Novel approach of using a cocktail of designed bacteriophages against gut pathogenic E. coli for bacterial load biocontrol.

BACKGROUND: This study was conducted to explore new approaches of animal biocontrol via biological control feed. METHOD: White rats were subjected to 140 highly lytic designed phages specific against E. coli. Phages were fed via drinking water, oral injection, and vegetable capsules. Phage feeding was applied by 24 h feeding with 11 d monitoring and 20 d phage feeding and monitoring. Group of rats received external pathogenic E. coli and another group did not, namely groups A and B. RESULTS: Phage feeding for 20 d via vegetable capsules yielded the highest reduction of fecal E. coli, 3.02 and 4.62 log, in rats group A and B respectively. Second best, feeding for 20 d via drinking water with alkali yielded 2.78 and 4.08 log in rats groups A and B respectively. The peak reduction in E. coli output was 5-10 d after phage feeding. Phage control declined after 10th day of feeding. CONCLUSIONS: The use of cocktail of designed phages succeeded in suppressing flora or external E. coli. The phage feed biocontrol is efficient in controlling E. coli at the pre-harvest period, precisely at the 6th-8th day of phage feeding when the lowest E. coli output found.

Influence of environmental variables in the efficiency of phage therapy in aquaculture.

Aquaculture facilities worldwide continue to experience significant economic losses because of disease caused by pathogenic bacteria, including multidrug-resistant strains. This scenario drives the search for alternative methods to inactivate pathogenic bacteria. Phage therapy is currently considered as a viable alternative to antibiotics for inactivation of bacterial pathogens in aquaculture systems. While phage therapy appears to represent a useful and flexible tool for microbiological decontamination of aquaculture effluents, the effect of physical and chemical properties of culture waters on the efficiency of this technology has never been reported. The present study aimed to evaluate the effect of physical and chemical properties of aquaculture waters (e.g. pH, temperature, salinity and organic matter content) on the efficiency of phage therapy under controlled experimental conditions in order to provide a basis for the selection of the most suitable protocol for subsequent experiments. A bioluminescent genetically transformed Escherichia coli was selected as a model microorganism to monitor real-time phage therapy kinetics through the measurement of bioluminescence, thus avoiding the laborious and time-consuming conventional method of counting colony-forming units (CFU). For all experiments, a bacterial concentration of ≈ 10(5) CFU ml(-1) and a phage concentration of ≈ 10(6-8) plaque forming unit ml(-1) were used. Phage survival was not significantly affected by the natural variability of pH (6.5-7.4), temperature (10-25 °C), salinity (0-30 g NaCl l(-1) ) and organic matter concentration of aquaculture waters in a temperate climate. Nonetheless, the efficiency of phage therapy was mostly affected by the variation of salinity and organic matter content. As the effectiveness of phage therapy increases with water salt content, this approach appears to be a suitable choice for marine aquaculture systems. The success of phage therapy may also be enhanced in non-marine systems through the addition of salt, whenever this option is feasible and does not affect the survival of aquatic species being cultured.

Coverage of diarrhoea-associated Escherichia coli isolates from different origins with two types of phage cocktails.

Eighty-nine T4-like phages from our phage collection were tested against four collections of childhood diarrhoea-associated Escherichia coli isolates representing different geographical origins (Mexico versus Bangladesh), serotypes (69 O, 27 H serotypes), pathotypes (ETEC, EPEC, EIEC, EAEC, VTEC, Shigella), epidemiological settings (community and hospitalized diarrhoea) and years of isolation. With a cocktail consisting of 3 to 14 T4-like phages, we achieved 54% to 69% coverage against predominantly EPEC isolates from Mexico, 30% to 53% against mostly ETEC isolates from a prospective survey in Bangladesh, 24% to 61% against a mixture of pathotypes isolated from hospitalized children in Bangladesh, and 60% coverage against Shigella isolates. In comparison a commercial Russian phage cocktail containing a complex mixture of many different genera of coliphages showed 19%, 33%, 50% and 90% coverage, respectively, against the four above-mentioned collections. Few O serotype-specific phages and no broad-host range phages were detected in our T4-like phage collection. Interference phenomena between the phage isolates were observed when constituting larger phage cocktails. Since the coverage of a given T4-like phage cocktail differed with geographical area and epidemiological setting, a phage composition adapted to a local situation is needed for phage therapy approaches against E. coli pathogens.

T4-like phage Bp7, a potential antimicrobial agent for controlling drug-resistant Escherichia coli in chickens.

Chicken-pathogenic Escherichia coli is severely endangering the poultry industry in China and worldwide, and antibiotic therapy is facing an increasing problem of antibiotic resistance. Bacteriophages can kill bacteria with no known activity in human or animal cells, making them an attractive alternative to antibiotics. In this study, we present the characteristics of a novel virulent bacteriophage, Bp7, specifically infecting pathogenic multidrug-resistant E. coli. Phage Bp7 was isolated from chicken feces. Bp7 belongs to the family Myoviridae, possessing an elongated icosahedral head and contractile sheathed tail. It has a 168-kb double-stranded DNA genome. For larger yields, its optimal multiplicity of infection (MOI) to infect E. coli was about 0.001. The latent period was 10 to 15 min, and the burst size was 90 PFU/infected cell. It was stable both at pH 5.0 to 10.0 and at 40°C or 50°C for at least 1 h. Bp7 could infect 46% of pathogenic clinical E. coli strains. Bp7 harbored 791 open reading frames (ORFs) and 263 possible genes. Among the 263 genes, 199 possessed amino acid sequence identities with ORFs of phage T4, 62 had identities with other T4-like phages, and only one lacked any database match. The genome of Bp7 manifested obvious division and rearrangement compared to phages T4, JS98, and IME08. Bp7 is a new member of the "T4-like" genus, family Myoviridae. Its wide host range, strong cell-killing activity, and high stability to pH make it an alternative to antimicrobials for controlling drug-resistant E. coli in chickens.

Growth and reactivation of single stranded DNA phage phiX174 in E. coli undergoing "thymineless death".

The thymine requirement of the E. coli strain HF 4704 (uvr A-, rec A+) is thermosensitive i.e. these cells require for their growth 2 microng thymine per ml at 37 degrees C but not at 30 degrees C. Such cells when starved for thymine for 3 h at 37 degrees C are capable of sustaining growth of single stranded DNA phage phiX174 without any diminution of burst size under nonpermissive conditions. Thymine starved HF 4704 cells also reactivate UV-irradiated phiX174 by about 3fold. To test if the thymine necessary for phage growth under "thymineless" conditions was supplied by host DNA degradation products, the transfer of 32P label from the host DNA to mature progeny phages was measured by means of sucrose density gradient analysis. It was found that only about 0.7% of 32P of the host DNA was transferred to the progeny phages growing in normal cells whereas the corresponding value was 7.8% in the case of thymine starved cells.

Prophage lambda induction of Escherichia coli K12 envA uvrB: a highly sensitive test for potential carcinogens.

A simple, inexpensive, and sensitive test for potential carcinogens based upon the property of carcinogens to induce prophage lambda is described. By using chemicals activated with microsomal enzymes and E. coli K12 permeable (envA) tester bacteria also deficient in DNA repair (uvrB), the range of carcinogens detected in a lysogenic induction test (inductest) has been extended. We have provided the evidence that, after activation, carcinogenic polycyclic hydrocarbons such as benzo[a5pyrene and 7,12-dimethylbenz[a]anthracene induce prophage lambda. Three variants of the test have been developed (inductests I, II, and III), which are as sensitive as the mutagenicity test of Ames et al. [Ames, B. N., McCann, J. and Yamasaki, E. (1975) Mutat. Res. 31, 347-364]. Inductests II and III provide a quantitative estimation of the inducing activity of a carcinogen. With the latter test, one can determine: (i) the cellular toxic effect of a carcinogen and (ii) the kinetics of appearance and disappearance of active metabolites. For two series of chemicals, aflatoxins and benz[a]anthracenes, there is a good correlation between their carcinogenic activity in rodents and their prophage inducing activity in bacteria. The fact that the majority of the cell population is induced makes it possible to test the inducing activity of carcinogens at the biochemical level, e.g., by measuring lambda repressor inactivation.

Growth of ribonucleic acid bacteriophage f2 in a conditional putrescine auxotroph of Escherichia coli: evidence for a polyamine role in translation.

The ribonucleic acid (RNA) bacteriophage, f2, grows poorly in a conditional putrescine auxotroph during polyamine starvation. The addition of putrescine simultaneously with f2 enhances phage growth, shortens the latent period, and increases the burst size. The stimulation of f2 growth is reflected in higher rates of phage RNA and protein syntheses as measured by radioactive labeling of infected cells in the presence of rifampin. Putrescine does not affect f2 adsorption or the penetration of its RNA. Rather, in vitro assays demonstrate that in putrescine-supplemented cells more molecules of f2 replicase are made per incoming parental RNA than in polyamine-starved cultures. The ability of polyamines to stimulate the translation of a preformed messenger suggests a physiological role for these organic cations in normal protein synthesis.

Properties of metK mutants of Escherichia coli K-12.

Some of the properties of three metK mutants of Escherichia coli K-12 have been examined. All three strains have lower than normal levels of SAM (S-adenosyl-l-methionine) synthetase and elevated levels of cystathionine synthetase and cystathionase. One strain (RG73) appears to have an unstable SAM synthetase, suggesting that it carries a structural gene mutation. The two strains (RG62 and RG109) which have the lowest levels of SAM synthetase when grown on minimal medium have appreciably higher levels of enzyme when grown on complete medium. Growth on defined media supplemented with leucine or methionine causes a several-fold increase in the specific activity of SAM synthetase with associated decreases in cystathionine synthetase and cystathionase, but the changes are not as large as those seen in cells grown on LB broth. The SAM pools of strains RG62 and RG109 are markedly lower than normal while that of strain RG73 is slightly below normal. The methionine pools of all three strains are elevated several-fold. The metK strains are able to synthesize cyclopropane fatty acids, but the rate of their formation is slowed. Modification and restriction of phage 21 appears to be normal, suggesting that these strains are able to methylate DNA.

Electrophoretic and other properties of bacteriophage Q : the effect of a variable number of read-through proteins.

When subjected to electrophoresis in polyacrylamide gels, the virions of wild-type Qbeta bacteriophage are found in a single, major, anomalously wide band. With Qbeta mutant 27-2, this wide band is replaced by a set of narrow, well-defined bands. The most rapidly migrating band of the mutant, comprising less than 10% of the total, contains defective virions. These virions have sedimentation coefficients ranging from 70 to 100% of the bulk of the unfractionated mutant, they contain no read-through protein (protein IIb), and they are deficient in maturation protein and contain fragmented RNA. The second band, comprising less than 3% of the total virus, has not been well characterized. The virions in the remaining electrophoretic bands are infective. Their distribution into bands is believed due to differences in their effective volume resulting from differences in their content of protein IIb. The most rapidly migrating band of this series contains virions with a few molecules of IIb protein, whereas the more slowly migrating bands contain virions with a larger number of IIb molecules. The adjacent bands in the series contain virions which differ by approximately three IIb molecules. Wild-type Qbeta virus is similar to the mutant in that the more slowly migrating virions contain more protein IIb than the more rapidly migrating virions. Their failure to resolve into distinct bands upon electrophoresis is believed due to a less restricted packing of protein IIb into their virions. Both wild-type Qbeta and mutant 27-2 also have 1 to 5% of the virions in the form of dimers which migrate with approximately one-half the mobility of the respective monomer forms. When the average amount of IIb per virion is increased by growth of the virus in a UGA suppressor strain, the electrophoretic pattern is altered. In the case of wild-type Qbeta, the single band is wider, whereas with Qbeta mutant 27-2 there occurs an increased number of partially resolved narrow bands. We suggest that the structural feature responsible for the difference in electrophoretic pattern between mutant 27-2 and wild-type Qbeta is the mode of IIb packing in the virions. In the mutant, the IIb proteins are found in the virions only in multiples of three, whereas wild-type virions may differ by only a single IIb protein.

Polyamines in the synthesis of bacteriophage deoxyribonucleic acid. II. Requirement for polyamines in T4 infection of a polyamine auxotroph.

Polyamine depletion produced by exogenous arginine in Escherichia coliK-12 cultures defective in agmatine ureohydrolase activity resulted in a marked inhibition of the rates of growth and nucleic acid synthesis. Addition of putrescine or spermidine to such depleted cultures restored the control rate of growth and nucleic acid accumulation. The omission of lysine resulted in a further decrease in the rates of growth and nucleic acid synthesis in polyamine-depleted cells. The addition of exogenous cadaverine increased the rates of growth and ribonucleic acid synthesis to those observed in lysine-supplemented cultures, suggesting that lysine or a derivative of lysine serves a function similar to cadaverine. Addition of lysine to polyamine-depleted cultures at neutral pH results in the synthesis of cadaverine and a new spermidine analogue, both containing lysine carbon. This new metabolite has been isolated and identified as N-3-aminopropyl-1, 5-diaminopentane. T4D infection of the polyamine-depleted mutant resulted in a very low rate of DNA synthesis and phage maturation. The addition of putrescine or spermidine 15 min before infection restored phage DNA synthesis and phage maturation to control rates, i.e., rates observed in infected cells grown in the absence of arginine.

Replication of T4rII bacteriophage in Escherichia coli K-12 (lambda).

The defect of T4rII replication in Escherichia coli K-12 (lambda) can be phenotypically reversed by various supplements to the growth medium. Arginine, lysine, spermidine, and a number of diamines allowed varying levels of rII replication. The best reversion was obtained with 0.4 m sucrose in 0.002 to 0.005 m Ca(++). Monovalent cations severely inhibited reversion. A cell surface site of polyamine action is consistent with the fact that spermidine inhibits phage ghost-induced cell lysis and with the finding that sufficient polyamine is available within the cells to allow normal patterns of neutralization of phage deoxyribonucleic acid, as detected by the polyamine content of progeny phage. In the absence of effective supplements, rII-infected cells swelled and lost refractility. The data indicate that a leaky cell envelop is involved. No difference in mucopeptides of uninfected K-12 (lambda) and K-12 was detected and, because the mucopeptide in r(+) infected cells was found to be at least partially hydrolyzed midway through the lytic cycle, it did not appear that the rII defect concerned mucopeptide synthesis. The pattern of cell phospholipid synthesis changes after phage infection, but no difference was detected between r(+) and rII with regard to biosynthesis of phosphatidylethanolamine and phosphatidylglycerol.

Growth of a dihydrofolate reductaseless mutant of bacteriophage T4.

A mutant of bacteriophage T4 was isolated which was unable to induce virus-specific dihydrofolate reductase in infected cells. The mutant was able to form several other early enzymes of pyrimidine metabolism. Growth of the mutant in a wild-type host, Escherichia coli B, was compared with that of the parent strain, T4BO(1), and T4td8, a mutant which lacks the ability to induce thymidylate synthetase. Growth studies were carried out in minimal medium, which gave higher growth rates and phage yields than the supplemented media used in previous studies. The reductase mutant formed deoxyribonucleic acid and plaque-forming particles at a rate slightly higher than the synthetase mutant but 1.5-to 2-fold lower than that of the wild-type phage under all conditions studied. The addition of thymine to a culture infected by the mutant increased the growth rate significantly, suggesting that the genetic lesion leads to a partial thymidylate deficiency. Like other viral genes controlling steps in thymidylate metabolism, the dihydrofolate reductase gene appears to be useful but not completely essential for growth.