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PubMed2004

Occurrence of Escherichia coli, noroviruses, and F-specific coliphages in fresh market-ready produce.

Forty samples of fresh produce collected from retail food establishments were examined to determine the occurrence of Escherichia coli, F-specific coliphages, and noroviruses. An additional six samples were collected from a restaurant undergoing investigation for a norovirus outbreak. Nineteen (48%) of the retail samples and all outbreak samples were preprocessed (cut, shredded, chopped, or peeled) at or before the point of purchase. Reverse transcription-PCR, with the use of primers JV 12 and JV 13, failed to detect norovirus RNA in any of the samples. All six outbreak samples and 13 (33%) retail samples were positive for F-specific coliphages (odds ratio undefined, P = 0.003). Processed retail samples appeared more likely to contain F-specific coliphages than unprocessed samples (odds ratio 3.8; 95% confidence interval 0.8 to 20.0). Only two (5.0%) retail samples were positive for E. coli; outbreak samples were not tested for E. coli. The results of this preliminary survey suggest that F-specific coliphages could be useful conservative indicators of fecal contamination of produce and its associated virological risks. Large-scale surveys should be conducted to confirm these findings.

Coliphages↗

PubMedDate not supplied

[Research of the RNA-coliphages, in comparison with usual microbial and chemical indexes of biological pollution, for hygienic and sanitary evaluations of coast-waters (author's transl)].

Hygienic significance of the usual microbial (E. coli, Enterococci, Clostridia) and chemical (COD, NH3, NO2) indexes of sea-water biological pollution was investigated, in comparison with RNA-coliphages assay. E. coli was confirmed as an easily measurable and significant index of biological pollution (at least for routinary investigations), while the other microbial and chemical parameters do not significantly increase the hygienic selectivity of the E. coli assay. RNA-coliphages assay, on the contrary, is able to increase the hygienic selectivity of sanitary investigations, particularly in lowly polluted coast-water. This behaviour of RNA-coliphages is in accordance with their great persistance in sea-water, due to their survival-time, closely comparable with human viruses. RNA-coliphages assay is therefore proposed as a simple and useful index of "viral pollution" for a complete hygienic evaluation of sea-water environment in addition to routinary E. coli assay.

Coliphages↗

PubMed1983

A simplified method for coliphage detection in natural waters.

The ARCAT (A Rapid Coliphage Analysis Technique) method for detecting coliphages in water has been modified. Modifications to the original method include media optimization, the use of frozen host cultures, the use of a single agar coliphage assay and optimized plaque resolution with 2, 3, 4-triphenyltetrazolium chloride. Detection of 5 coliphages per 100 ml of water sample is accomplished in 6 hours for rapid estimation of water quality.

Coliphages↗

PubMed1997

Comparison of PCR and plaque assay for detection and enumeration of coliphage in polluted marine waters.

A total of 68 marine samples from various sites impacted by sewage and storm waters were analyzed by both the plaque assay and a reverse transcriptase (RT) PCR technique for F(sup+)-specific coliphage. The coliphage levels detected by the plaque assay averaged 1.90 x 10(sup4) PFU/100.0 ml. Using a most probable number (MPN) PCR approach, the levels averaged 2.40 x 10(sup6) MPN-PCR units/100.0 ml. Two samples were positive by RT-PCR but negative by plaque assay, and 12 samples were positive by plaque assay but negative by RT-PCR (levels lower than 11.00 PFU/100.0 ml). The host system used for the plaque assay may detect somatic coliphage in addition to the F(sup+)-specific coliphage. When it is used as an indicator of pollution, contamination may be missed with more restrictive systems. The difference in results may be due to the sensitivity, specificity, or inhibition of RT-PCR in marine samples. This study provides information on quantifying PCR results by an MPN method and insights into interpretation of PCR data for detection of viruses in marine environments.

Journal Article↗

PubMed2002

Fate of coliphage in a wastewater treatment process.

The fate of coliphage in a wastewater treatment plant in the central part of Japan was investigated from March to December 2001. A relative abundance of coliphage, 1000-10,000 PFU/ml determined with three different Escherichia coli strains, was detected in the influent. But, no remarkable seasonal change in the phage concentration in the influent was observed during the ten-month test period. Almost ten times higher coliphage concentration was detected by the F+ E. coli strain than by the other two F- strains. The RNA phage was more stable than the DNA phage against aerobic treatment using activated sludge. Most of the phages in the influent and primary settling tank were detected as suspended forms. Anaerobic-aerobic treatment enhanced adsorption of the phage by the solid particles. Almost no phage was detected in the effluent. Aerobic treatment using activated sludge and/or the addition of flocculants such as PAC was effective for the removal of coliphage, an index of enteric viral pollution.

Journal Article↗

PubMed1991

Secondary structure of coliphage Q beta RNA. Analysis by electron microscopy.

The secondary structure of genomic RNA from the coliphage Q beta has been examined by electron microscopy in the presence of varying concentrations of spermidine using the Kleinschmidt spreading technique. The size and position of structural features that cover 70% of the viral genome have been mapped. The structural features that are visualized by electron microscopy in Q beta RNA are large. They range in size from 170 to 1600 nucleotides. A loop containing approximately 450 nucleotides is located at the 5' end of the RNA. It includes the initiation region for the viral maturation protein. A large hairpin containing approximately 1600 nucleotides is located in the center of the molecule. It is multibranched and includes most of the viral coat gene, the readthrough region of the A1 gene, and approximately one third of the viral replicase gene. Within the central hairpin, the initiation region for the viral replicase gene pairs with a region within the distal third of the viral coat gene. This structure may participate in the regulation of translational initiation of the viral replicase gene. Two structural variants of the central hairpin were observed. One of them brings the internal S and M viral replicase binding regions into juxtaposition. These observations suggest that the central hairpin may also participate in the regulation of translation of the viral coat gene. The secondary structures that are observed in Q beta RNA differ significantly from structures that we described previously in the genomic RNA of coliphage MS2 but are similar to structures we observed by electron microscopy in the related group B coliphage SP.

Coliphages↗

PubMed1992

Improved method for coliphage detection based on beta-galactosidase induction.

An improved method for coliphage detection based on the induction of beta-galactosidase in Escherichia coli is described. Upon infection by coliphages, the cells are lysed and a stable indolyl product that is dark blue becomes visible within each plaque. The improved method is compared to the proposed coliphage detection procedure described in Standard Methods for the Examination of Water and Wastewater.

Coliphages↗

PubMed2004

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.

Adaptation, Physiological↗

PubMed2002

F-specific RNA coliphages: occurrence, types, and survival in natural waters.

A small, well-defined watershed was investigated over a 2-year period to determine the prevalence of F-specific RNA coliphage (F + RNA) serotypes as indicators of animal fecal contamination. Sampling sites collected runoff from areas of urban and agricultural land use patterns. F-specific coliphages were concentrated from 2-L freshwater samples by polyethylene glycol precipitation, isolated using the double agar layer (DAL) method, confirmed as F + RNA by RNAse suppression, and serotyped. A subset of serotyped F + RNA were confirmed by genotyping. To determine relative survival, 10 confirmed F + RNA field isolates and 5 prototypic F + RNA were spiked into surface water and incubated at 25 degrees C for 36 days. F-specific coliphage isolation was strongly associated with rainfall events and was infrequent from primarily animal impacted surface waters. Field isolates were predoffiinantly Type I F + RNA (81%) and raw sewage isolates were predominantly Type III F + RNA (57%). Genotyping from either the watershed or raw sewage samples never positively identified Type IV F + RNA. Results from laboratory studies showed that F + RNA differ in their survival in water and that Type IV strains were the least persistent. Type III F + RNA were found to be reliably related to the release of uncontrolled human fecal material in the watershed, but the results of this study suggest that further study is required before utilizing for fecal source identification in natural waters.

Agriculture↗

PubMed1993

Sensitivity of naturally occurring coliphages to type I and type II restriction and modification.

Protection against lethal infections by bacteriophage may seem the most likely role of restriction-modification (R-M) systems in bacteria and the reason for their evolution. There are, however, phenomena which question this phage-mediated selection hypothesis for the maintenance of extant R-M systems. Most prominent among these are the mechanisms phage have to avoid or otherwise limit the effects of the restriction endonucleases produced by their host bacteria. To evaluate the importance of these antirestriction mechanisms in Escherichia coli, we have examined the sensitivity of coliphage from natural and laboratory sources to a series of type I and II R-M systems. The results of our study indicate that, in vivo, restriction endonucleases have no effect on a substantial fraction of naturally occurring coliphage. The absence of restriction sites appears to be the most common reason why these phage are unaffected by type II restriction endonucleases, but other antirestriction mechanisms also operate. On the other hand, the frequency of naturally occurring coliphage sensitive to restriction appears sufficiently great for phage-mediated selection to be a viable hypothesis for the maintenance of R-M in E. coli and its accessory elements.

Biological Evolution↗

PubMed1981

Recovery of coliphages from wastewater effluents and polluted lake water by the magnetite-organic flocculation method.

A magnetite-organic flocculation method was developed for the concentration of coliphages from wastewater effluents and polluted lake water. A high percent (68 to 100%) recovery of coliphages from sewage effluents was achieved by this procedure. Coliphage recovery from Lake Alice, a sewage-contaminated lake, showed phage concentrations ranging from 2.3 X 10(2) to 1.9 X 10(3) plaque-forming units per liter. This method is simple and inexpensive and may be carried out under field conditions.

Coliphages↗

PubMed1983

Concentration of coliphage from water and sewage with charge-modified filter aid.

Methods of detecting and concentrating animal viruses from large volumes of water and wastewater have experienced rapid development in recent years, but only a few methods are available for the concentration of bacteriophages. The present study describes the use of a charge-modified (Zeta Plus) filter aid (AMF Cuno, Meriden, Conn.) for the concentration of coliphages from large volumes of water and sewage. Coliphages MS-2 and f2 were efficiently adsorbed from water and sewage to the positively charged filter aid. Elution was accomplished with 4% beef extract--0.5 M NaCl adjusted to pH 9.5. The recovery of f2 from 10- to 20-liter volumes of tap water ranged between 11 and 70%, and the recovery of MS-2 ranged between 43 and 70%. The efficiency of recovery of naturally occurring coliphages from secondarily treated sewage ranged between 16 and 44%. This technique appears to be promising because it requires low-cost equipment (47-mm polypropylene filter housing), is easy to handle, and can filter large volumes of water (greater than or equal to 20 liters) with good recoveries. Filtrations can be conducted at the ambient pH of the water, and the unit cost per filtration (i.e., the cost of filter aid) comes to less than three cents per sampling. The technique could be useful in evaluation of viral water quality, study of ecology and occurrence of phages in natural waters, and isolation of rare phages from natural waters.

Coliphages↗

PubMed1994

Effect of distance from the polluting focus on relative concentrations of Bacteroides fragilis phages and coliphages in mussels.

Concentrations of fecal bacteria, somatic and F-specific coliphages, and phages infecting Bacteroides fragilis in naturally occurring black mussels (Mytilus edulis) were determined. Mussels were collected over a 7-month period at four sampling sites with different levels of fecal pollution. Concentrations of both fecal bacteria and bacteriophages in mussel meat paralleled the concentration of fecal bacteria in the overlying waters. Mussels bioaccumulated efficiently, although with different efficiencies, all of the microorganisms studied. Ratios comparing the levels of microorganisms in mussels were determined. These ratios changed in mussels collected at the different sites. They suggest that bacteriophages infecting B. fragilis and somatic coliphages have the lowest decay rates among the microorganisms studied, with the exception of Clostridium perfringens. On the contrary, concentrations of F-specific coliphages showed a greater rate of decay than the other bacteriophages at sites more distant from the focus of contamination. Additionally, levels of enteroviruses were studied in a number of samples, and in these samples, the B. fragilis bacteriophages clearly outnumbered the enteroviruses. The results of this study indicate that, under the environmental conditions studied, the fate of phages infecting B. fragilis released into the marine environment resembles that of human viruses more than any other microorganism examined.

Animals↗

PubMed2006

Sequence variation among group III F-specific RNA coliphages from water samples and swine lagoons.

Typing of F-specific RNA (FRNA) coliphages has been proposed as a useful method for distinguishing human from animal fecal contamination in environmental samples. Group II and III FRNA coliphages are generally associated with human wastes, but several exceptions have been noted. In the present study, we have genotyped and partially sequenced group III FRNA coliphage field isolates from swine lagoons in North Carolina (NC) and South Carolina (SC), along with isolates from surface waters and municipal wastewaters. Phylogenetic analysis of a region of the 5' end of the maturation protein gene revealed two genetically different group III FRNA subclusters with 36.6% sequence variation. The SC swine lagoon isolates were more closely related to group III prototype virus M11, whereas the isolates from a swine lagoon in NC, surface waters, and wastewaters grouped with prototype virus Q-beta. These results suggest that refining phage genotyping systems to discriminate M11-like phages from Q-beta-like phages would not necessarily provide greater discriminatory power in distinguishing human from animal sources of pollution. Within the group III subclusters, nucleotide sequence diversity ranged from 0% to 6.9% for M11-like strains and from 0% to 8.7% for Q-beta-like strains. It is demonstrated here that nucleotide sequencing of closely related FRNA strains can be used to help track sources of contamination in surface waters. A similar use of phage genomic sequence information to track fecal pollution promises more reliable results than phage typing by nucleic acid hybridization and may hold more potential for field applications.

Animals↗

PubMed1974

Synthesis of indicator strains and density of ribonucleic acid-containing coliphages in sewage.

Escherichia coli strains freshly isolated from natural sources are inefficient indicators of coliphages present in sewage. Four E. coli strains recently isolated from clinical specimens were mutagenized to obtain lac(-) mutants. Such mutants were infected with an F'lac(+) sex factor of E. coli K-12. Pairs of isogenic lac(-) and lac(-)/F'lac(+) strains were used as indicators of coliphages present in sewage, and it was found that such strains can be effectively used for a direct and almost selective enumeration of F-specific coliphage contents of sewage samples. Serological tests were applied to a number of F-specific phages isolated. All the isolates that were tested fell into two distinguishable antigenic classes: members of one class being related to ribonucleic acid (RNA) phage MS2 and those of the other being related to another RNA phage, namely, Qbeta. MS2-related phages have been found to be more widely distributed than the Qbeta related phages. Most habitats sampled were found to yield only one or the other kind of phage. Single-stranded deoxyribonucleic acid-containing F-specific phages were not detectable by the methods employed by us.

Anti-Bacterial Agents↗

PubMed1976

Role of lipopolysaccharide in adsorption of coliphage T4D to Escherichia coli B.

Coliphage T4D was strongly adsorbed to intact lipopolysaccharides and alkaline and lipase-treated lipopolysaccharides from cells of Escherichia coli B, but was not so adsorbed to heat-treated cells. In contrast, coliphage T2h was not adsorbed to lipopolysaccharides and the heat-treated cells. Acid hydrolysate of lipopolysaccharides strongly inhibited the adsorption of phage T4D to acetone and ether-treated cells. The adsorption of phage T4D to the acetone and ether-treated cells was markedly inhibited by authentic D-glucosamine, N-acetyl-D-glucosamine, alpha-methyl-N-acetyl-D-glucosaminide, alpha-methyl-D-glucoside, and D-maltose. Authentic D-glucose and E,L-2,6-diaminopimelic acid also showed similar activity. These compounds did not affect the adsorption of phage T2h to the acetone- and ether-treated cells. Concanavalin A and wheat-germ agglutinin inhibited phage T4D adsorption to the acetone and ether-treated cells probably by blocking the phage-receptor sites on the cell wall. The blocking by concanavalin A and by wheat-germ agglutinin was reversed by alpha-methyl-D-glucoside and by alpha-methyl-N-acetyl-D-glucosaminide, respectively. Results suggested the possibility that coliphage T4D requires N-acetyl-D-glucosaminyl-glucose or glucosyl-D-glucosamine residues of the core of lipopolysaccharides for the initial attachment to the cell wall of Escherichia coli B.

Acetone↗

PubMed1994

[Evaluation of the hygienic and sanitary quality of water from shallow wells situated in an urban area: a comparison between the use of coliphages and bacterial indicators of fecal pollution].

One hundred and four water samples from eight private shallow wells situated in the urban area of Jaboticabal city, State of S. Paulo, Brazil, were submitted to coliphage, total coliform, fecal coliform and fecal streptococcus counts, for the purpose of discovering their hygienic and sanitary quality and of verifying the correlations between the coliphage numbers and the fecal pollution indicator bacteria. Ninety-six (92.3%) of the samples were not up to the microbiological potability standards. This result demonstrates the unsatisfactory hygienic and sanitary quality of the water samples. The results show also the absence of correlations among coliphages, and the fecal pollution indicator bacteria.

Brazil↗

PubMed2004

Contribution of the colmation layer to the elimination of coliphages by slow sand filtration.

River bank or slow sand filtration is a major procedure for processing surface water to drinking water in central europe. In order to model the performance of river bank and slow sand filtration plants, we are studying the different mechanisms by which the elimination of pathogens is realized. An important question concerning the mode of action of slow sand filters and river bank filtration units is the role of the colmation layer or "schmutzdecke" on the elimination of human pathogens. The schmutzdecke is an organic layer which develops at the surface of the sand filter short after the onset of operation. We have inoculated a pilot plant for slow sand filtration with coliphages and determined their rate of breakthrough and their final elimination. In the first experiment, with a colmation layer still missing, the breakthrough of the coliphages in the 80 cm mighty sandy bed amounted to ca. 40 %. In contrast, less than 1 % of coliphages escaped from the filter as the same experiment was repeated two months later, when a substantial colmation layer had developed. Our preliminary conclusions are that the colmation layer is extremely efficient in eliminating of viruses.

Coliphages↗