Due to the rapid spread of antibiotic resistance, and the difficulties of treating biofilm-associated infections, alternative treatments for S. aureus infections are urgently needed. We tested the lytic activity of several wild type phages against a panel of 110 S. aureus strains (MRSA/MSSA) composed to reflect the prevalence of S. aureus clonal complexes in human infections. The plaquing host ranges (PHR) of the wild type phages were in the range of 51% to 60%. We also measured what we called the kinetic host range (KHR), i.e., the percentage of strains for which growth in suspension was suppressed for 24 h. The KHR of the wild type phages ranged from 2% to 49%, substantially lower than the PHRs. To improve the KHR and other key pharmaceutical properties, we bred the phages by mixing and propagating cocktails on a subset of S. aureus strains. These bred phages, which we termed evolution-squared (ε2) phages, have broader KHRs up to 64% and increased virulence compared to the ancestors. The ε2-phages with the broadest KHR have genomes intercrossed from up to three different ancestors. We composed a cocktail of three ε2-phages with an overall KHR of 92% and PHR of 96% on 110 S. aureus strains and called it PM-399. PM-399 has a lower propensity to resistance formation than the standard of care antibiotics vancomycin, rifampicin, or their combination, and no resistance was observed in laboratory settings (detection limit: 1 cell in 1011). In summary, ε2-phages and, in particular PM-399, are promising candidates for an alternative treatment of S. aureus infections.

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.

Alternative treatments for Escherichia coli infections are urgently needed, and phage therapy is a promising option where antibiotics fail, especially for urinary tract infections (UTI). We used wastewater-isolated phages to test their lytic activity against a panel of 47 E. coli strains reflecting the diversity of strains found in UTI, including sequence type 131, 73 and 69. The plaquing host range (PHR) was between 13 and 63%. In contrast, the kinetic host range (KHR), describing the percentage of strains for which growth in suspension was suppressed for 24 h, was between 0% and 19%, substantially lower than the PHR. To improve the phage host range and their efficacy, we bred the phages by mixing and propagating cocktails on a subset of E. coli strains. The bred phages, which we termed evolution-squared ε2-phages, of a mixture of Myoviridae have KHRs up to 23% broader compared to their ancestors. Furthermore, using constant phage concentrations, Myoviridae ε2-phages suppressed the growth of higher bacterial inocula than their ancestors did. Thus, the ε2-phages were more virulent compared to their ancestors. Analysis of the genetic sequences of the ε2-phages with the broadest host range reveals that they are mosaic intercrossings of 2–3 ancestor phages. The recombination sites are distributed over the whole length of the genome. All ε2-phages are devoid of genes conferring lysogeny, antibiotic resistance, or virulence. Overall, this study shows that ε2-phages are remarkably more suitable than the wild-type phages for phage therapy.

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 plasma and 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. This phage inhibition is due to a 99% reduction of phage adsorption, already at 10% plasma concentration. Coagulation factors bind S. aureus resulting in the formation of aggregates and blood clots that protect the bacterium from the phages. This was confirmed by the finding that purified fibrinogen 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 may be fundamentally challenging, and more investigation is needed prior to proceeding to in-human trials.

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.