Summary Phage therapy is a promising solution for bacterial infections that are not eradicated by conventional antibiotics. A crucial element of this approach is appropriate matching of bacteriophages and antibiotics to the bacterial target according to the clinical setting. However, there is currently little consistency in the protocols used for the laboratory evaluation of bacteriophages intended for antibacterial treatment. In this Personal View, we suggest a framework aimed to match appropriate bacteriophage-based treatments in clinical microbiology laboratories. This framework, which we have termed Clinical Phage Microbiology, is based on the current research on phage treatments. In addition, we discuss special cases that might require additional relevant evaluation, including bacteriophage interactions with the host immune response, biofilm-associated infections, and polymicrobial infections. The Clinical Phage Microbiology pipeline could serve as the basis for future standardisation of laboratory protocols for personalised phage therapy.

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

http://manuscript.elsevier.com/S1879625722000086/pdf/S1879625722000086.pdf

Mitigation of evolved bacterial resistance to phage therapy.

Bacteriophage therapy is a potential adjunctive treatment for periprosthetic joint infections (PJIs) given the capabilities of bacteriophages to degrade biofilms, self-replicate, and lyse bacteria. However, many aspects of this therapeutic are ill-defined, and the narrow spectrum of bacteriophage activity along with limited available bacteriophage strains curb potential use for specific bacteria such as Staphylococcus aureus at the present time. Therefore, the aim of this study was to determine the feasibility of using bacteriophages for PJI by (1) categorizing the causative organisms in hip and knee PJI at a tertiary academic center and (2) evaluating in vitro activity of a group of bacteriophages against clinical S. aureus PJI isolates.Patients with chronic hip or knee PJI after undergoing the first stage of a 2-stage revision protocol from 2017 to 2020 were identified retrospectively by a query of the hospital billing database. The causative pathogens in 129 cases were reviewed and categorized. From this cohort, preserved S. aureus isolates were tested against a library of 15 staphylococcal bacteriophages to evaluate for bacterial growth inhibition over 48 hours.S. aureus was the most common pathogen causing PJI (26% [33] of 129 cases). Of 29 S. aureus samples that were analyzed for bacteriophage activity, 97% showed adequate growth inhibition of the predominant planktonic colonies by at least 1 bacteriophage strain. However, 24% of the 29 samples demonstrated additional smaller, slower-growing S. aureus colonies, none of which had adequate growth inhibition by any of the initial 14 bacteriophages. Of 5 secondary colonies that underwent subsequent testing with another bacteriophage with enhanced biofilm activity, 4 showed adequate growth inhibition.Effective bacteriophage therapeutics are potentially available for S. aureus PJI isolates. The differences in bacteriophage activity against the presumed small-colony variants compared with the planktonic isolates have important clinical implications. This finding suggests that bacteriophage attachment receptors differ between the different bacterial morphologic states, and supports future in vitro testing of bacteriophage therapeutics against both planktonic and stationary states of PJI clinical isolates to ensure activity. 

Assessment of Staphylococcal Clinical Isolates from Periprosthetic Joint Infections for Potential Bacteriophage Therapy

Despite occurring at the microscopic scale, the armed race between phages and their bacterial hosts involves multiple mechanisms, some of which are just starting to be understood. On the one hand, bacteria have evolved strategies that can stop the viral infection at different stages (adsorption, DNA injection and replication, biosynthesis and assembly of the viral progeny and/or release of the newly formed virions); on the other, phages have gradually evolved counterattack strategies that allow them to continue infecting their prey. This co-evolutionary process has played a major role in the development of microbial populations in both natural and man-made environments. Notably, understanding the parameters of this microscopic war will be paramount to fully benefit from the application of phage therapy against dangerous, antibiotic-resistant human pathogens. This review gathers the current knowledge regarding the mechanisms of phage resistance in the Staphylococcus genus, which includes Staphylococcus aureus, one of the most concerning microorganisms in terms of antibiotic resistance acquisition. Some of these strategies involve permanent changes to the bacterial cell via mutations, while others are transient, adaptive changes whose expression depends on certain environmental cues or the growth phase. Finally, we discuss the most plausible strategies to limit the impact of phage resistance on therapy, with a special emphasis on the importance of a rational design of phage cocktails in order to thwart therapeutic failure.

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Understanding the Mechanisms That Drive Phage Resistance in Staphylococci to Prevent Phage Therapy Failure

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 e2-phages, of a mixture of Myoviridae have KHRs up to 23% broader compared to their ancestors. Furthermore, using constant phage concentrations, Myoviridae e2-phages suppressed the growth of higher bacterial inocula than their ancestors did. Thus, the e2-phages were more virulent compared to their ancestors. Analysis of the genetic sequences of the e2-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 e2-phages are devoid of genes conferring lysogeny, antibiotic resistance, or virulence. Overall, this study shows that e2-phages are remarkably more suitable than the wild-type phages for phage therapy.

Natural Bred ε2-Phages Have an Improved Host Range and Virulence against Uropathogenic Escherichia coli over Their Ancestor 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 (e2) phages, have broader KHRs up to 64% and increased virulence compared to the ancestors. The e2-phages with the broadest KHR have genomes intercrossed from up to three different ancestors. We composed a cocktail of three e2-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, e2-phages and, in particular PM-399, are promising candidates for an alternative treatment of S. aureus infections.

https://www.mdpi.com/1424-8247/14/4/325/pdf

Marcela Restrepo-Córdoba

Papers

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