphages, and their use thus gave quantitative data on the degree of viral pollution in any given water sample. Ninety-two water samples were tested in parallel for the presence of enteroviruses, by using a flocculation method to concentrate the viruses followed by isolation in cultures of a buffalo green monkey (BGM) kidney continuous cell line. The enterovirus and coliphage recovery rates showed similar differences when waters with different levels of pollution were compared. Seasonal fluctuations of both the coliphage and enterovirus (mostly poliovirus) levels in river water were demonstrated by statistical analysis of the data collected. The levels increased in the winter and sharply declined in the summer months as the river water temperature increased. Chemical pollution did not seem to influence the survival of either the coliphages or the enteroviruses in the observed rivers.

Coliphages

Effects of pyrrol-carboxylic acid derivatives on the growth of coliphages.

Effects of 14 pyrrol-carboxylic acid derivatives and analogues (PY-compounds) on the growth of coliphage MS2 using E. coli E102 (Hfr) as the host were measured by the agar double-layer method. Enlargements of plaque size were observed with 7 PY-compounds but increase in plaque numbers was not induced. These enlargements of plaque size were specific to RNA coliphages MS2, GA and qbeta and not found with DNA coliphages delta AC and T4. Furthermore, the interaction between PY-compound PY-10 and the coliphage MS2 was dependent on the host bacterium (indicator strain). When E102 (Hfr) was used, the enlargement was marked, in the case of substrain W1895 (Hfr) it was less, while in the case of substrain W6 (F+) it was undetectable. The one-step growth of the phage MS2 and the production of intracellular phage MS2 were little affected by the PY-compound PY-10. However, the rate of one-step growth was increased in the early stage after infection. Accordingly, the enlargements of plaque size by the PY-compounds might be correlated with an increase in rate of release of phage particles.

Coliphages

A liquid, colorimetric presence-absence coliphage detection method.

A liquid, colorimetric presence-absence coliphage detection method based on the induction of beta-galactosidase by Escherichia coli is described. The release of beta-galactosidase in the medium due to lytic cell infections by coliphages permits the hydrolysis of a yellow chromogenic substrate that develops into a distinct red coliphage positive sample, while a coliphage negative sample remains yellow. This method has proven to be rapid, simpler to perform than an agar medium assay, easy to read and interpret, inexpensive, and highly sensitive.

Chlorophenols

Elution and reconcentration of coliphages in water from positively charged membrane filters with urea-arginine phosphate buffer.

Coliphages in drinking water and waste water samples have been adsorbed onto positively charged membrane filters, eluted with urea-arginine phosphate buffer (UAPB), reconcentrated, and detected with Escherichia coli C (ATCC 13706). The proposed membrane filter-based UAPB method for concentration and detection of coliphages compares favorably with the beef extract elution and reconcentration procedure and also with the proposed coliphage detection procedure described in Standard Methods for the Examination of Water and Wastewater. The higher recovery of coliphages with UAPB elution from positively charged membrane filters is attributed to testing the whole volume of concentrated sample, rather than partial analysis of the sample as in the procedure described in Standard Methods for the Examination of Water and Wastewater, especially when the titre is very low.

Arginine

Physical properties of single- and double-stranded coliphage ribonucleic acid.

1. The physical characteristics of single- and double-stranded coliphage RNA with regard to their sedimentation behaviour in gradients of sucrose in high or low ionic conditions were examined. The effect of heat on their sedimentation characteristics was also determined. 2. Single-stranded coliphage RNA was found to exist in three different forms having sedimentation coefficients 28s, 20s and 12s. The latter two were interchangeable, depending on ionic strength. All three were almost equally infectious to spheroplasts. 3. Double-stranded coliphage RNA was found to be non-infectious to spheroplasts and had sedimentation coefficients 15s and 12s. Thermal denaturation gave rise to infectious single-stranded 12s RNA. 4. Four possible hypotheses on the mechanism of replication of coliphage RNA are discussed.

Carbon Isotopes