2nd Place Global Finalist, SARC 2026

Abstract:

Methicillin-resistant Staphylococcus aureus (MRSA) causes over 130,000 deaths annually and leads global pathogen-attributable mortality growth (Murray et al., 2024). Biofilm formation reduces vancomycin susceptibility up to 1,000-fold through matrix-mediated diffusion restriction and persister cell dormancy, a structural problem dose escalation cannot resolve (Sharma et al., 2023). Bacteriophage K, a polyvalent myovirus with characterised depolymerase activity against staphylococcal matrices, offers a mechanistically distinct adjunct, yet optimal sequencing relative to vancomycin remains undefined. This proposal compares phage-first, simultaneous, and antibiotic-first administration strategies against mature MRSA biofilms in vitro, quantified via crystal violet staining, CFU enumeration, and LIVE/DEAD confocal microscopy. Given that phage depolymerases disrupt the matrix prior to antibiotic exposure and sub-inhibitory vancomycin promotes phage replication via phage-antibiotic synergy (PAS), it is hypothesised that phage-first administration will yield the greatest reductions in biofilm biomass and viable cell counts, with synergistic interaction (FICI ≤ 0.5) exclusive to this group.

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Introduction:

AMR is projected to cause over 39 million deaths between 2025 and 2050, with MRSA the leading single pathogen-drug combination driving rising attributable mortality (Murray et al., 2024). In low- and middle-income countries (LMICs), this burden is compounded by restricted second-line antibiotic access and limited stewardship capacity, making MRSA biofilm infections, particularly prosthetic device infections , frequently managed with protocols that have demonstrably failed (Tacconelli et al., 2018; Visperas et al., 2022). MRSA's clinical threat stems not from resistance alone but from resistance combined with biofilm formation: its extracellular polymeric matrix restricts antibiotic penetration, promotes persister formation, and shields bacteria from immune clearance (Arshad et al., 2024; Bhattacharya & Horswill, 2024), driving minimum biofilm eradication concentrations beyond safe systemic vancomycin doses (Sharma et al., 2023). Bacteriophages re-emerge here as a mechanistically orthogonal strategy: phages actively replicate within biofilms and encode depolymerases that degrade the matrix, reopening diffusion channels before antibiotic action (Guo et al., 2023). Compassionate-use cases demonstrate clinical feasibility against refractory S. aureus (Dedrick et al., 2019; Petrovic Fabijan et al., 2020), yet systematic evidence on whether administration sequence alters outcome is absent. This study addresses that gap.

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Literature Review:​ 

Vancomycin monotherapy fails against MRSA biofilms because protein-eDNA scaffold architecture imposes a diffusion barrier reducing effective intrabiofilm antibiotic concentration by an order of magnitude or more, while simultaneously selecting metabolically dormant persisters refractory to all activity-targeting agents (Arshad et al., 2024; Sharma et al., 2023). This is a structural failure, not a dosing failure. Phage-encoded depolymerases cleave both polysaccharide and eDNA matrix components, physically dismantling this barrier before antibiotic exposure; progeny virions then penetrate inward, amplifying matrix degradation iteratively (Guo et al., 2023). This temporal logic: disruption first, antibiotic second, is mechanistically incompatible with simultaneous administration, where both agents compete through an intact matrix.

PAS adds a further rationale: sub-inhibitory antibiotics amplify phage replication via antibiotic-induced bacterial filamentation, enlarging adsorption surface area (Comeau et al., 2007; Hosseinidoust et al., 2023). Loganathan et al. (2024) found phage-first strategies consistently outperformed antibiotic-first approaches across MRSA clinical isolates; Chaudhry et al. (2018) reported up to 3-log CFU/mL reductions under sequential versus simultaneous administration, most pronounced with sub-MIC vancomycin. Both studies, however, used non-MRSA strains or non-Phage K phages, leaving the specific pairing examined here systematically uncharacterised. Kebriaei et al. (2023) showed that multi-phage cocktails outperform single-phage approaches, yet sequence optimisation of a single-phage regimen is considerably more implementable in LMICs, where cocktail manufacturing and regulatory approval are prohibitive barriers. With MRSA prosthetic joint infection failure rates of 31--63% (Visperas et al., 2022), protocol-level optimisation is both mechanistically justified and directly actionable.

 

Methodology:
A controlled in vitro experiment will compare three treatment sequences against mature MRSA biofilms (one untreated control), conducted in biological triplicate.

Bacterial culture and biofilm formation. MRSA strain ATCC BAA-1717 will be cultured in TSB at 37°C, 180 rpm. Cultures diluted to 0.5 McFarland (~1×10⁸ CFU/mL) will be seeded into 96-well polystyrene microtitre plates and incubated statically at 37°C for 24 hours; non-adherent cells removed by PBS washes.

Treatment groups. Group A (phage-first): Bacteriophage K (MOI 1) for 2 h, PBS wash, then vancomycin (0.5× MIC) for 22 h. Group B (simultaneous): Phage K (MOI 1) + vancomycin (0.5× MIC) concurrently for 24 h. Group C (antibiotic-first): vancomycin (0.5× MIC) for 2 h, PBS wash, then Phage K (MOI 1) for 22 h. Group D: sterile PBS. Vancomycin MIC determined by broth microdilution per EUCAST guidelines. Sub-inhibitory concentrations are used to isolate phage-driven matrix disruption and enable PAS-mediated phage amplification.

Outcome measurements. Biofilm biomass: crystal violet staining with OD₅₉₅ absorbance (Tecan Spark). Bacterial viability: sonication-resuspended biofilm plated on Mannitol Salt Agar for CFU enumeration. Structural integrity: LIVE/DEAD BacLight confocal microscopy (Zeiss LSM 800, 488/561 nm). Statistical analysis: one-way ANOVA with Tukey's HSD (α = 0.05) in GraphPad Prism 10; CFU data log₁₀-transformed. Phage-antibiotic interaction classified by FICI (≤0.5 = synergy; 0.5–2.0 = additivity; >2.0 = antagonism) per EUCAST standards.

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Conclusion:

This study will determine whether Bacteriophage K pre-treatment yields superior MRSA biofilm eradication compared to simultaneous and antibiotic-first protocols. Confirmation of phage-first synergy (FICI ≤ 0.5) would provide the first systematic sequencing evidence for this phage-antibiotic-pathogen triad — translatable to clinical practice without novel drug development, new manufacturing infrastructure, or additional regulatory pathways beyond existing compassionate-use frameworks (Dedrick et al., 2019). This positions sequencing optimisation as the most immediately actionable intervention for the 31–63% treatment failure burden in MRSA prosthetic joint infections (Visperas et al., 2022), particularly in LMICs where critical-priority MRSA is least tractable (Tacconelli et al., 2018). Limitations include the absence of immune microenvironment and pharmacokinetic complexity in the microtitre plate model, use of a single reference strain limiting generalisability, and the inability to capture phage-resistant mutant emergence within a 24-hour window (Hampton et al., 2020). Future work should validate findings in murine implant infection models across clinical MRSA isolate panels.

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