Richa, R. | bioRxiv Science

bioRxiv2025

Optimizing phage-antibiotic combinations: impact of administration order against daptomycin non-susceptible (DNS) MRSA clinical isolates

BackgroundThe rise of bacterial resistance has driven the exploration of novel therapies, such as bacteriophage-antibiotic cocktails (PACs), which have shown in vitro promise against resistant pathogens, including daptomycin non-susceptible (DNS-MRSA) strains. While daptomycin has been a cornerstone for treating MRSA bacteremia and vancomycin-refractory infective endocarditis, the emergence of DNS-MRSA presents a significant challenge due to high morbidity, mortality, and rapid intrinsic resistance development. MethodsPhages Intesti13 and Sb-1, were selected for their unique host ranges and activity against sixteen DNS-MRSA strains. Synergy with antibiotics was assessed via growth suppression curves and 24-hour time-kill assays (TKAs) across administration sequences and MIC increments. Selected regimens were further assessed in an ex-vivo simulated endocardial vegetation (SEV) models, with pharmacokinetic analyses confirming target antibiotic concentrations. ResultsIn the ex-vivo SEV model, simultaneous PAC administration using daptomycin{+/-}phage, showed superior bactericidal activity over sequential treatments in isolate C6 (p<0.01). Similarly, in the same model, C2 reached detection limits within 48h and remained suppressed for 120h (p<0.0037). Sequential outcomes varied by phage-antibiotic order and antibiotic choice. Simultaneous and phage-first regimens outperformed antibiotic-first, especially in 24h TKAs, but showed variability at lower MICs and between in-vitro and ex-vivo settings. ConclusionThis study highlights PACs potential for DNS-MRSA treatment, emphasizing the importance of administration timing. The observed differences across clinical strains emphasize the need for strain-specific evaluations and a deeper understanding of phage-antibiotic interactions to optimize therapy. Future research must focus on expanding phage diversity, refining protocols, and clinically validating sequential strategies to enhance PAC efficacy.

microbiology↗

bioRxiv2024

In vivo Treatment of a Severe Vascular Disease via a Bespoke CRISPR-Cas9 Base Editor

Genetic vascular disorders are prevalent diseases that have diverse etiologies and few treatment options. Pathogenic missense mutations in the alpha actin isotype 2 gene (ACTA2) primarily affect smooth muscle cell (SMC) function and cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection, and death in childhood. Here, we explored genome editing to correct the most common MSMDS-causative mutation ACTA2 R179H. In a first-in-kind approach, we performed mutation-specific protein engineering to develop a bespoke CRISPR-Cas9 enzyme with enhanced on-target activity against the R179H sequence. To directly correct the R179H mutation, we screened dozens of configurations of base editors (comprised of Cas9 enzymes, deaminases, and gRNAs) to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We then created a murine model of MSMDS that exhibits phenotypes consistent with human patients, including vasculopathy and premature death, to explore the in vivo therapeutic potential of this base editing strategy. Delivery of the customized base editor via an engineered SMC-tropic adeno-associated virus (AAV-PR) vector substantially prolonged survival and rescued systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta, and brain. Together, our optimization of a customized base editor highlights how bespoke CRISPR-Cas enzymes can enhance on-target correction while minimizing bystander edits, culminating in a precise editing approach that may enable a long-lasting treatment for patients with MSMDS.

bioengineering↗