Isolation and Characterization of Bacteriophages That Infect Citrobacter rodentium, a Model Pathogen for Intestinal Diseases

Enteropathogenic Escherichia coli (EPEC) is a major pathogen for diarrheal diseases among children. Antibiotics, when used appropriately, are effective; however, their overuse and misuse have led to the rise of antibiotic resistance worldwide. Thus, there are renewed efforts into the development of phage therapy as an alternative antibacterial therapy. Because EPEC in vivo models have shortcomings, a surrogate is used to study the mouse pathogen Citrobacter rodentium in animal models. In this study, two new phages CrRp3 and CrRp10, which infect C. rodentium, were isolated and characterized. CrRp3 was found to be a new species within the genus Vectrevirus, and CrRp10 is a new strain within the species Escherichia virus Ime09, in the genus Tequatrovirus. Both phages appear to have independently evolved from E. coli phages, rather than other Citrobacter spp. phages. Neither phage strain carries known genes associated with bacterial virulence, antibiotic resistance, or lysogeny. CrRp3 is more potent, having a 24-fold faster adsorption rate and shorter lytic cycle when compared to the same properties of CrRp10. However, a lysis curve analysis revealed that CrRp10 prevented growth of C. rodentium for 18 h, whereas resistance developed against CrRp3 within 9 h. We also show that hypoxic (5% oxygen) conditions decreased CrRp3 ability to control bacterial densities in culture. In contrast, low oxygen conditions did not affect CrRp10 ability to replicate on C. rodentium. Together, CrRp10 is likely to be the better candidate for future phage therapy investigations.

Characterization of the biological properties of bacteriophages Staphylococcus aureus variant bovis

Cattle mastitis is the main cause of economic losses in milk production worldwide, and Staphylococcus aureus is the pathogen that causes it most. Bacteriophages may be an alternative treatment for this disease. In this study, we studied the effect of temperature and pH on the lytic activity of bacteriophages isolated from cows with signs of mastitis. The isolation and production of pure phage lines were performed on an indicator culture of Staphylococcus aureus var. bovis 1491f conventional methods. The following phages were isolated and labeled: Phage SAvB07, Phage SAvB08, Phage SAvB12, Phage SAvB14. To determine the effect of temperature and pH, aliquots after the action of these factors were sown by the double agar method at regular intervals. The study found that phage lytic activity was temperature dependent. Thus, under the influence of temperature 45 °C after 30 minutes of action, it decreased by 3.0–3.4 times for bacteriophages Phage SAvB07, Phage SAvB08, Phage SAvB12 and after one hour was 2.4–12.6%. Phage SAvB14 strain was more resistant to temperature. Its activity decreased by only 67.6% during the analyzed period. With higher temperatures (55–65 °C), the intensity of phage infection decreased significantly, but remained stable. The most resistant to the effects of temperature was Phage SAvB14 – its activity was on average higher by 15.6–33.9% compared with other phages taken in the experiment. The results of our studies on the effect of pH on the reproduction of phages showed that the maximum number of phage virions was observed at pH 6 7. However, the most resistant to acidity was the phage strain Phage SAvB14, compared with other strains taken in the experiment. Therefore, the bacteriophage Phage SAvB14 exhibited the greatest stability and has considerable potential for in vivo use in the treatment of mastitis of cows caused by Staphylococcus aureus.

Modifying Plaque assay and Clearance test as tools in determination of phage typing for E. Coli bacterial interspecies

Bacteriophage of E. Coli interspecies from sewage samples were isolated , the phage particles were isolated from two different sewage samples . The first sample was collected from sewage sample of Baghdad university and the second sample was isolated from domestic sewage sample , first sample showed phages specialized for three E. Coli interspecies bacteria (first plate ) and two E. Coli interspecies bacteria (second plate ) , meanwhile second sample showed phage specialized for two E. Coli. interspeciesThe study of appearance of E coli phages from first sample showed three types of E. coli phages with different size of inhibition zone ( 1 , 0.7,0.5 )Cm respectively ( first plate ) , meanwhile E. Coli interspecies bacteria showed phages related with two interspecies with size of inhibition zone ( 0.5 ,0.4) Cm respectively ( second plate ), on other hand , the second sample showed also two interspecies E. coli with inhibition zone (1,0.8)Cm . experimental method has been designed which showed the modifying method of phage assay to determine phage typing assay . phage has been tested particles with different bacterial strains ( E. coli , shagilla and Serratia ) from different sources and the control was the host of each bacteriophages by taking the O.D for all the tests and controls , to setup new criteria for phage typing .:and this test is called ( Clearance Test ) The result showed that O.D for Test 1 , 2 , 3, was ( 1.6 , 1.2 . 1.7) for ( E. coli , shagilla and Serratia ) bacterial strains , meanwhile the control tests was ( 0.3 , 0.2, 0.4 ) for strains isolated from first sample (first and second plate ) and second samples with different interspecies respectively . This result can predict high specificity of phage strain and this method can be used to determine interspecies strains .So from this experiment we can identify only Clearance Test by measuring only O.D. of bacterial strain with different phages instead of going through plaque assay .

Bacteriophage-Based Genetic System for Selection of Nonsplicing Inteins

ABSTRACT A genetic selection system that detects splicing and nonsplicing activities of inteins was developed based on the ability to rescue a T4 phage strain with a conditionally inactive DNA polymerase. This phage defect can be complemented by expression of plasmid-encoded phage RB69 DNA polymerase. Insertion of an intein gene into the active site of the RB69 DNA polymerase gene renders polymerase activity and phage viability dependent on protein splicing. The effectiveness of the system was tested by screening for thermosensitive splicing mutants. Development of genetic systems with the potential of identifying protein splicing inhibitors is a first step towards controlling proliferation of pathogenic microbes harboring inteins in essential proteins.

Sampling Natural Viral Communities from Soil for Culture-Independent Analyses

ABSTRACT An essential first step in investigations of viruses in soil is the evaluation of viral recovery methods suitable for subsequent culture-independent analyses. In this study, four elution buffers (10% beef extract, 250 mM glycine buffer, 10 mM sodium pyrophosphate, and 1% potassium citrate) and three enumeration techniques (plaque assay, epifluorescence microscopy [EFM], and transmission electron microscopy [TEM]) were compared to determine the best method of extracting autochthonous bacteriophages from two Delaware agricultural soils. Beef extract and glycine buffer were the most effective in eluting viable phages inoculated into soils (up to 29% recovery); however, extraction efficiency varied significantly with phage strain. Potassium citrate eluted the highest numbers of virus-like particles from both soils based on enumerations by EFM (mean, 5.3 × 108 g of dry soil−1), but specific soil-eluant combinations posed significant problems to enumeration by EFM. Observations of virus-like particles under TEM gave confidence that the particles were, in fact, phages, but TEM enumerations yielded measurements of phage abundance (mean, 1.5×108 g of dry soil−1) that were about five times lower. Clearly, the measurement of phage abundance in soils varies with both the extraction and enumeration methodology; thus, it is important to assess multiple extraction and enumeration approaches prior to undertaking ecological studies of phages in a particular soil.

Bacteriophage Therapy Rescues Mice Bacteremic from a Clinical Isolate of Vancomycin-Resistant Enterococcus faecium

ABSTRACT Colonization of the gastrointestinal tract with vancomycin-resistant Enterococcus faecium (VRE) has become endemic in many hospitals and nursing homes in the United States. Such colonization predisposes the individual to VRE bacteremia and/or endocarditis, and immunocompromised patients are at particular risk for these conditions. The emergence of antibiotic-resistant bacterial strains requires the exploration of alternative antibacterial therapies, which led our group to study the ability of bacterial viruses (bacteriophages, or phages) to rescue mice with VRE bacteremia. The phage strain used in this study has lytic activity against a wide range of clinical isolates of VRE. One of these VRE strains was used to induce bacteremia in mice by intraperitoneal (i.p.) injection of 109 CFU. The resulting bacteremia was fatal within 48 h. A single i.p. injection of 3 × 108 PFU of the phage strain, administered 45 min after the bacterial challenge, was sufficient to rescue 100% of the animals. Even when treatment was delayed to the point where all animals were moribund, approximately 50% of them were rescued by a single injection of this phage preparation. The ability of this phage to rescue bacteremic mice was demonstrated to be due to the functional capabilities of the phage and not to a nonspecific immune effect. The rescue of bacteremic mice could be effected only by phage strains able to grow in vitro on the bacterial host used to infect the animals, and when such strains are heat inactivated they lose their ability to rescue the infected mice.

Impact of Acid on Survival of Vibrio vulnificus and Vibrio vulnificus Phage†

Three strains of Vibrio vulnificus and V. vulnificus phages were tested for acid sensitivity at 21°C. V. vulnificus strain 304 was more resistant to pH 4.0 than strains CVD-1 and A-9, whereas acid sensitivities of V. vulnificus strains at pH 3.0 and 2.0 were similar. V. vulnificus phage strain 110A-7 was more resistant to pH 4.0 than strain 153A-7, whereas acid sensitivities of phage strains at pH 3.5 and 3.0 were similar. Numbers of V. vulnificus and its phage were close to the limit of detection after 100 s at pH 2.0 and after 24 min at pH 3.0. Acid D-values at 21°C decreased as pH decreased for both V. vulnificus and phages. D-values of phage strains at pH 3.5 were 10-fold greater than those of host strain at pH 4.0. D-values of phage strains were slightly greater than those of host strain at pH 3.0. These results suggest that V. vulnificus and its phage were very sensitive to pH of less than 3.0, although V. vulnificus phages were more resistant to acid than their host.

CORRELATION BETWEEN MAP POSITION AND PHENOTYPE OF CTI MUTANTS IN THE C CISTRON OF RHIZOBIUM MELILOTI PHAGE 16—3

ABSTRACT Nine temperature-sensitive clear mutations (Cti) in the C cistron (coding for the repressor protein) of Rhizobium meliloti temperate phage 16—3 were characterized according to the inductive temperature, the immunity of cells lysogenic for these mutant phages to superinfection by homoimmune weak virulent mutants, the phenotype of double-ti mutants and interallelic complementation. The results indicate that mutations of similar phenotypic expression are clustered on the genetic map. Furthermore, it seems probable that the C cistron of the original phage 16—3 is identical to that of the independently isolated phage strain 36.

Evidence that HT mutant strains of bacteriophage P22 retain an altered form of substrate specificity in the formation of transducing particles in Salmonella typhimurium

SUMMARYThe effects of two different deletions of the tryptophan operon on the cotransduction linkage of the nearby cysB and pyrF markers were studied using three sets of donor lysates, each produced by a different HT mutant P22 phage strain. Each trp operon deletion (present in both donor and recipient to preserve homology) caused changes in the cotransduction frequencies. This indicated that the HT mutant phage encapsulating mechanism, whose ability to discriminate phage DNA from host-cell DNA is absent or diminished, could still distinguish among nucleotide sequences in selecting bacterial chromosome sites at which to initiate transducing particle formation. The three HT mutant phage strains each produced different sets of cotransduction linkage values, indicating that this aspect of substrate specificity was altered differently and uniquely by each HT mutation.