T-even-related bacteriophages as candidates for treatment of Escherichia coli urinary tract infections.

Multidrug-resistant uropathogenic Escherichia coli (UPEC) is increasing gradually on a worldwide scale. We therefore examined the possibility of bacteriophage (phage) therapy for urinary tract infections (UTIs) caused by the UPEC strains as an alternative to chemotherapy. In addition to the well-known T4 phage, KEP10, which was newly isolated, was used as a therapeutic phage candidate. KEP10 showed a broader bacteriolytic spectrum (67%) for UPEC strains than T4 (14%). Morphological and genetic analyses showed that KEP10 resembles phage T4. Phages T4 and KEP10 injected into the peritoneal cavity of mice were distributed immediately to all organs examined and maintained a high titer for at least 24 h. They were stable in the urine of both mice and humans for 24 h at 37 degrees C. Administration of these phages into the peritoneal cavity caused a marked decrease in the mortality of mice inoculated transurethrally with a UPEC strain, whereas most of the control mice died within a few days of bacterial infection. Inoculation with phage alone produced no adverse effects attributable to the phage per se. The present study experimentally demonstrated the therapeutic potential of phage for E. coli-induced UTIs, and T-even-related phages may be suitable candidates with which to treat them.

Isolation and characterization of a new T-even bacteriophage, CEV1, and determination of its potential to reduce Escherichia coli O157:H7 levels in sheep.

Bacteriophage CEV1 was isolated from sheep resistant to Escherichia coli O157:H7 colonization. In vitro, CEV1 efficiently infected E. coli O157:H7 grown both aerobically and anaerobically. In vivo, sheep receiving a single oral dose of CEV1 showed a 2-log-unit reduction in intestinal E. coli O157:H7 levels within 2 days compared to levels in the controls.

Enhancement of solar water pasteurization with reflectors.

A simple and reliable method that could be used in developing countries to pasteurize milk and water with solar energy is described. A cardboard reflector directs sunshine onto a black jar, heating water to pasteurizing temperatures in several hours. A reusable water pasteurization indicator verifies that pasteurization temperatures have been reached.

The removal of phages T1 and PP7, and poliovirus from fluids with hollow-fiber ultrafilters with molecular weight cut-offs of 50,000, 13,000, and 6000.

We tested the ability of hollow-fiber ultrafilters with molecular weight cut-offs (MWCOs) of 50,000, 13,000, and 6000 to remove and detect viral agents (phage T1, 50-150 nm; phage PP7, poliovirus, 28-30 nm) from ultrapure water, 0.85% saline with 1% trypticase soy broth, and Dulbecco's modified Eagle minimum essential medium with 10% fetal bovine serum (DMEM-10). Virus diluted in saline and DMEM-10 were tested to evaluate filter performance under conditions that minimize the adsorption of viral particles to the filter matrix. During filtration, the retentate was returned to the input reservoir, and the permeate was removed to a separate vessel. Thus, the virus concentration in the feed increased over the course of filtration. Filter performance was evaluated by comparing the concentration of infectious virus in the initial virus suspension with the virus concentration in the permeate and retentate. Very efficient removal of phages T1 and PP7 was observed with the filters with MWCOs of 13,000 and 6000 (titer reduction > 7 logs) for all three fluids tested. No poliovirus was detected in the permeate of the ultrafilters with MWCOs of 13,000 or 6000 (titer reduction > 6 logs). These results indicate that the ultrafilters with MWCOs of 13,000 and 6000 were very effective in removing small viral particles (25-30 nm) by size exclusion. The recovery efficiency of the virus in the retentate varied by fluid type. However, filtration with virus diluted in DMEM-10 resulted in consistent recovery of the viruses tested. The results suggest that these ultrafilters may have the dual potential of removing viral contaminants from fluids and concentrating virus in the retentate.

Mutations that eliminate the requirement for the vertex protein in bacteriophage T4 capsid assembly.

The capsid of bacteriophage T4 is composed of two essential structural proteins, gp23, the major constituent of the capsid, and gp24, a less prevalent protein that is located in the pentameric vertices of the capsid. gp24 is required both to stabilize the capsid and to allow it to be further matured. This requirement can be eliminated by bypass-24 (byp24) mutations within g23. We have isolated, cloned and sequenced several new byp24 mutations. These mutations are cold-sensitive in the absence of gp24, and are located in regions of g23 not known to contain any other mutations affecting capsid assembly. The cold-sensitivity of the byp24 mutations can be reduced by further mutations within g23 (trb mutations). Cloning and sequencing of these trb mutations has revealed that they lie in regions of g23 that contain clusters of mutations that cause the production of high levels of petite and giant phage (ptg mutations). Despite the proximity of the trb mutations to the ptg mutations, none of the ptg mutations has a Trb phenotype. The mutation ptE920g, which is also located near one of the ptg clusters, and which produces only petite and wild-type phage, has been shown to confer a Trb but not a Byp24 phenotype. The relevance of these observations to our understanding of capsid assembly is discussed.

Removal of micro-organisms by filtration through unwoven cloth coated with a pyridinium-type polymer.

Unwoven cloth coated with 32 mg/g of a copolymer of N-benzyl-4-vinyl-pyridinium chloride and styrene was found to be effective in removing micro-organisms from water. In experiments demonstrating removal of Escherichia coli by filtration through ten sheets of the unwoven cloth, the rate of removal was 99.99% at a filtration rate of 2.6 cm/h, and remained at 99% even at a high filtration rate of 300 cm/h and a low influent concentration of the bacterial cells such as 10(3) cells/ml. The rate of removal tended to increase with a decrease in the influent bacterial concentration. Seven other bacteria and two yeasts were effectively removed by filtration through the unwoven cloth. Filtration through the unwoven cloth was also effective in removing spores of fungi from water but was not very effective in removing bacteriophage T4 from aqueous solution.

Important factors for testing barrier materials with surrogate viruses.

This study evaluated bacteriophages phi X174, T7, PRD1, and phi 6 as possible surrogates for pathogenic human viruses to challenge barrier materials and demonstrated some important factors for their use. Chemical incompatibility with test material was demonstrated when lipid-enveloped phi 6 was inactivated by an aqueous eluate of vinyl gloves, but 0.5% calf serum protected phi 6 from the eluate. Low concentrations (2%) of calf serum also prevented the exaggerated binding of the bacteriophages to filters. Recovery of viruses from surfaces decreased with increasing time before recovery. Penetration through punctures displayed different types of kinetics. The combined data indicate that (i) some bacteriophages may serve as surrogate viruses, (ii) experimental conditions determine whether a particular virus is appropriate as a challenge, and (iii) phi X174 is an excellent choice as a surrogate virus to test barrier materials. The data further indicate that before barrier materials are challenged with viruses, adequate tests should be performed to ensure that the virus is compatible with the test material and test conditions, so that meaningful data will result.

Concentration of viruses and dissolved DNA from aquatic environments by vortex flow filtration.

Vortex flow filtration (VFF) was used to concentrate viruses and dissolved DNA from freshwater and seawater samples taken in Florida, the Gulf of Mexico, and the Bahamas Bank. Recoveries of T2 phage and calf thymus DNA added to artificial seawater and concentrated by VFF were 72.8 and 80%, respectively. Virus concentrations determined by transmission electron microscopy of VFF-concentrated samples ranged from 3.4 x 10(7)/ml for a eutrophic Tampa Bay sample to 2.4 x 10(5) for an oligotrophic oceanic surface sample from the southeastern Gulf of Mexico. Viruslike particles were also observed in a sample taken from a depth of 1,500 m in the subtropical North Atlantic Ocean. Filtration of samples through Nuclepore or Durapore filters (pore size, 0.2 micron) prior to VFF reduced phage counts by an average of two-thirds. Measurement of dissolved-DNA content by Hoechst 33258 fluorescence in environmental samples concentrated by VFF yielded values only ca. 35% of those obtained for samples concentrated by ethanol precipitation (the standard dissolved-DNA method). However, ethanol precipitation of VFF-concentrated extracts resulted in an increase in measurable DNA, reaching 80% of the value obtained by the standard method. These results indicate that a portion of the naturally occurring dissolved DNA is in a form inaccessible to nucleases and Hoechst stain, perhaps bound to protein or other polymeric material, and is released upon ethanol precipitation. Viral DNA contents estimated from viral counts averaged only 3.7% (range, 0.9 to 12.3%) of the total dissolved DNA for samples from freshwater, estuarine, and offshore oligotrophic environments.(ABSTRACT TRUNCATED AT 250 WORDS)

[DNA-protein cross-links as a possible reason for genomic damage during its protonation]. / DNK-belkovye sshivki kak vozmozhnaia prichina povrezhdenii genoma pri ego protonirovanii.

The change in survival of bacteriophages with DNA of different GC-contents after their incubation in media of different acidities with subsequent neutralization was studied. It was shown that the higher the GC-content, the more sensitive is the phage to the action of H(+)-ions. Evidence is presented that the acidic inactivation of virions is not connected with the helix-coil transition of the intraphage DNA due to its protonation. The extractability of DNA from phages subjected to different concentrations of H(+)-ions with subsequent neutralization of the medium to pH 8 was determined. The changes in: transfection ability, UV-spectra, the quantity of the residual proteins, and the contents of glutamic and lysine amino acid residues in these proteins were investigated. The effect of glutamic acid on the parameters of DNA melting curves was followed for different pH values. Proceeding from the data obtained, we concluded that acidification of the medium from neutral tp pH congruent to 4 leads to formation of non-covalent DNA-protein cross-links due to interaction of the GC base pairs of DNA with glutamic and aspartic amino acid residues, whereas acidification of the medium to pH less than 4 with subsequent neutralization to pH 8 results in the formation of covalent DNA-protein cross-links of Schiff base type. The influence of non-covalent DNA-protein cross-links on the properties of DNA and their regulatory role in genome functioning are discussed.

Evaluation of an Escherichia coli host strain for enumeration of F male-specific bacteriophages.

A method was developed for the selective enumeration of F male-specific bacteriophages in samples of environmental waters. The host strain for the phages, Escherichia coli HS(pFamp)R, has three antibiotic resistance markers, ampicillin on the Famp plasmid, which codes for pilus production, and streptomycin and nalidixic acid on the chromosome. The strain is resistant to coliphages T2 to T7 and phi X174. More than 95% of the phages from environmental samples which plaqued on the host strain were F male specific. The host bacterium had a higher plaquing efficiency than E. coli K-12 Hfr for F-specific phages in stock suspensions and sewage effluents. The F male-specific phage levels in prechlorinated, secondary-treated sewage effluents generally were about 10(3) to 10(4) PFU/100 ml. The levels in the influents to the sewage treatment plants and in septic tank contents were about 10(5) PFU/100 ml. RNA-containing phages composed about 90% of the total F-specific phage population in sewage effluents.

Bacteriophage T7 DNA packaging. I. Plasmids containing a T7 replication origin and the T7 concatemer junction are packaged into transducing particles during phage infection.

Bacteriophage T7 DNA is a linear duplex molecule with a 160 base-pair direct repeat (terminal redundancy) at its ends. During replication, large DNA concatemers are formed, which are multimers of the T7 genome linked head to tail through recombination at the terminal redundancy. We define the sequence that results from this recombination, a mature right end joined to the left end of T7 DNA, as the concatemer junction. To study the processing and packaging of T7 concatemers into phage particles, we have cloned the T7 concatemer junction into a plasmid vector. This plasmid is efficiently (at least 15 particles/infected cell) packaged into transducing particles during a T7 infection. These transducing particles can be separated from T7 phage by sedimentation to equilibrium in CsCl. The packaged plasmid DNA is a linear concatemer of about 40 x 10(3) base-pairs with ends at the expected T7 DNA sequences. Thus, the T7 concatemer junction sequence on the plasmid is recognized for processing and packaging by the phage system. We have identified a T7 DNA replication origin near the right end of the T7 genome that is necessary for efficient plasmid packaging. The origin, which is associated with a T7 RNA polymerase promoter, causes amplification of the plasmid DNA during T7 infection. The amplified plasmid DNA sediments very rapidly and contains large concatemers, which are expected to be good substrates for the packaging reaction. When cloned in pBR322, a sequence containing only the mature right end of T7 DNA is sufficient for efficient packaging. Since this sequence does not contain DNA to the right of the site where a mature T7 right end is formed, it was expected that right ends would not form on this DNA. In fact, with this plasmid the right end does not form at the normal T7 sequence but is instead formed within the vector. Apparently, the T7 packaging system can also recognize a site in pBR322 DNA to produce an end for packaging. This site is not recognized solely by a "headful" mechanism, since there can be considerable variation in the amount of DNA packaged (32 x 10(3) to 42 x 10(3) base-pairs). Furthermore, deletion of this region from the vector DNA prevents packaging of the plasmid. The end that is formed in vector DNA is somewhat heterogeneous. About one-third of the ends are at a unique site (nucleotide 1712 of pBR322), which is followed by the sequence 5'-ATCTGT-3'. This sequence is also found adjacent to the cut made in a T7 DNA concatemer to produce a normal T7 right end.

Temperature-sensitive gel for virus concentration from urine.

Cross-linked, partially hydrolyzed polyacrylamide gels (temperature-sensitive gels) with the property to swell at 4 degrees C and collapse at higher temperatures (greater than 19 degrees C) were used to concentrate bacteriophage T-2 from urine. Samples of urine, 50 ml, seeded with bacteriophage T-2 were reduced to approximately 5 ml, with an average virus recovery of 53%. Subsequent experiments with feline cell-associated herpes virus resulted in a 6-fold decrease in volume with 54% virus recovery. The gel could be used repeatedly without any loss in efficiency.

A simple method of distinguishing the bacterial viruses T3 and T7, and a critical reevaluation of their heterologous and homologous exclusion.

A method is presented allowing a clear distinction between bacterial viruses T3 and T7 by plating on selectively permissive host cells. The indicator strains are Escherichia coli cells containing either cloned pif genes (exclusively permissive for T3) or the EcoRV DNA restriction system (permissive only for T7): The efficiencies of plating of the two phages on these hosts differ by more than 8 orders of magnitude. This method was applied to reinvestigate the controversial question of mutual exclusion between T3 and T7. Under single-burst conditions, about 50% of coinfected cells (permissive for both viruses) produced T3 and T7 progeny while about 25% reproduced only T3 and about 25% only T7. The burst size of co-infected cells was slightly reduced, compared to controls infected with only one virus type. Homologous exclusion among T3 phages was also not seen; rather, there was a gene dosage effect: T3-encoded RNA polymerase activity as well as T3-specific RNA synthesis increased proportionally to the multiplicity of infection (2.5-20 plaque-forming units/cell).

A comparison of Selas and membrane filters for the sterilization of bacteriophage preparations.

Lysates of three different coliphage were sterilized by filtration through Selas, Millipore GVWP, and Millipore GS filters. Phage titers were comparable when either the Selas or Millipore GVWP (hydrophilic) filters were used; however, the GVWP filters were faster and could accommodate more lysate before the filters clogged. The Millipore GS (hydrophobic) filters were unsatisfactory.

Reactions of fish to microorganisms in wastewater.

Fish were inoculated with various microorganisms present in wastewater. A threshold concentration was determined over which these microorganisms were recovered from the muscles. The threshold concentrations were different for bacteria, bacteriophages, and polio 1 LSc virus. The threshold values were lower when fish were inoculated than when they were immersed in water containing these organisms. Depuration experiments were efficient when the fish did not contain high concentrations of bacteria in their muscles. As the threshold concentrations are an expression of the capability of the immune system of the fish, these values can be useful for the design and management of fishponds in which treated wastewater is used.

Specificity of virus adsorption to clay minerals.

Competitive adsorption studies indicated that reovirus type 3 and coliphage T1 did not share common adsorption sites on kaolinite and montmorillonite. Compounds in the minimal essential medium (e.g., fetal bovine serum, amino acids) in which the reovirus was maintained blocked adsorption of coliphage T1 to kaolinite and partially to montmorillonite in synthetic estuarine water, but they had no effect on coliphage adsorption to montmorillonite in distilled water or on the adsorption of the reovirus to either clay. The blockage of positively charged sites on kaolinite or montmorillonite by treatment of the clays with sodium metaphosphate or with the supernatants from montmorillonite or kaolinite, respectively, had no effect on adsorption of the reovirus. These data indicate that there was a specificity in adsorption sites for mixed populations of reovirus type 3 and coliphage T1 and emphasize the importance of using more than one type of virus, especially in combination, to predict virus behavior (e.g., adsorption, loss of infectivity) in soils and sediments containing clay minerals.

[Isolation of highly purified RNA ligase from bacteriophage T4]. / Vydelenie vysokoochishchennoi RNK-ligazy bakteriofaga T4.

A technique for isolation of RNA-ligase of bacteriophage T4 was proposed. It is mainly based on the using of Soviet materials and sorbents and includes seven purification stages. The technique enables to isolate about 80 000 units of active enzyme from 100 g of E. coli B cells infected with the phage. T4am N82; that makes up 20% of the activity of the cell extract. The obtained preparations of RNA-ligase are homogeneous by the data of electrophoresis and practically, free of endo- and exonuclease admixtures.

[Isolation of biologically active halves of the long tail fibers and whiskers of bacteriophage T4]. / Vydelenie biologicheski aktivnykh polovinok dlinnykh khvostovykh fibrill i bakenbard bakteriofaga T4.

The following substructural elements of bacteriophage T4 have been isolated in homogeneous and biologically active state: the whole long tail fibrils, distal and proximal halves of the long tail fibrils and whiskers. It has been shown that during the infection of Escherichia coli with T4 amber-mutants (defective in the genes coding for heads and tails), the major part of distal and proximal fibril halves found within the cell appears to be unassociated. The interaction of distal and proximal fibril halves with the bacteriophage particle is necessary for the whole tail fibril formation to proceed effectively.

[Changes in the nature of the sensitivity of an E. coli M strain carrying Inc-group R plasmids, to coliphages T3 and T7]. / Izmenenie prirody chuvstvitel'nosti shtamma E. coli M, nesushchego R-plazmidy nekotorykh Inc-grupp, k koli-fagam T i T7.

After the transfer of prototype plasmids R6K (IncX), R387 (IncK), R27 (IncH1) and T (IncN) to E. coli M nalr the appearance of histidine-dependent mutants (R27, T), histidine-leucine-dependent mutants (R6K), methionine-proline-dependent mutants (R387) was observed among the resulting transconjugates. The mutations of E. coli M nalr R+ cells induced by the introduction of the plasmids were accompanied by the transformation of the cells from the S-form into the R-form. In contrast to the prototrophs E. coli M nalr, the auxotrophs carrying plasmids R6K, R27, T acquired sensitivity to phage T7, and the methionine-proline-dependent mutant became sensitive to phages T and T7. The above-mentioned plasmids rendered E. coli M cells capable of synthetizing the donor pili. But the adsorption of phages T3 and T7 on the auxotrophic cells, both with and without plasmids, occurred due to their interaction with the cell-wall receptors.