In vitro and in vivo assessment of phage therapy against Staphylococcus aureus causing bovine mastitis.

OBJECTIVE: The aim of this study was to assess the efficacy of lytic bacteriophages on Staphylococcus aureus causing bovine mastitis, by in vitro and in vivo assays using Galleria mellonella and murine mastitis models. METHODS: Between May and December 2016, ten S. aureus (five methicillin-resistant and five methicillin-sensitive) isolates were isolated from milk samples of cattle with mastitis in Belgium and Norway. The isolates were assessed in vitro for their susceptibility to four lytic bacteriophages (Romulus, Remus, ISP and DSM105264) and subsequently in vivo in G. mellonella larvae and in murine mastitis model. RESULTS: Romulus, Remus and ISP showed a lytic activity against the S. aureus isolates in vitro. A larvae survival rate below 50% was observed at 4 days post-inoculation (DPI) in the groups infected with a methicillin-sensitive S. aureus isolate and treated with these three phages in vivo. An incomplete recovery of the mouse mastitis was observed at 48h post-inoculation (HPI) in the groups infected and treated with the ISP phage in vivo. CONCLUSIONS: The observations are much more pronounced statistically between the infected- phosphate buffered saline (PBS)-treated and infected-phage-treated groups in G. mellonella and the murine mastitis model demonstrating an effect of the phages against S. aureus associated with bovine mastitis.

REVIVAL OF MICROBIAL THERAPEUTICS, WITH EMPHASIS ON PROBIOTIC LACTOBACILLUS (REVIEW).

The idea to use living microorganisms for disease prevention and treatment was introduced a century ago, but yet the full potential and benefits of microbial therapeutics has not been entirely understood. In the light of developments of human microbiome studies, probiotics are gaining new momentum, where health benefit conferring by Lactobacillus are emerging as one of the novel approaches in the treatment and prophylactics of dysbiosis. The present review focuses on the origin and development of the probiotic's concept, mechanisms of action and anticipated use of probiotic Lactobacillus as well as of microbial therapeutics. The required regulatory frameworks associated with probiotic use and marketing are discussed.

Antibiofilm potential of purified environmental bacteriophage preparations against early stage Pseudomonas aeruginosa biofilms.

AIMS: This paper presents the potential of environmentally sourced bacteriophages to affect the growth of clinical isolates of Pseudomonas aeruginosa biofilms, and assesses the respective plaque morphotypes presented by each bacteriophage, in vitro. METHODS AND RESULTS: Bacterial host strains were typed for their ability to produce the quorum sensing-controlled virulence factor pyocyanin, and then tested for bacteriophage susceptibility using the spot test method. The bacteriophages were co-administered with ciprofloxacin in order to determine whether the bacteriophages would demonstrate synergistic or antagonistic behaviour to the antibiotic in vitro. Results suggest a potential relationship between the bacteriophage plaque size and biofilm inhibition, where those producing smaller plaques appear to be more effective at reducing bacterial biofilm formation. CONCLUSIONS: This phenomenon may be explained by a high adsorption rate leading to the rapid formation of smaller plaques, and greater biofilm reduction associated with the loss of viable bacterial cells before the cells can adhere to the surface and form a biofilm. Results from the co-administration of bacteriophage and ciprofloxacin suggest that the two work synergistically to affect P. aeruginosa biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: The data indicate enhanced efficacy of ciprofloxacin by ≥50%. This could offer an alternative strategy for targeting antibiotic-resistant infections.

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.

Bacteriophage-derived enzyme that depolymerizes the alginic acid capsule associated with cystic fibrosis isolates of Pseudomonas aeruginosa.

AIMS: To identify enzymes associated with bacteriophages infecting cystic fibrosis (CF) strains of Pseudomonas aeruginosa that are able to degrade extracellular alginic acids elaborated by the host bacterium. METHODS AND RESULTS: Plaques produced by 21 Ps. aeruginosa-specific phages were screened for the presence of haloes, an indicator of capsule hydrolytic activity. Four phages produced haloed plaques, and one (PT-6) was investigated further. PT-6 was shown by electron microscopy to belong to Podoviridae family C1, to reduce the viscosity of four alginate preparations using a rolling ball viscometer and to release uronic acid-containing fragments from the polymers, as judged by spectrophotometry and thin layer chromatography. The alginase was partially purified by gel filtration chromatography and shown to be a 37 kDa polypeptide. CONCLUSIONS: Infection of CF strains of Ps. aeruginosa by phage PT-6 involves hydrolysis of the exopolysaccharide secreted by the host. SIGNIFICANCE AND IMPACT OF THE STUDY: The alginase produced by PT-6 has the potential to increase the well-being of CF suffers by improving the surface properties of sputum, accelerating phagocytic uptake of bacteria and perturbing bacterial growth in biofilms.