# P-1268. Phage Sensitivity Profiles and their Association with Biofilm-mediated Antibiotic Tolerance in DNS-MRSA Isolates

## TL;DR

This study explores how phage sensitivity relates to antibiotic tolerance in daptomycin-resistant MRSA strains, suggesting phage profiling could help identify difficult-to-treat infections.

## Contribution

The study identifies a potential link between phage resistance and biofilm-mediated antibiotic tolerance in DNS-MRSA isolates.

## Key findings

- Phage-resistant DNS-MRSA strains showed higher biofilm-associated antibiotic tolerance, as indicated by elevated MBIC values.
- Strains susceptible to all three phages had uniformly low MBICs, suggesting reduced biofilm tolerance.
- Dual mutations in gyrA and parC were found in phage-resistant strains with elevated MBICs.

## Abstract

Daptomycin non-susceptible MRSA (DNS-MRSA) presents a significant therapeutic challenge due to resistance to multiple antibiotic agents and biofilm-associated antibiotic tolerance. While phage therapy is a promising alternative, resistance mechanisms and strain-specific differences in biofilm formation may hinder efficacy.Table 1.List of DNS-MRSA strains with corresponding MIC/MBIC and phage susceptibilitiesMIC and MBIC tests were determined via microbroth dilution assays*Daptomycin (DAP), Levofloxacin (LEV), Rifampicin (RIF), Delafloxacin (DEL)Bacteriophages: Intesti13, Sb-1, and RomulusS: Sensitive, clear or high sensitivity to Sb-1 with distinct plaque formation (EOP of 0.1-1 in comparison to reference strain)I: Turbid or medium sensitivity to phage with distinct plaque formation (EOP of 0.0001-0.01 in comparison to reference strain)R: Resistant or no disturbance of bacterial lawn and no plaque formation

List of DNS-MRSA strains with corresponding MIC/MBIC and phage susceptibilities

MIC and MBIC tests were determined via microbroth dilution assays

*Daptomycin (DAP), Levofloxacin (LEV), Rifampicin (RIF), Delafloxacin (DEL)

Bacteriophages: Intesti13, Sb-1, and Romulus

S: Sensitive, clear or high sensitivity to Sb-1 with distinct plaque formation (EOP of 0.1-1 in comparison to reference strain)

I: Turbid or medium sensitivity to phage with distinct plaque formation (EOP of 0.0001-0.01 in comparison to reference strain)

R: Resistant or no disturbance of bacterial lawn and no plaque formation

We characterized the antimicrobial susceptibility and biofilm-associated tolerance of several DNS-MRSA clinical isolates and two transposon mutagenized S. aureus strains: wild-type JE2 (wt) and NE766 (deficient in poly-N-acetylglucosamine-mediated biofilm production). MIC and MBIC values were determined via broth microdilution for daptomycin (DAP), levofloxacin (LEV), rifampin (RIF), and delafloxacin (DEL). Phage susceptibility was previously determined via plaque assays. Antibiotic and phage resistance mechanisms, including mutations in gyrA, gyrB, qacC, and parC were identified through whole-genome sequencing.

Among the DNS-MRSA isolates, we observed discordance between MIC and MBIC values, particularly for rifampin and delafloxacin, in Kayvirus phage-resistant strains C10 and C23. These strains also harbored dual gyrA and parC mutations. In contrast, NE766 displayed notably lower MBICs across all antibiotics tested (Table 1), despite comparable MICs to JE2 (wt). Strains with susceptibility to all three phages demonstrated uniformly low MBICs, suggesting a potential correlation between phage susceptibility and reduced biofilm-associated tolerance.

These results suggest that phage resistance, particularly to Kayvirus-type phages, may serve as a potential marker of biofilm-associated antibiotic tolerance in certain clinical DNS-MRSA strains. While phage susceptibility alone does not guarantee improved antibiotic activity, combined phage-antibiotic profiling and biofilm characterization may offer a more comprehensive approach to identifying recalcitrant phenotypes and tailoring therapy.

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