Appelmans protocol – A directed in vitro evolution enables induction and recombination of prophages with expanded host range

Infections caused by carbapenem-resistant Acinetobacter baumannii (CRAB) present significant healthcare challenges due to limited treatment options. Bacteriophage (phage) therapy offers potential as an alternative treatment. However, the high host specificity of phages poses challenges for their therapeutic application. To broaden the phage spectrum, laboratory-based phage training using the Appelmans protocol was employed in this study. As a result, the protocol successfully expanded the host range of a phage cocktail targeting CRAB. Further analysis revealed that the expanded host range phages isolated from the output cocktail were identified as recombinant derivatives originating from prophages induced from encountered bacterial strains. These findings provide valuable genetic insights into the protocol's mechanism when applied to phages infecting A. baumannii strains that have never been investigated before. However, it is noteworthy that the expanded host range phages obtained from this protocol exhibited limited stability, raising concerns about their suitability for therapeutic purposes

A retrospective, observational study of 12 cases of expanded access customized phage therapy: production, characteristics, and clinical outcomes

BACKGROUNDAntibiotic resistance (AMR) is undermining modern medicine, a problem compounded by bacterial adaptation to antibiotic pressures. Phages are viruses that infect bacteria. Their diversity and evolvability offer the prospect of their use as a therapeutic solution. Reported are outcomes of customized phage therapy for patients with difficult-to-treat AMR infections. METHODSWe retrospectively assessed 12 cases of customized phage therapy from a phage production center. Phages were screened, purified, sequenced, characterized, and FDA-approved via the IND compassionate care route. Outcomes were assessed as favorable or unfavorable by microbiologic and clinical standards. Infections were device-related or systemic. Other experiences such as time to treatment, antibiotic synergy and immune responses were recorded. RESULTSFifty requests for phage therapy were received. Customized phages were generated for twelve patients. After treatment, 42% (5/12) of cases showed bacterial eradication and 58% (7/12) showed clinical improvement, with two-thirds of all cases (66%) showing favorable responses. No major adverse reactions were observed. Antibiotic-phage synergy in vitro was observed in most cases. Immunological neutralization of phage was reported in five cases. Several cases were complicated by secondary infections. Complete characterization of the phages (morphology, genomics, and activity) and their production (methods, sterility, and endotoxin tests) are reported. CONCLUSIONSCustomized phage production and therapy was safe and yielded favorable clinical or microbiological outcomes in two-thirds of cases. A center or pipeline dedicated to tailoring the phages against a patient's specific AMR bacterial infection may be a viable option where standard treatment has failed