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.

ε2-Phages Are Naturally Bred and Have a Vastly Improved Host Range in Staphylococcus aureus over Wild Type Phages.

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.

Characterization of the temperate phage vB_RleM_PPF1 and its site-specific integration into the Rhizobium leguminosarum F1 genome.

Bacteriophages may play an important role in regulating population size and diversity of the root nodule symbiont Rhizobium leguminosarum, as well as participating in horizontal gene transfer. Although phages that infect this species have been isolated in the past, our knowledge of their molecular biology, and especially of genome composition, is extremely limited, and this lack of information impacts on the ability to assess phage population dynamics and limits potential agricultural applications of rhizobiophages. To help address this deficit in available sequence and biological information, the complete genome sequence of the Myoviridae temperate phage PPF1 that infects R. leguminosarum biovar viciae strain F1 was determined. The genome is 54,506 bp in length with an average G+C content of 61.9 %. The genome contains 94 putative open reading frames (ORFs) and 74.5 % of these predicted ORFs share homology at the protein level with previously reported sequences in the database. However, putative functions could only be assigned to 25.5 % (24 ORFs) of the predicted genes. PPF1 was capable of efficiently lysogenizing its rhizobial host R. leguminosarum F1. The site-specific recombination system of the phage targets an integration site that lies within a putative tRNA-Pro (CGG) gene in R. leguminosarum F1. Upon integration, the phage is capable of restoring the disrupted tRNA gene, owing to the 50 bp homologous sequence (att core region) it shares with its rhizobial host genome. Phage PPF1 is the first temperate phage infecting members of the genus Rhizobium for which a complete genome sequence, as well as other biological data such as the integration site, is available.

Genomic and phenotypic characterization of Rhizobium gallicum phage vB_RglS_P106B.

The phage P106B (vB_RglS_P106B) is a Siphoviridae phage with a narrow spectrum of infectivity, which has been isolated from soils with a history of pea cultivation. The trapping host of P106B is an indigenous strain of Rhizobium gallicum (SO14B-4) isolated from soils associated with Vicia cracca. Phenotypic characterization of the phage revealed that P106B has an approximate burst size of 21 p.f.u. per infected cell with 60 min and 100 min eclipse and latent periods, respectively. Phage P106B was unable to transduce under the conditions tested. The genome of P106B is 56 024 bp in length with a mean DNA G+C content of 47.9 %. The complete genome sequence contains 95 putative ORFs and a single tRNA gene coding for leucine with the anticodon TTA. Putative functions could only be assigned to 22 of the predicted ORFs while a significant number of ORFs (47) shared no sequence similarities to previously characterized proteins. The remaining 26 putative protein-coding genes exhibited a sequence resemblance to other hypothetical proteins. No lysogeny-related genes were found in the P106B genome.