Staphylococcus aureus is a gram-positive pathogen that causes a variety of diseases, including bovine mastitis, which has severe economic consequences. Standard antibiotic treatment results in selection of resistant strains, leading to a need for alternative treatments, such as bacteriophage therapy. Forty-nine S. aureus isolates were obtained from the milk of mastitic cows for use in screening of staphylococcal phages. Fifteen isolates which were positive for both coagulase and hemolysin were assayed by PCR for variation in the X region and the immunoglobulin G-binding region of the protein A gene (spa) and in the carboxy terminus of the coagulase gene (coa) and for the presence of enterotoxin C, G, H, and I genes. The host ranges of 52 phages isolated from sewage influent were determined by performing spot tests with the 15 S. aureus isolates, and two phages were subsequently chosen for further analysis. ⌽SA039 had the widest host range, producing clear plaques on 13 of the 15 isolates (87%), while ⌽SA012 produced clear plaques on 8 isolates (53%) and was the only phage that produced a clear plaque on a nonmastitic S. aureus strain. Transmission electron microscopy revealed that the phages were similar sizes and belonged to the Myoviridae family. Measurement of optical densities during coculture with S. aureus isolates confirmed the breadth of the ⌽SA039 host range and showed that ⌽SA012 had potent lytic capability. ⌽SA012-resistant bacteria did not appear for three of seven isolates tested (43%) after 65 h of incubation. These two phages are proposed as candidates for phage therapy of bovine mastitis.In the dairy industry, mastitis is a widespread problem responsible for important decreases in milk production. Economic losses of $100 million per year have been estimated for farms in Hokkaido, one of the largest milk-producing areas in Japan (28). Mastitis can be caused by over 150 different microorganisms, and one of the most important of these organisms is Staphylococcus aureus (22). After diagnosis of mastitis, the standard treatment regimen consists of isolating the diseased cow and treating it with antibiotics. However, this approach has drawbacks, such as its high cost and the eradication of harmless or beneficial organisms due to the lack of specificity of antibiotics. Additionally, the incidence of antibioticresistant bacteria has increased in recent years (4). As a result, there has been renewed interest in the use of other natural or engineered antimicrobial agents as an alternative or supplementary treatment for staphylococcal diseases such as mastitis (11,21,26). One group of alternatives with great potential involves bacteriophages (phages) and their derivatives, and a number of promising candidates have been described (2,5,7,13,17,18,27), notably bacteriophage K.One of the main obstacles to successful treatment of mastitis using phages is the fact that most phages are able to infect only a very narrow range of hosts. Given the plural etiology of many mastitis cases, it is desirable to find a phag...
The application of bacteriophages (phages) in therapy urgently requires the production of wide-host-range recombinant phages that possess strong lytic activity. The wide-host-range IP008 phage was classified by transmission electron microscopy analysis as an A2 morphotype member of the Myoviridae family of the order Caudovirales. IP008 showed a high homology (99.4% similarity in the amino acid alignment of the major capsid protein Gp 23) with KEP10, another wide-host-range phage. The long tail fiber genes (genes 37 and 38) from the genome of T2 were replaced with those of the IP008 phage by homologous recombination. The host range of the recombinant phages was identical to that of IP008. Furthermore, the recombinant phage bacterial lytic activity was restored. Future analyses of host-range mutants of the closely related phages T2 and IP008 could lead to a more precise localization of the genetic factors responsible for receptor specificity.
Culture-independent PCR-DGGE fingerprinting was used to reveal the bacterial composition and diversity associated with raw milk of mastitis cows from Hokkaido, Japan for the first time. All the mastitis milk samples were diagnosed as solely infection by Coliforms using the classical microbiological method based on on-farm culturing. Our results revealed that the bovine mastitis-associated bacteria were host-specific because community structure varied between each sample. Klebsiella pseudomoniae, Lactococcus lactis Staphylococcus aureus and Escherichia sp were found to be the widely distributed species. Furthermore, more than one mastitis-causing pathogen was found to be present in some mastitis samples. These pathogens may not only act as etiology agents but also play a role in disrupting the natural microbial ecology in mastitis bovine. This finding highlights the limitation of the traditional identification and characterization strategy. Therefore, it is suggested that the methodology applied in this study might be a valuable addition to mastitis control and prevention.
Patient-derived tumor xenograft (PDTX) mouse models were used to discover new therapies for naïve and drug resistant BRAFV600E-mutant melanoma. Tumor histology, oncogenic protein expression, and antitumor activity were comparable between patient and PDTX-matched models thereby validating PDTXs as predictive preclinical models of therapeutic response in patients. PDTX models responsive and non-responsive to BRAF/MEK standard of care (SOC) therapy were used to identify efficacious combination therapies. One such combination includes a CDK4/6 inhibitor that blocks cell cycle progression. The rationale for this is that the retinoblastoma protein (pRb) is 95% wildtype in BRAF mutant melanoma. We discovered that 77/77 stage IV metastatic melanoma tissues were positive for inactive phosphorylated pRb (pRb-Ser780). Rb is hyperphosphorylated and inactivated by CDK4/6:cyclin D1 and when restored to its hypophosphorylated active form blocks cell cycle progression. The addition of a CDK4/6 inhibitor to SOC therapy was superior to SOC. Importantly, triple therapy in an upfront treatment and salvage therapy setting provided sustained durable response. We also showed that CDK4/6 blockade resensitized drug resistant melanoma to SOC therapy. Durable response was associated with sustained suppression of pRb-Ser780. Thus, reactivation of pRb may prove to be a clinical biomarker of response and the mechanism responsible for durable response. In light of recent clinical trial data using this triple therapy against BRAFV600E-mutant melanoma, our findings demonstrating superior and prolonged durable response in PDTX models portend use of this therapeutic strategy against naïve and SOC resistant BRAF V600E-mutant metastatic melanoma coupled with pRB-Ser780 as a biomarker of response.
Salmonella typhimurium antigens were displayed on the capsid of a T2 bacteriophage to explore the potential of phage display for an oral vaccine. Segments of the flagellin proteins FliC (H1 antigen) and FljB (H2) were fused to the N-terminal of T2 phage SOC to give two recombinant phages, T2FliCm and T2FljBm. Over 14 days, 19 BALB/c mice were orally administered twice, either with purified recombinant FliCm and FljBm protein, or T2FliCm and T2FljBm with or without host Escherichia coli. Feces were sampled over 10 weeks and examined for phage by plaque assay and for the presence of mucosal IgA by ELISA. Relatively few phages were detected relative to the amount administered (up to 8.21 x 10(3) PFU/g faeces) and none were detected five days after initial administration. The administration of a large number of phages appeared to cause no clinical symptoms. IgA concentration in feces peaked around four weeks after the second administration and subsided after eight weeks. The highest relative titers were observed in the protein group (0.37% for anti-FliCm and 0.22% for anti-FljBm) and the mouse group which received no E. coli (0.33% and 0.35%) despite the theoretical amount of protein contained in a phage dose being at least 80-465 times lower than the protein dose administered. The possibility that the immuno-stimulatory properties of the phage create an adjuvant effect to enhance the immunogenic properties of the displayed proteins is discussed. We conclude that phage may be valuable as a vector for oral vaccines.
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