SELECTION OF THE ACTIVE PHAGES AGAINST B.FRAGILIS FOR FURTHER STUDY OF THRAPEUTIC PERPECTIVES.

B.fragilis is an obligate anaerobic commensal colonizing human intestines and carries number of physiological functions. At the same time B.fragilis is commonly isolated from the septic clinical samples and due to its capsule represents one of the provoking agents for abscess development. Enterotoxigenic B.fragilis (ETBF) strains also increase the likelihood of developing colon cancer. Increasing incidence of antibiotic-resistant pathogens led to the high demand to alternative antimicrobials. Bacteriophage (phage) therapy already practiced for a century in some of the Post-Soviet countries including Georgia has been suggested as a substitute of antibiotics. It should be noted that this study is the first attempt to isolate virulent B.fragilis phages for further therapeutic application as all phages known up until now were used for detection of fecal water contamination only. The aim of the study was to isolate B.fragilis specific phages for their further use against infections caused by this bacteria Eighteen B.fragilis strains were isolated from human feces using conventional microbiological methods and precise identification was done via MULDI-TOF mass spectrometry. Three ETBF strains were provided by the University of Ghent (Belgium). Three lytic phages (ФVA7, ФMTK and ФUZ-1) of Siphoviridae family were isolated from the waste water samples collected in Tbilisi and in Ghent using conventional phage isolation and enumeration techniques. Electron microscopy was used for the visualization of the phage particles. To determine lytic activity of the isolated phages and estimate their antimicrobial efficacy the spot test assay and efficiency of plating (EOP) were studied using 18 clinical strains of B.fragilis and 12 intestinal commensal strains related to Bacterioides spp. and Parabacterioides spp.. Although according to the spot test results two of the isolated phages expressed high specificity to B.fragilis demonstrating broad host range within this species, however EOP results showed that only ФVA7 can be selected as the best candidate for the model in vitro tissue culture experiments aiming demonstration of the therapeutic and prophylactic potential of phages against ETBF and/or NETBF.

Staphylococcus aureus controls interleukin-5 release in upper airway inflammation.

Staphylococcus aureus is a frequent colonizer of the upper airways in chronic rhinosinusitis with nasal polyps, but also resides intramucosally; it has been shown that secreted staphylococcal proteins such as enterotoxins and serine proteases induce the release of cytokines such as IL-5. We have analyzed nasal polyp tissue freshly obtained during routine surgery, which did or did not contain cultivatable S. aureus, to study spontaneous IL-5 production by nasal polyp tissue over 24 and 72h in tissue culture. In S. aureus-positive samples we interfered by killing the bacteria using antibiotics or S. aureus specific intravenous staphylococcal phages (ISP), active or heat-inactivated. Phage-neutralizing antibodies were used to demonstrate the specificity of the phage-mediated effects. We monitored S. aureus colony forming units, and identified S. aureus proteins by mass spectrometry. We demonstrate that cultivatable S. aureus may be found in type-2 inflamed nasal polyps; the pathogen is replicating within 24h and secretes proteins, including enterotoxins and serine proteases. The presence of S. aureus was associated with a significantly higher release of IL-5. Killing of S. aureus by antibiotics or specific ISP significantly reduced the IL-5 release. The suppressive activity of the bacteriophage on IL-5 be abolished by heat inactivation or anti-phage antibodies. BIOLOGICAL SIGNIFICANCE: In this study, we used high resolution mass spectrometry to identify S. aureus proteins directly in infected nasal polyp tissue and nasal polyp tissue incubated over 24 and 72h in culture. We discovered bacterial proteins including enterotoxins and serine proteases like proteins. These experiments indicate a direct role of S. aureus in the regulation of IL-5 production in nasal polyps and may suggest the involvement of bacterial proteins detected in the tissues.

Pre-adapting parasitic phages to a pathogen leads to increased pathogen clearance and lowered resistance evolution with Pseudomonas aeruginosa cystic fibrosis bacterial isolates.

Recent years have seen renewed interest in phage therapy--the use of viruses to specifically kill disease-causing bacteria--because of the alarming rise in antibiotic resistance. However, a major limitation of phage therapy is the ease at with bacteria can evolve resistance to phages. Here, we determined whether in vitro experimental coevolution can increase the efficiency of phage therapy by limiting the resistance evolution of intermittent and chronic cystic fibrosis Pseudomonas aeruginosa lung isolates to four different phages. We first pre-adapted all phage strains against all bacterial strains and then compared the efficacy of pre-adapted and nonadapted phages against ancestral bacterial strains. We found that evolved phages were more efficient in reducing bacterial densities than ancestral phages. This was primarily because only 50% of bacterial strains were able to evolve resistance to evolved phages, whereas all bacteria were able to evolve some level of resistance to ancestral phages. Although the rate of resistance evolution did not differ between intermittent and chronic isolates, it incurred a relatively higher growth cost for chronic isolates when measured in the absence of phages. This is likely to explain why evolved phages were more effective in reducing the densities of chronic isolates. Our data show that pathogen genotypes respond differently to phage pre-adaptation, and as a result, phage therapies might need to be individually adjusted for different patients.