Theoretical and Empirical Contribution to Understanding Persister Cell Mechanisms in MRSA Biofilm Infections Under Clinical Antibiotic Exposure Conditions in Nigeria

Antibiotic persister cells represent a phenotypically distinct, metabolically dormant subpopulation within MRSA biofilms that survive lethal antibiotic concentrations and repopulate infections upon antibiotic withdrawal, constituting a central yet incompletely understood mechanism of recurrent chronic infections. Existing persister cell theories have been validated primarily in laboratory reference strains, and their applicability to clinical MRSA isolates from resource-limited tropical settings with distinct antibiotic exposure histories has not been empirically tested. This study combined original theoretical framework development with empirical investigation of persister cell mechanisms in MRSA clinical isolates from tertiary hospitals in Enugu, Kano, and Lagos States. MRSA isolates were phenotypically screened for persister formation capacity using the standard biphasic killing assay under clinical vancomycin and daptomycin concentrations. Transcriptomics of sorted persister and non-persister populations was performed using single-cell RNA sequencing (scRNA-seq) to characterize persister-specific gene expression programs. Metabolomic profiling of persister-enriched biofilms was conducted by untargeted NMR spectroscopy. A novel theoretical model, the Persistence-Reactivation-Tolerance (PRT) cascade, was formulated incorporating clinical antibiotic pharmacokinetic profiles and biofilm spatial heterogeneity. Results showed significant inter-isolate variation in persister fractions (0.001 to 2.3% of biofilm population). scRNA-seq identified transcriptional states associated with toxin-antitoxin systems (hipA, mazF) and oxidative stress response suppression as persister-defining signatures. Metabolomics revealed ATP depletion and TCA cycle downregulation as metabolic hallmarks. The PRT cascade model successfully predicted recurrence risk in retrospective clinical outcome data. This work constitutes an original theoretical and empirical contribution to MRSA persister biology with direct clinical translational implications. Keywords: MRSA persisters, biofilm, single-cell transcriptomics, persistence mechanism, antibiotic tolerance.