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PubMed2004

Fate of Salmonella following application of swine manure to tile-drained clay loam soil.

Land application of animal manure is an important means of utilization of nitrogen and phosphorus. However, the presence of pathogens in manure and their occasional leaching into subsurface water has become a topic of concern during the past few years. This study was undertaken to determine the extent to which Salmonella anatum may leach through tile-drained clay loam soil on which swine manure has been applied. For this purpose, swine manure was experimentally contaminated with S. anatum and applied to three tile-drained plots in winter of 2001 while another three plots served as negative controls. Following rainfall events in the spring of 2002 the tiles started to flow and leachate samples of subsurface water were collected at various time intervals and tested for S. anatum. Salmonella anatum was not found to leach into the subsurface drainage water indicating that it was either retained in the upper layers of soil or did not survive over winter. The leaching of faecal coliforms and coliphages was also tested. Faecal coliforms and coliphages were detected in the subsurface water from both manure and control plots, indicating the ability of the tile drainage system to transport these organisms to groundwater as the water percolates through the soil. Additional temporal studies over a longer time period are needed to determine the survival and leaching of pathogens and indicators into subsurface water.

Agriculture↗
PubMed1990

[Comparative evaluation of Escherichia coli strains as markers for the study of bacteriophages].

The count of coliphages in polluted waters was found to be dependent on many experimental factors. The host-strain used for their enumeration is among the most important. In this paper we report a comparative investigation on the variability of counts of coliphages in sewage as a result of variations in host-strains of Escherichia coli and in methodologies. Two methods were used for enumerating them: the M.P.N. and the direct count. E. coli C, B1, Hfr and E36 consistently produced more plaques than any other host tested.

Blood Coagulation Factors↗
PubMed1994

[Reduction of bacteria and viruses in purification plant discharges using microfiltration in a model plant].

Microfiltration through a membrane matrix of a nominal pore size of 0.2 microns has been applied for advanced treatment of mechanically and biologically treated wastewater. Elimination of bacteria and coliphages as well as the decrease in some chemical constituents were studied at a flow rate of 80 l/h. Microfiltration resulted in a reduction of E. coli, coliform bacteria, fecal streptococci and of coliphages by more than 4 logs in the filtrate. Thus, the quality requirements of EC Directives for bathing water (EC, 76/160) could be maintained. As a result of microfiltration, a 43 +/- 13% removing of the total phosphorous compounds (Pt) in the pre-filtered secondary effluent from 11 +/- 2 mmol/m3 (0.34 +/- 0.06 mg/l) to 6 +/- 2 mmol/m3 (0.19 +/- 0.06 mg/l) was measured. The dosage of FeCl3 (between 30 and 150 mmol/m3) as coagulant before microfiltration improved the reduction of Pt to result in an average value of 75 +/- 16%. After adding FeCl3, orthophosphates (PO4-P) could be efficiently reduced by microfiltration. Thus, PO4-P concentrations in effluent samples were, in most cases, below the detection limit (0.01 mmol/m3).

Bacteria↗
PubMed2002

Compact form of DNA induced by DNA-binding protein HU.

Interaction of DNA-binding protein HU from Bacillus stearothermophilus (HUBst) with coliphage T2 DNA was investigated by means of a single-duplex DNA chain visualization method using fluorescence microscopy. Fluorescence microscopic images of coliphage T2 DNA molecules were observed as a function of HUBst concentration. The average fluorescence image size of T2 DNA decreased with increase in HUBst concentration to a size comparable to that of a DNA globule induced by polyethylene glycol (PEG) and multivalent cation (MVC). The change to globule-like DNA proceeded gradually and monotonously, in contrast to the coil-globule transition of DNA induced by PEG and MVC. The histogram of the fluorescence image length was essentially a single-modal one throughout the process of conformational change. These results indicate that the process of shrinking of DNA from a random coil to a globule-like one is not of a transitional nature. The interaction of HUBst with DNA and the mechanism of shrinkage are concluded to be different from those of PEG-induced and MVC-induced coil-globule transition of DNA.

Bacterial Proteins↗
PubMed1995

Secondary structure model for the last two domains of single-stranded RNA phage Q beta.

We have determined the nucleotide sequence of three positive single-stranded RNA coliphages and have used this information, together with the known sequences of the related phages Q beta and SP, to construct a secondary structure model for the two distal domains of Q beta RNA. The 3' terminal domain, which is about 100 nucleotides long, contains most of the 3' untranslated region and folds into four short, regular hairpins. The adjacent 3' replicase domain contains about 1100 nucleotides. Hairpins in this protein-coding domain are much longer and more irregular than in the 3' untranslated region. Both domains are defined by long-distance interactions. The secondary structure is not a collection of hairpin structures connected by single-stranded regions. Rather, the RNA stretches between the stem-loop structures are all involved in an extensive array of long-distance interactions that contract the molecule to a rigid structure in which all hairpins are predicted to have a fixed position with respect to each other. A general feature of the model is that helices tend to be organized in four-way junctions with little or no unpaired nucleotides between them. As a result, there is a potential for coaxial stacking of adjacent stems. The essential features of the model are supported by the S1 nuclease cleavage pattern. Viral RNA sequences are strongly constrained by their coding function. As a result, structural evolution by simple base-pair substitution is not always possible, as this usually requires the juxtaposition of the codon wobble positions in stems. Rather, we often observe co-ordinate base substitutions that maintain the stem, but tend to change the position at which bulges or internal loops are found. Structures that differ in this way are apparently equally fit. Also, the relative position of hairpin loops can shift several nucleotides through an alignment based on maximal sequence identity i.e. amino acid homology. The fact that these structural irregularities do not occur at the 3' untranslated region suggests indeed that the coding function of the RNA constrains the secondary structure. Hairpins with the stable tetraloop motif GNRA and UNCG or their complement are over-represented. This suggests their involvement in segregation of plus and minus strand. The genome of the coliphages contains a well-defined high affinity binding site for the coat protein, which serves to suppress replicase translation and also acts as a nucleation point in capsid formation. Close to the 3' end we find an additional conserved helix that meets the described consensus criteria for coat-protein binding.

Allolevivirus↗
PubMed2001

Phylogeny, genome evolution, and host specificity of single-stranded RNA bacteriophage (family Leviviridae).

Bacteriophage of the family Leviviridae have played an important role in molecular biology where representative species, such as Q beta and MS2, have been studied as model systems for replication, translation, and the role of secondary structure in gene regulation. Using nucleotide sequences from the coat and replicase genes we present the first statistical estimate of phylogeny for the family Leviviridae using maximum-likelihood and Bayesian estimation. Our analyses reveal that the coliphage species are a monophyletic group consisting of two clades representing the genera Levivirus and Allolevivirus. The Pseudomonas species PP7 diverged from its common ancestor with the coliphage prior to the ancient split between these genera and their subsequent diversification. Differences in genome size, gene composition, and gene expression are shown with a high probability to have changed along the lineage leading to the Allolevivirus through gene expansion. The change in genome size of the Allolevivirus ancestor may have catalyzed subsequent changes that led to their current genome organization and gene expression.

Allolevivirus↗
PubMed1990

Detection of protein-DNA complex formation by time-resolved fluorescence depolarization of bound ethidium bromide.

We introduce the use of time-resolved fluorescence spectroscopy to probe the interaction between gene regulatory proteins and DNA. Changes in the decay kinetics of fluorescence polarization anisotropy of ethidium bromide bound to DNA segments report changes in hydrodynamic volume and shape which occurs upon complex formation between protein and DNA. We have used the decay of fluorescence polarization anisotropy as a spectroscopic handle on the interaction between several site-specific DNA-binding proteins involved in transcriptional regulation (the cro repressor of coliphage lambda, the lac repressor of Escherichia coli, and the RNA polymerase of coliphage T7) and their target DNA fragments ranging in length from 17 to 36 base pairs. The technique allows one to follow complex formation while varying solution conditions such as temperature, pH, ionic strength, and presence of effector molecules. Macromolecular concentrations ranging from 10(-7) to 10(-4) M can be used, allowing estimates of relative binding affinities. The magnitude of the observed rotational correlation times (phi obs) can be used to infer information about the size and shape of the complexes.

Base Sequence↗
PubMed1990

The presence of bacterial virus in groundwater and treated drinking water.

Ten raw urban well water samples and twelve water samples collected from distribution lines after the well waters were treated were examined for bacteriological and coliphage/bacteriophage populations. The raw well waters were found to contain <1/100 ml total coliforms and fecal streptococci, but they all contained varying concentrations of coliphage and bacteriophage. The treated waters were all found to have <1 total coliforms and fecal streptococci per 100 ml with the exception of one treated water sample from Community C. However, even though the treated water samples contained free and total chlorine levels varying from 0.05 to 1.5 ppm, they were all found to contain usually greater amounts of coliphage and bacteriophage than the raw well waters.

Journal Article↗
PubMed2006

Plasticity of the gene functions for DNA replication in the T4-like phages.

We have completely sequenced and annotated the genomes of several relatives of the bacteriophage T4, including three coliphages (RB43, RB49 and RB69), three Aeromonas salmonicida phages (44RR2.8t, 25 and 31) and one Aeromonas hydrophila phage (Aeh1). In addition, we have partially sequenced and annotated the T4-like genomes of coliphage RB16 (a close relative of RB43), A. salmonicida phage 65, Acinetobacter johnsonii phage 133 and Vibrio natriegens phage nt-1. Each of these phage genomes exhibited a unique sequence that distinguished it from its relatives, although there were examples of genomes that are very similar to each other. As a group the phages compared here diverge from one another by several criteria, including (a) host range, (b) genome size in the range between approximately 160 kb and approximately 250 kb, (c) content and genetic organization of their T4-like genes for DNA metabolism, (d) mutational drift of the predicted T4-like gene products and their regulatory sites and (e) content of open-reading frames that have no counterparts in T4 or other known organisms (novel ORFs). We have observed a number of DNA rearrangements of the T4 genome type, some exhibiting proximity to putative homing endonuclease genes. Also, we cite and discuss examples of sequence divergence in the predicted sites for protein-protein and protein-nucleic acid interactions of homologues of the T4 DNA replication proteins, with emphasis on the diversity in sequence, molecular form and regulation of the phage-encoded DNA polymerase, gp43. Five of the sequenced phage genomes are predicted to encode split forms of this polymerase. Our studies suggest that the modular construction and plasticity of the T4 genome type and several of its replication proteins may offer resilience to mutation, including DNA rearrangements, and facilitate the adaptation of T4-like phages to different bacterial hosts in nature.

Amino Acid Sequence↗
PubMed2005

Inactivation of particle-associated viral surrogates by ultraviolet light.

This study investigated whether colloid-sized particles can enmesh and protect viruses from 254-nm ultraviolet (UV) light and sought to determine the particle characteristics (e.g. size, chemical composition) that are most relevant in causing a protective effect. Two viral surrogates (MS2 coliphage and bacteriophage T4), three types of particles (kaolin clay, humic acid powder, and activated sludge), two coagulants (alum and ferric chloride), two filtration conditions (none and 0.45 microm), and two UV doses (40 and 80 mJ/cm2 for MS2 coliphage; 2 and 7 mJ/cm2 for bacteriophage T4) were considered in a series of bench-scale UV collimated beam experiments. Transmission electron microscopy was used to qualitatively confirm the phage particle-association after coagulation. Humic acid and activated sludge floc particles shielded both viral surrogates to a statistically significant degree (with >99% confidence) relative to particle-free control conditions, while the kaolin clay particles provided no significant protection. The results of the study suggest that particles <2 microm in diameter are large enough to protect viruses from UV light and that particulate chemical composition (e.g. UV-absorbing organic content) may be a critical factor in the survival of particle-associated viruses during UV disinfection.

Alum Compounds↗
PubMed2002

Complete nucleotide sequence and likely recombinatorial origin of bacteriophage T3.

We report the complete genome sequence (38,208 bp) of bacteriophage T3 and provide a bioinformatic comparative analysis with other completely sequenced members of the T7 group of phages. This comparison suggests that T3 has evolved from a recombinant between a T7-like coliphage and a yersiniophage. To assess this, recombination between T7 and the Yersinia enterocolitica serotype O:3 phage phiYeO3-12 was accomplished in vivo; coliphage progeny from this cross were selected that had many biological properties of T3. This represents the first experimentally observed recombination between lytic phages whose normal hosts are different bacterial genera.

Amino Acid Sequence↗
PubMed2002

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.

Bacteriophages↗
PubMed2003

Faecal contamination of greywater and associated microbial risks.

The faecal contamination of greywater in a local treatment system at Vibyåsen, 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.

Bacteria↗
PubMed2003

Fate of physical, chemical, and microbial contaminants in domestic wastewater following treatment by small constructed wetlands.

In order to evaluate the efficacy of constructed wetlands for treatment of domestic wastewater for small communities located in rural areas, small-scale wetland mesocosms (400 L each) containing two treatment designs (a mixture of Typha, Scirpus, and Juncus species; control without vegetation) were planted into two depths (45 or 60 cm) with pea gravel. Each mesocosm received 19 L/day of primary-treated domestic sewage. Mesocosms were monitored (inflow and outflow samples) on a monthly basis over a 2-year period for pH, total suspended solids (TSS), 5-day biochemical oxygen demand (BOD(5)), total Kjeldahl nitrogen (TKN), dissolved oxygen (DO), and conductivity. Microbiological analyses included enumeration of fecal coliforms, enterococci, Salmonella, Shigella, Yersinia, and coliphage. Significant differences between influent and effluent water quality for the vegetated wetlands (p<0.05) were observed in TSS, BOD(5), and TKN. Increased DO and reduction in fecal coliform, enterococcus, Salmonella, Shigella, Yersinia, and coliphage populations also were observed in vegetated wetlands. Greatest microbial reductions were observed in the planted mesocosms compared to those lacking vegetation. Despite marked reduction of several contaminants, wetland-treated effluents did not consistently meet final discharge limits for receiving bodies of water. Removal efficiencies for bacteria and several chemical parameters were more apparent during the initial year compared to the second year of operation, suggesting concern for long-term efficiency and stability of such wetlands.

Ecosystem↗
PubMed2002

Depuration dynamics of viruses in shellfish.

The consumption of shellfish has been associated with viral infections even in cases where shellfish complied with the current regulation, which is based on bacterial analysis. In this study, depuration rates of potential indicators and human viruses have been analysed in order to study the use of complementary parameters for evaluating the microbiological quality of depurated shellfish. Depuration of naturally highly polluted mussels has been evaluated and analyses for Escherichia coli, Clostridium perfringens, somatic coliphages, F-RNA phages and bacteriophages infecting Bacteroides fragilis RYC2056 and HSP40, human adenovirus, enterovirus have been done. Seawater of the depuration tank was disinfected by UV irradiation, ozone and passed through a skimmer and a biological filter. The correct functioning of the depuration tank was monitored by the quantification of total organic carbon (TOC), NH4+ and total aerobic bacteria in the seawater. To study the relation between the bacteriophages and the human viruses analysed, a logistic regression model was applied. F-RNA phages are significantly related to human adenoviruses and enteroviruses. Thus, they can be used as a complementary parameter for evaluating the efficiency of the depuration treatment. Somatic coliphages are also significantly associated with enteroviruses. Bacteriophages infecting B. fragilis HSP40 were analysed by the double-agar-layer (DAL) method, which quantifies infectious viruses, and by nested PCR, which detects the presence of the genome of these phages. The highest sensitivity of the molecular techniques was demonstrated and the results obtained are an indicator of a close relation between positive results by PCR and the presence of infectious viral particles in shellfish. All shellfish samples were negative for human viruses by PCR after 5 days of depuration treatment and the results obtained applying a regression model also showed negative results or nearly for F-RNA phages and bacteriophages infecting B. fragilis RYC2056. Thus, in this specific depuration treatment, 5 days may be necessary to assess the sanitary quality of shellfish.

Adenoviridae↗
PubMed1997

Molecular analysis of a filamentous phage (fsl) of Vibrio cholerae O139.

A filamentous bacteriophage from Vibrio cholerae O139 strain A1-4450 was isolated (fsl). The phage fsl had a ssDNA genome and dsDNA as a replicative form (RF) in lysogenic host cell. The DNA sequence of fsl RF was determined. It consisted of 6340 bp and had a G + C content of 43.5%. Fifteen possible ORFs were found in fsl. One of them, ORF384, was estimated to encode 384 amino acid residues (44.6 kDa) and had homologous regions with the zot gene of V. cholerae and gene I of the coliphage group. ORF104, located upstream of ORF384, was homologous to gene 93 protein of Pf3 (filamentous phage of Pseudomonas sp.) corresponding to gene VI of coliphage. Other than ORF384 and ORF104, the ORF81, ORF44, ORF29, and ORF193 were speculated to correspond to gene V, gene VII, gene IX, and gene III, respectively, in the order as reported on f1 phage.

Amino Acid Sequence↗
PubMed2003

Bacteriophages as viral indicators for radiation processing of water: a chemical approach.

Inactivation of the bacteriophages PHI X 174 (somatic coliphage), MS2 (F-specific coliphage) and B40-8 (phage infecting Bacteroides fragilis) suspended in tap water was studied applying gamma and electron beam irradiation as well. PHI X 174 phage was found to be a suitable viral indicator for water disinfection by means of ionizing radiation. The nutrient broths introduced simultaneously with the bacteriophages into the water when it is spiked with the phages for the experiments did not significantly change the scavenging capacity of the water matrix. No dose rate effect was observed with MS2 and B40-8 phages but PHI X 174 phage showed a clear dose rate effect. It was found that in water MS2 phage is significantly more sensitive to ionizing radiation than Escherichia coli.

Bacteriophages↗
PubMed2005

Microbial characterization and population changes in nonpotable reclaimed water distribution systems.

Changes in the microbial quality of nonpotable reclaimed water distribution systems in seven metropolitan areas of the southwestern United States were investigated by performing pathogen monitoring and bacterial growth. Water samples were collected from tertiary-treated effluents at point of entry and point of use in the distribution systems. The samples were analyzed for Cryptosporidium oocysts, Giardia cysts, enteroviruses, microbial indicators, and assimilable organic carbon (AOC). Cryptosporidium and Giardia were detected in 16% (12/77) and 43% (33/77) of nonpotable reclaimed water samples, whereas no infectious Cryptosporidium parvum were detected in any of the samples. No infectious enteroviruses were detected in any sample (0/27). At point of entry total coliform and fecal coliforms ranged from 0.7 to 280 and from 0 to 1.9 colony-forming units (cfu)/100 mL, respectively. Increases in the number of coliforms were observed as water passed through the reclaimed water distribution systems. However, no such increase in the number of coliphages was found. The chlorination practices at some utilities were not sufficient to inactivate coliforms and coliphages, but supplemental ultraviolet disinfection resulted in lower numbers of these microbial indicators. AOC levels decreased by 3-fold as water passed through the distribution systems, which inversely correlated with bacterial regrowth.

Animals↗