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

Abstract

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.

Clinical Phage Microbiology: a suggested framework and recommendations for the in-vitro matching steps of phage therapy

“…Nevertheless, the use of phage cocktails is unavoidable if no bacteriophage has shown sufficient efficacy when tested individually 70 or in the case of polymicrobial infections. 10 Furthermore, if a discrepancy between the results of different phage evaluation techniques arises, 71,72 phage cocktails with increased coverage could be considered. 70 In cases of polymicrobial infections, or any use of phage cocktails, we recommend assessing the combined effect of all planned treatments on a mixture of all relevant targets with a growth kinetics checkerboard.…”

Natural Bred ε2-Phages Have an Improved Host Range and Virulence against Uropathogenic Escherichia coli over Their Ancestor Phages

“…Comparing the plaque formation and the killing of planktonic bacteria in suspension revealed significant differences in the determined host range with less efficacy of phages in the kinetic measurements. Recently, we observed this phenomenon for S. aureus phages as well [ 29 ]. The KHR after 6 h was comparable to the PHR, indicating that initial phage efficiency is comparable in both assays.…”

“…To improve the host range of the wild-type phages, mixtures of two different sets of phages were bred by incubation with 24 E. coli strains ( Table S2 ) in artificial urine for 20 rounds, essentially as described [ 29 ]. The first breeding cocktail contained the myoviruses CHD16UKE1, CHD94UKE2, 101117BS1, and G3G7.…”

“…Recently, using a similar method, we created natural ε 2 -phages of S. aureus showing a host range twice as broad as their ancestors [ 29 ]. The fact that the host range improvement in the ε 2 -phages of E. coli is not as large could be related to the fact that E. coli strains are more diverse.…”

“…The principle of ‘Cross-breeding’ was already described in 1947 for E. coli phages with different plaque morphologies [ 25 ], and in nature, genetic intercrossing is one of the drivers of phage evolution, which leads to the mosaicism of phage genomes [ 26 ]. Some authors have used this method to generate phages with a wider host range [ 27 , 28 ] and we recently described the optimization of this method to generate Staphylococcus aureus phages with a vastly improved virulency and host range [ 29 ].…”