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Survival of T3 coliphage in varied extracellular environments. I. Viability of the coliphage during storage and in aerosols.

The objective of this study was to determine the feasibility of using airborne T3 coliphage as a viral tracer in microbial aerosols. Although T3 coliphage was relatively stable when stored either at temperatures ranging from 21 to 37 C or in the frozen state at -20 C, there was a 2-log loss in infectivity when stored for 72 days at 4 C. Either agitation of stored coliphage suspensions held at 31 C or wide fluctuations in storage temperature produced an increased loss of infectivity. In the airborne state, freshly prepared coliphage and stored coliphage behaved similarly, with survival diminishing as the relative humidity (RH) was lowered. The greatest loss occurred during the first five min following aerosolization. The results showed that only under certain conditions of temperature and relative humidity can T3 coliphage be used as a satisfactory aerosol tracer.

Aerosols↗

Ribonucleic acid synthesis by Escherichia coli C 3000/L after infection by the ribonucleic acid coliphage ZIK/1, and properties of the coliphage-induced double-stranged ribonucleic acid.

1. The efficiency of extracting nucleic acids from Escherichia coli after five methods of obtaining cell lysis was determined. 2. The recovery of various nucleic acid species isolated after chromatography on methylated albumin-coated kieselguhr was also examined. 3. Double-stranded coliphage-induced RNA was isolated from infected bacteria and its resistance to ribonuclease digestion under various conditions determined. 4. The involvement of double-stranded RNA during the infection process was demonstrated. 5. The time-course of the syntheses in infected cells of double-stranded RNA, DNA, single-stranded coliphage and 16s ribosomal RNA, transfer RNA and ribosomal 23s RNA was examined. 6. It was demonstrated that the syntheses of DNA, transfer RNA and ribosomal RNA decreased 10-15min. after infection. 7. Synthesis of coliphage RNA commenced 10-15min. after infection and double-stranded RNA was also synthesized from about 10min. after coliphage adsorption.

Bacteriolysis↗

Replication of coliphage Q beta as affected by host cell number, nutrition, competition from insusceptible cells and non-FRNA coliphages.

F-specific RNA (FRNA) coliphages, which infect Escherichia coli by attachment to F pili, might serve as indicators of human enteric viruses in groundwater, provided these phages do not replicate in groundwater and replicate only to a limited extent in wastewater. Several factors that could influence phage replication in either of these environments were examined. Q beta did not replicate when host cells were fewer than 10(4) cfu ml-1. Replication selected for insusceptible cells when Q beta was incubated with its E. coli host. Loss of Q beta, presumably by inactivation, occurred in autoclaved on-site and urban wastewater, autoclaved groundwater, and in filter-sterilized spent LB broth. Replication did not occur in LB broth diluted with sterile saline to 1% of its original strength, which indicates that replication of FRNA coliphages cannot occur in such nutrient-poor environments as wastewater and groundwater. Competition from non-FRNA coliphages and insusceptible cells tended to reduce Q beta replication, as predicted, but phage yields unexpectedly increased significantly when Enterococcus faecalis was added to cultures.

Allolevivirus↗

Control of gene expression in P2-related coliphages: the in vitro transcription pattern of coliphage 186.

Transcription in vitro of coliphage 186 DNA generated four transcripts. The most abundant transcript was that of the late control gene B and an equivalent transcript was identified for the closely related phage P2. A second transcript was from the rightward promoter at 75% and predicted to be under CI repressor control. The remaining two transcripts initiated from the one promoter located at 95% and are apparently under LexA control in vivo. The significance of these transcripts is discussed in relation to coliphage 186.

Base Sequence↗

Partial characterization of coliphage WPK and a comparison with coliphage T3.

Coliphage WPK was originally isolated from sewage in Kiel, Germany, because its plaque diameter continued to expand for days. Electron microscopy revealed an isometric capsid with dimensions of 54 nm between opposite apices, and a short, noncontractile tail 16 nm long, placing phage WPK into morphogroup C1. The nucleic acid of phage WPK was linear double stranded DNA. The host ranges of phages WPK and T3 were identical. Of ten E. coli strains tested for host range, two were resistant and of eighteen other Enterobacteriaceae only four were susceptible. Seven gram-negative species which are not members of the Enterobacteriaceae were refractory. However, there were differences in plaque morphology and plaque expansion between the two phages. Phage T3 plaques expanded for at least seven days on E. coli B only, while phage WPK plaques expanded for at least seven days on four strains of E. coli. The buoyant density of WPK, determined by isopycnic density gradient centrifugation in CsCl, was 1,508 g/ml which was significantly different than that of T3 at 1.493 g/ml (P less than 0.05). Phage-encoded proteins were examined for each phage using [35S]methionine incorporation, SDS-PAGE, and autoradiography. Of thirty proteins identified in phage WPK and twenty-eight in phage T3, only fourteen were of the same size in both. We concluded that phage WPK was distinct, but related to T3.

Coliphages↗

phi gamma: A coliphage coliphage related to, but distinct from the phi 80 virion.

The coliphage phi gamma, though capable of genetic recombination with phi 80, is morphologically distinct from the phi 80 virion. It has a prolate head (58.4 X 46.7 nm) bearing a tail (143 nm) which is strikingly flexible. On the basis of their buoyant density in CsC1, both infectious and transducing phi gamma particles form a single population. This density value is slightly higher than that of the phi 80 virion. The phi gamma chromosome is a double-stranded linear DNA molecule of 13.4 mum in length (corresponding mol. wt.: 27.6 X 10(6)). From its melting temperature and its buoyant density in CsC1, phi gamma DNA appears to have a base composition very close to that of Escherichia coli DNA.

Capsid↗

Evaluation of F+ RNA and DNA coliphages as source-specific indicators of fecal contamination in surface waters.

Male-specific (F+) coliphages have been investigated as viral indicators of fecal contamination that may provide source-specific information for impacted environmental waters. This study examined the presence and proportions of the different subgroups of F+ coliphages in a variety of fecal wastes and surface waters with well-defined potential waste impacts. Municipal wastewater samples had high proportions of F+ DNA and group II and III F+ RNA coliphages. Bovine wastewaters also contained a high proportion of F+ DNA coliphages, but group I and IV F+ RNA coliphages predominated. Swine wastewaters contained approximately equal proportions of F+ DNA and RNA coliphages, and group I and III F+ RNA coliphages were most common. Waterfowl (gull and goose) feces contained almost exclusively F+ RNA coliphages of groups I and IV. No F+ coliphages were isolated from the feces of the other species examined. F+ coliphage recovery from surface waters was influenced by precipitation events and animal or human land use. There were no significant differences in coliphage density among land use categories. Significant seasonal variation was observed in the proportions of F+ DNA and RNA coliphages. Group I F+ RNA coliphages were the vast majority (90%) of those recovered from surface waters. The percentage of group I F+ RNA coliphages detected was greatest at background sites, and the percentage of group II F+ RNA coliphages was highest at human-impacted sites. Monitoring of F+ coliphage groups can indicate the presence and major sources of microbial inputs to surface waters, but environmental effects on the relative occurrence of different groups need to be considered.

Animals↗

Removal of coliphages in secondary effluent by microfiltration-mechanisms of removal and impact of operating parameters.

The efficacy of a microfiltration (MF) pilot plant in removing somatic coliphages (referred hereafter as coliphages) present in the secondary effluent was evaluated during this study. The impact of operating parameters such as feed coliphage concentrations, permeate flux and membrane fouling on the removal of coliphages by the MF plant was investigated. The study showed that membrane fouling was beneficial for removing coliphages by MF. It was also shown that the removal of coliphages by MF was initially governed by adsorption on membrane surface or in membrane pores. As the membrane fouled, however, the removal of coliphages was primarily governed by direct interception on the cake layer formed on the surface of the membrane. Increases in feed coliphage concentrations resulted in the passage of larger numbers of coliphages when the MF was clean but had little impact on the passage of coliphages when the membrane became fouled. Increasing permeate flux lowered log-removal values (LRVs) for the clean membrane but resulted in an initial increase in LRVs for the fouled membrane followed by a drop in LRVs with further increases in permeate flux.

Coliphages↗

Male-specific coliphages as an additional fecal contamination indicator for screening fresh carrots.

The objective of this study was to evaluate the efficacy of male-specific (F+) coliphages as a fecal-contamination indicator for fresh carrots. The prevalence of specific pathogens and indicator organisms on the surface of carrots obtained from a farm, truck, and processing shed was studied. Twenty-five carrot samples collected from each of these locations were washed, and aliquots of the wash were analyzed for the presence of F+ coliphages, Escherichia coli, Salmonella, and Shigella. Additionally, the Salmonella isolates were genotyped using pulsed-field gel electrophoresis (PFGE). Our studies detected the presence of F+ coliphages, E. coli, and Salmonella on carrots. All samples, however, tested negative for Shigella. Although none of the carrot samples from the field were positive for E. coli, one sample was positive for Salmonella, and another was positive for F+ coliphages. From the truck, two carrot samples (8%) were positive for Salmonella, four (16%) were positive for F+ coliphages, and four (16%) were positive for E. coli. None of the carrot samples from the processing shed were positive for Salmonella. However, 2 carrot samples (8%) were positive for E. coli, and 14 carrot samples (56%) were positive for F+ coliphages. The PFGE results suggest that there were three distinct Salmonella genotypes among the carrot samples from the truck and that the Salmonella isolates identified on carrot samples from the field and truck locations were different. Microbiological screening of fresh produce such as carrots (which can be exposed to fecal contaminants in soils and water) should ensure the detection of both viral and bacterial contaminants. Overall, in this study, F+ coliphages were detected in 25% of the carrot samples, compared to E. coli (8%), Salmonella (4%), and Shigella (0%). The results suggest F+ coliphages can serve as a conservative indicator of fecally associated viruses on carrots. This suggests that in addition to E. coli screening, F+ coliphages should be included when produce such as carrots that are vulnerable to fecal contaminants are screened. Since the detection of specific enteric viral pathogens is expensive, screening for viral indicators of fecal contamination using F+ coliphages can be an economical approach to providing an additional level of assurance about the microbiological quality of fresh carrots.

Coliphages↗

Pretreatment to reduce somatic Salmonella phage interference with FRNA coliphage assays : successful use in a one-year survey of vulnerable groundwaters.

Somatic salmonella (SS) phages were commonly found in higher numbers than F-specific RNA (FRNA) coliphages in a multi-site survey of contamination-vulnerable groundwaters. The relative abundance of SS phages required that a pretreatment procedure be implemented to reduce the SS phage content of samples before FRNA coliphage assay with Salmonella typhimurium WG49. Pretreatment involved selective SS phage removal by Salm. typhimurium WG45 cells. This pretreatment proved effective in producing interference-free samples throughout the one-year survey period and in seeded evaluation, was shown not to affect the detection of representative FRNA coliphage MS2. During the survey, 30 groundwater sites located in the continental United States, Puerto Rico and the Virgin Islands were examined for FRNA coliphages and SS phages at monthly intervals. FRNA coliphages were detected at six of the 30 sites and in 33 of 329 monthly samples. SS phages were also detected at six sites and in 28 of 329 monthly samples. Five of the phage-positive sites were positive for both phage groups. At those five sites, 58 monthly samples were collected during the survey period. Those 58 samples yielded an average FRNA coliphage concentration of 140 pfu per 100 l of groundwater as compared to an average SS phage concentration of 565 pfu per 100 l of groundwater. Twenty of the 58 samples were positive for both the FRNA coliphages and SS phages. In those samples, FRNA coliphages were more abundant in five samples; SS phages were more abundant in 15 samples. Because these results demonstrate that SS phage levels may often exceed FRNA coliphage levels in environmental waters, it is clear that SS phage removal procedures will greatly enhance the effectiveness of the WG49-based FRNA coliphage assay.

Bacteriophages↗

Nucleotide sequence of a single-stranded RNA phage from Pseudomonas aeruginosa: kinship to coliphages and conservation of regulatory RNA structures.

We report the complete nucleotide sequence of the single-stranded RNA phage PP7 from Pseudomonas aeruginosa. There are three open reading frames which code for apparent protein homologues of the single-stranded RNA coliphages, i.e., maturation protein, coat protein, and replicase. A fourth overlapping reading frame exists that probably encodes a lysis protein, similar to what has been found in the group A coliphages such as MS2. The genetic map of PP7 is colinear with group A coliphages and we accordingly classify the phage as a levivirus. There is, generally speaking, no significant nucleotide sequence identity between PP7 and the coliphages except for a few regions where homologous parts of proteins are encoded, most notable in the replicase gene. In these regions the nucleotide sequence similarity between PP7 and MS2 is no greater than between PP7 and the group B coliphages such as Q beta. Surprisingly, Q beta and MS2 are no closer to each other than they are to PP7. Several regulatory RNA secondary structure features that are present in the coliphages were identified also in PP7 RNA although the sequences involved cannot be aligned. Among these are the coat protein binding helix at the start of the replicase gene, structures at the 5' and 3' terminus of the RNA, a replicase binding site, and the structure of the coat protein cistron start. Some of these features resemble MS2 type coliphages but others the Q beta type. These findings suggest that PP7 is related to the coliphages but branched off before the coliphages diverged into separate groups.

Amino Acid Sequence↗

Methodology for enumeration of coliphages in foods.

The effects of eluent composition, pH, and chaotropic agents on the recovery of T2, MS2, and indigenous coliphages from various foods were investigated. Additionally, methods of sample suspension and clarification were evaluated for coliphage recovery and application to various foods. Clarified sample suspensions were assayed for coliphages with a modified agar layer technique and appropriate Escherichia coli hosts. Centrifugation and polypropylene mesh filtration were more rapid and effective than glass wool filtration for clarification of sample suspensions and subsequent recovery of coliphages. Blending, stomaching, and shaking procedures were generally comparable for sample liquefaction and release of coliphages from foods. Complex basal eluents, EC medium and 1% casein, were generally more effective than a less complex eluent, phosphate buffer, for elution of coliphages from foods. For most foods, incorporation of sodium chloride or chaotropic agents, i.e., sodium trichloroacetate, urea, Tween 80, Triton X-100, and sodium nitrate, into basal eluents did not enhance recovery of coliphages. Indigenous coliphage recovery was not affected by sample suspension pH over a range of 6.0 to 9.0. With an optimal procedure, i.e., EC medium eluent, blending, and centrifugation, the recovery of T2 and MS2 ranged from 48 to 81% and from 58 to 100%, respectively, depending on the food type.

Animals↗

Animal viruses, coliphages, and bacteria in aerosols and wastewater at a spray irrigation site.

Aerosol samples collected at the Muskegon County Wastewater Management System Number 1 spray irrigation site in Michigan by using the Army prototype XM2 Biological Sampler/Collector were examined for the presence of animal viruses, coliphages, and bacteria. Air samples, collected in Earle lactalbumen hydrolysate, and wastewater samples were filtered through a 0.45- and 1.2-micron membrane filter sandwich, pretreated with 10% beef extract (pH 7.0), and assayed for animal viruses by the plaque method on Buffalo green monkey kidney cells. Untreated air and wastewater samples were assayed for coliphages by the soft agar overlay method with three Escherichia coli hosts (ATCC 13706, 15597, and 11303) and for bacteria by the heterotrophic plate count method. Filtered air samples were assayed for coliphages by the most-probable-number method with the same three hosts. Although no animal viruses were detected in the aerosol samples, coliphages and bacteria were recovered. E. coli ATCC 13706 coliphage were recovered more often and in greater numbers than either of the other two types of coliphages. Concentrations of animal viruses, coliphages, and bacteria detected in the raw influent decreased as the wastewater was aerated and stored in the lagoons. No animal viruses were detected in the wastewater at the pump station just before distribution to the spray irrigation rigs. The most-probable-number method was more sensitive and consistent than the overlay procedure in detecting low levels of coliphages in air samples.

Aerosols↗