# P-1190. Targeting Phage Defense Mechanisms: Expanding Phage Activity in the Context of Suspected abiF-Mediated Resistance against MRSA

## TL;DR

This study explores how adding a third phage can overcome bacterial resistance in MRSA, particularly when the bacteria have the abiF gene.

## Contribution

The study identifies that the abiF gene may mediate resistance to certain phages and shows that Silviavirus-like phages can bypass this resistance.

## Key findings

- Adding a third phage significantly enhanced bacterial killing in abiF-positive MRSA isolates.
- abiF-negative isolates showed similar responses to two- and three-phage cocktails.
- Phage cocktails with diverse infection mechanisms may improve treatment of multidrug-resistant S. aureus.

## Abstract

Phage resistance mechanisms represent a critical challenge in the advancement of phage therapy. Staphylococcus aureus utilizes multiple defense strategies, including adsorption inhibition via wall teichoic acid modification, restriction-modification systems, CRISPR-Cas systems, and abortive infection (Abi) pathways. The Abi system has been proposed to trigger bacterial self-destruction to prevent Kayvirus phage propagation, although its functional role in S. aureus remains poorly characterized.Two versus three-phage combinations stratified by abiF and pdpSau genotypes

Two versus three-phage combinations stratified by abiF and pdpSau genotypes

We evaluated four well-characterized daptomycin non-susceptible MRSA (DNS-MRSA) isolates from the Cubist repository. Time-kill assays (24h) were performed using either a two-phage cocktail (Intesti13 + Sb-1) or a three-phage cocktail (Intesti13 + Sb-1 + Romulus) at a multiplicity of infection (MOI) of 1.0. Change in bacterial burden was measured as log10 CFU/mL reduction. Statistical analysis was conducted using two-way ANOVA with Tukey's post hoc test (p < 0.05). Whole genome sequences were used to assess the presence of known phage defense systems.

While the two-phage cocktail modestly reduced bacterial burden in most DNS-MRSA isolates, adding Romulus significantly enhanced killing in select strains. The most pronounced effect was in C31, which harbors the abiF gene; this isolate was resistant to the two-phage cocktail (+1.14 ± 0.04 log10 CFU/mL) but exhibited a > 4.0 log10 reduction with the three-phage combination (–2.75 ± 0.29, p < 0.0001), suggesting Romulus may evade abiF-mediated defense. In contrast, abiF-negative isolates (C2, C4, C6) responded similarly to both cocktails, indicating a potential K-phage resistance mechanism.

These findings suggest that abiF may mediate resistance specific to Kayirus-like phages, with Silviavirus-like phages remaining unaffected. Incorporating phages with distinct infection mechanisms may help bypass such resistance and enhance therapeutic efficacy. A diverse phage cocktail could be key to overcoming intrinsic defenses and optimizing empiric treatment for multidrug-resistant S. aureus.

Michael J. Rybak, PharmD, PhD, MPH, Abbvie: Grant/Research Support|Innoviva: Grant/Research Support|Melina: Grant/Research Support|Merck: Grant/Research Support|Shionogi: Grant/Research Support