Phage activity against Staphylococcus aureus is impaired in plasma and synovial fluid.

S. aureus is a pathogen that frequently causes severe morbidity and phage therapy is being discussed as an alternative to antibiotics for the treatment of S. aureus infections. In this in vitro and animal study, we demonstrated that the activity of anti-staphylococcal phages is severely impaired in 0.5% plasma or synovial fluid. Despite phage replication in these matrices, lysis of the bacteria was slower than phage propagation, and no reduction of the bacterial population was observed. The inhibition of the phages associated with a reduction in phage adsorption, quantified to 99% at 10% plasma. S. aureus is known to bind multiple coagulation factors, resulting in the formation of aggregates and blood clots that might protect the bacterium from the phages. Here, we show that purified fibrinogen at a sub-physiological concentration of 0.4 mg/ml is sufficient to impair phage activity. In contrast, dissolution of the clots by tissue plasminogen activator (tPA) partially restored phage activity. Consistent with these in vitro findings, phage treatment did not reduce bacterial burdens in a neutropenic mouse S. aureus thigh infection model. In summary, phage treatment of S. aureus infections inside the body may be fundamentally challenging, and more investigation is needed prior to proceeding to in-human trials.

Effective reduction of Salmonella Enteritidis in broiler chickens using the UPWr_S134 phage cocktail.

Salmonella is a poultry-associated pathogen that is considered one of the most important zoonotic bacterial agents of contaminated food of animal origin including poultry products. Many efforts are taken to eliminate it from the food chain, and phages are one of the most promising tools to control Salmonella in poultry production. We investigated the usefulness of the UPWr_S134 phage cocktail in reducing Salmonella in broiler chickens. For this purpose, we analyzed the survivability of phages in the harsh environment encountered in the chicken gastrointestinal tract, which has low pH, high temperatures, and digestive activity. Phages in the cocktail UPWr_S134 showed the ability to remain active after storage at temperatures ranging from 4 to 42°C, reflecting temperatures of storage conditions, broiler handling, and the chicken body, and exhibited robust pH stability. We found that although simulated gastric fluids (SGF) caused phage inactivation, the addition of feed to gastric juice allows maintenance of UPWr_S134 phage cocktail activity. Further, we analyzed UPWr_S134 phage cocktail anti-Salmonella activity in live animals such as mice and broilers. In an acute infection model in mice, the application of doses of 107 and 1014 PFU/ml UPWr_S134 phage cocktail resulted in delaying symptoms of intrinsic infection in all analyzed treatment schedules. In Salmonella-infected chickens orally treated with the UPWr_S134 phage cocktail the number of pathogens in internal organs in comparison to untreated birds was significantly lower. Therefore we concluded that the UPWr_S134 phage cocktail could be an effective tool against this pathogen in the poultry industry.

Engineering of cellobiose phosphorylase for the defined synthesis of cellotriose.

Cellodextrins are non-digestible oligosaccharides that have attracted interest from the food industry as potential prebiotics. They are typically produced through the partial hydrolysis of cellulose, resulting in a complex mixture of oligosaccharides with a varying degree of polymerisation (DP). Here, we explore the defined synthesis of cellotriose as product since this oligosaccharide is believed to be the most potent prebiotic in the mixture. To that end, the cellobiose phosphorylase (CBP) from Cellulomonas uda and the cellodextrin phosphorylase (CDP) from Clostridium cellulosi were evaluated as biocatalysts, starting from cellobiose and α-D-glucose 1-phosphate as acceptor and donor substrate, respectively. The CDP enzyme was shown to rapidly elongate the chains towards higher DPs, even after extensive mutagenesis. In contrast, an optimised variant of CBP was found to convert cellobiose to cellotriose with a molar yield of 73%. The share of cellotriose within the final soluble cellodextrin mixture (DP2-5) was 82%, resulting in a cellotriose product with the highest purity reported to date. Interestingly, the reaction could even be initiated from glucose as acceptor substrate, which should further decrease the production costs.Key points• Cellobiose phosphorylase is engineered for the production of cellotriose.• Cellotriose is synthesised with the highest purity and yield to date.• Both cellobiose and glucose can be used as acceptor for cellotriose production.

Performance of Leuconostoc citreum FDR241 during wheat flour sourdough type I propagation and transcriptional analysis of exopolysaccharides biosynthesis genes.

This study focused on the performance of the dextran producer Leuconostoc citreum as starter culture during 30 days of wheat flour type I sourdough propagation (back-slopping). As confirmed by RAPD-PCR analysis, the strain dominated throughout the propagation procedure, consisting of daily fermentations at 20 °C. The sourdoughs were characterized by consistent lactic acid bacteria cell density and acidification parameters, reaching pH values of 4.0 and mild titratable acidity. Carbohydrates consumption remained consistent during the propagation procedure, leading to formation of mannitol and almost equimolar amount of lactic and acetic acid. The addition of sucrose enabled the formation of dextran, inducing an increase in viscosity of the sourdough of 2-2.6 fold, as well as oligosaccharides. The transcriptional analysis based on glucosyltransferases genes (GH70) showed the existence in L. citreum FDR241 of at least five different dextransucrases. Among these, only one gene, previously identified as forming only α-(1-6) glycosidic bonds, was significantly upregulated in sourdough fermentation conditions, and the main responsible of dextran formation. A successful application of a starter culture during long sourdough back-slopping procedure will depend on the strain robustness and fermentation conditions. Transcriptional regulation of EPS-synthetizing genes might contribute to increase the efficiency of industrial processes.