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Despite over a century of collecting bacteriophages, there has been a persistent lack of interest in systematically cataloging resulting phage banks. The result was a situation in which the ongoing growth of phage infrastructures was paralleled by an increasing fragmentation of knowledge about collections' contents and existence. Over the last two decades, renewed interest in phage therapy and phage biology has further exacerbated confusion amid a rapid increase in the number of large and small phage collections and an ongoing dearth of coordination and standardized cataloging. Whatever the modalities (isolated phages or genomes), the time has undoubtedly come to create sustainable, interconnected, and equitable phage banking infrastructures. This article reviews both the history and current status of microbial collections, provides a nonexhaustive overview of relevant phage collections, and reflects on the challenges and potential of centralizing therapeutically relevant collections ahead of likely paradigm shifts caused by synthetic biology and artificial intelligence.

Introduction : Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of sight-threatening infections in the US. These strains pose a significant challenge in managing ocular infections, as they frequently exhibit resistance to first-line empirical antibiotics. To assess the potential of bacteriophages as innovative topical therapies for treatment of recalcitrant ocular infections, we evaluated the in vitro antimicrobial activity of a set of anti-S. aureus phages against a collection of ocular MRSA clinical isolates collected in the US. Methods : The host range of six phages (V4SA2, V1SA9, V1SA12, V1SA19, V1SA20 and V1SA22) was assessed using the spot assay on a panel of 50 multidrug-resistant (MDR) ocular MRSA isolates selected to be representative of clones circulating in the US. Subsequently, liquid culture-based host range assay was performed for the three most active phages using different multiplicity of infection (MOI of 10-2, 1 or 100 phages/bacteria). Results : In total, 90.0% of bacterial isolates were susceptible to at least one of the six phages. The spot host range assay showed that phages V1SA19, V1SA20 and V1SA22 had the broadest spectrum, being active against 86%, 84% and 82% of the isolates, respectively, including the MDR-MRSA CC5 and the community-associated CC8 lineages. A phage dose effect was observed across the liquid culture-based host range assay. Conclusion : Phages V1SA19, V1SA20 and V1SA22 exhibited high antimicrobial activity against ocular MRSA. Bacteriophages represent a promising anti-infective strategy in ophthalmology that could be explored for improved topical therapy of recalcitrant MRSA infections.

Phage therapy uses viruses (phages) against antibiotic resistance. Tailoring treatments to specific patient strains requires stocks of various highly concentrated purified phages. It, therefore, faces challenges: titration duration and specificity to a phage/bacteria couple; purification affecting stability; and highly concentrated suspensions tending to aggregate. To address these challenges, interferometric light microscopy (ILM), characterising particles (size, concentration, and visual homogeneity) within minutes, was applied herein to anti-Staphylococcus aureus myovirus phage suspensions. Particle concentration was linearly correlated with phage infectious titre (R2 > 0.97, slope: 3 particles/plaque forming units (PFU)) at various degrees of purification, allowing to approximate the infectious titre for suspensions ≥ 3 × 108 PFU/mL, thereby encompassing most therapeutic doses. Purification narrowed and homogenised particle distribution while maintaining therapeutic concentrations. When compared to dynamic light scattering, electrophoretic mobility, and UV/Visible-spectroscopy, ILM best detected aggregates according to our homemade scoring. Although ILM has certain limitations, such as the inability to detect podoviruses (hydrodynamic diameter < 80 nm), or to measure particles in low-concentrated suspensions (< 108 particles/mL), the present proof-of-concept positions this technique as a valuable quality control tool, as a complement to titration rather than a replacement for this technique, for phage suspensions, paving the way for further investigations.

Phage therapy is a highly promising approach to address the challenge that is presented by the global burden of antimicrobial resistance. Given the natural specificity of phages, phage susceptibility testing (PST) is a prerequisite for successful personalized therapy, allowing the selection of active phages from large and diverse collections. However, the issue of an easy-to-use and standardized technique remains. In this review, we describe the principles, advantages and drawbacks of two routinely used PST techniques: plaque and growth kinetic assays. These are labour-intensive and time-consuming methods that require automation of one or more steps, including preparation of test panels, incubation, reading and analysis of results. In addition to automation, there is an urgent need to establish a reference method to enable efficient of PST techniques selection of therapeutic phages. We discuss knowledge gaps and parameters that need to be investigated to work towards this goal.

Phage therapy appears to be a promising approach to tackle multidrug-resistant bacteria, including staphylococci. However, most anti-staphylococcal phages have been characterized in Staphylococcus aureus, while a limited number of studies investigated phage activity against S. epidermidis. We studied the potential of phage training to extend the host range of two types of anti-S. aureus phages against S. epidermidis isolates. The Appelmans protocol was applied to a mixture of Kayvirus and a mixture of Silviavirus phages repeatedly exposed to seven S. epidermidis strains representative of nosocomial-associated sequence types (ST), including the world-wide disseminated ST2. We observed increased activity only for the Kayvirus mixture against two of these strains (ST2 or ST35). Phage subpopulations isolated from the training mixture using these two strains (five/strain) exhibited different evolved phenotypes, active only against their isolation strain or strains of the same ST. Of note, 16/47 ST2 strains were susceptible to one of the groups of trained phages. A comparative genomic analysis of ancestral and trained phage genomes, conducted to identify potential bacterial determinants of such specific activity, found numerous recombination events between two of the three ancestors. However, a small number of trained phage genes had nucleotide sequence modifications impacting the corresponding protein compared to ancestral phages, two to four of them per phage genome being specific of each group of phage subpopulations exhibiting different host range. The results suggest that anti-S. aureus phages can be adapted to S. epidermidis isolates but with inter- and intra-ST specificity.ImportanceS. epidermidis is increasingly recognized as a threat for public health. Its clinical importance is notably related to multidrug resistance. Phage therapy is one of the most promising alternative therapeutic strategies to antibiotics. Nonetheless, only very few phages active against this bacterial species have been described. In the present study, we showed that phage training can be used to extend the host range of polyvalent Kayvirus phages within the Staphylococcus genera to include S. epidermidis species. In the context of rapid development of phage therapy, in vitro forced adaptation of previously characterized phages could be an appealing alternative to fastidious repeated isolation of new phages to improve the therapeutic potential of a phage collection.

The resurgence of phage therapy, once abandoned in the early 20th century in part due to issues related to the purification process and stability, is spurred by the global threat of antibiotic resistance. Engineering advances have enabled more precise separation unit operations, improving overall purification efficiency. The present review discusses the physicochemical properties of impurities commonly found in a phage lysate, e.g., contaminants, phage-related impurities, and propagation-related impurities. Differences in phages and bacterial impurities properties are leveraged to elaborate a four-step phage purification process: clarification, capture and concentration, subsequent purification and polishing. Ultimately, a framework for rationalising the development of a purification process is proposed, considering three operational characteristics, i.e., scalability, transferability to various phages and duration. This guide facilitates the preselection of a sequence of unit operations, which can then be confronted with the expected impurities to validate the theoretical capacity of the process to purify the phage lysate.

Bacteriophages are naturally occurring viruses that specifically target bacteria. They are widely distributed in the environment. The concept of phage therapy is to isolate phages, characterize them, cultivate them and then purify them to treat bacterial infections. There is currently a revival of phage therapy, and its implementation presupposes the availability of active phages of pharmaceutical quality. From a regulatory point of view, the status of phages is not yet clearly defined by the authorities. The availability of phages produced by the pharmaceutical industry and through academic development programs such as the PHAGEinLYON program represents a breakthrough in the development of phage therapy. Prosthetic joint infections and digestive diseases seem to be relevant indications, but preclinical studies and randomized clinical trials are now needed to be done.

Introduction : In neonatal intensive care units (NICUs), the standard chemical-based disinfection procedures do not allow a complete eradication of pathogens from environmental surfaces. In particular, the clone Staphylococcus capitis NRCS-A, a significant pathogen in neonates, was shown to colonize neonatal incubators. The aim of this study was to evaluate the in vitro effect of a bacteriophage cocktail on NRCS-A eradication. Methods : Three bacteriophages were isolated, genetically characterized and assessed for their host range using a collection of representative clinical strains (n=31) belonging to the clone NRCS-A. The efficacy of a cocktail including these three bacteriophages to eradicate the reference strain S. capitis NRCS-A CR01 was determined in comparison or in combination with the chemical disinfectant Surfanios Premium on either dry inoculum or biofilm-embedded bacteria. The emergence of bacterial resistance against the bacteriophages alone or in cocktail was evaluated by growth kinetics. Results : The three bacteriophages belonged to two families and genera, namely Herelleviridae/Kayvirus for V1SC01 and V1SC04 and Rountreeviridae/Andhravirus for V1SC05. They were active against 17, 25 and 16 of the 31 tested strains respectively. Bacteriophage cocktails decreased the bacterial inoculum of both dry spots and biofilms, with a dose dependent effect. The sequential treatment with bacteriophages then Surfanios Premium did not show enhanced efficacy. No bacterial resistance was observed when using the bacteriophage cocktail. Discussion : This study established a proof-of-concept for the use of bacteriophages to fight against S. capitis NRCS-A. Further investigations are needed using a larger bacterial collection and in real-life conditions before being able to use such technology in NICUs.

Phage therapy a promising antimicrobial strategy to address antimicrobial resistance for infections caused by the major human pathogen Staphylococcus aureus. Development of therapeutic phages for human use should follow pharmaceutical standards, including selection of strictly lytic bacteriophages with high therapeutic potential and optimization of their production process. Results: Here, we describe three novel Silviavirus phages active against 82% of a large collection of strains (n = 150) representative of various methicillin-susceptible and -resistant S. aureus clones circulating worldwide. We also investigated the optimization of the efficiency and safety of phage amplification protocols. To do so, we selected a well-characterized bacterial strain in order to (i) maximize phage production yields, reaching phage titres of 1011 PFU/mL in only 4 h; and (ii) facilitate phage purity while minimizing the risk of the presence of contaminants originating from the bacterial host; i.e., secreted virulence factors or induced temperate phages.