High level of persister frequency in clinical staphylococcal isolates

**Background:** Persister cells are a bacterial subpopulation that exhibits transient, non-heritable antibiotic tolerance without genetic mutation, contributing to treatment failure in biofilm-associated infections. Staphylococcus aureus and coagulase-negative staphylococci (CNS) are major causes of device-associated biofilm infections that are notoriously difficult to treat. Despite their clinical significance, studies on persister cells in clinical isolates are limited. This study aimed to evaluate the association between persister cell formation and biofilm production, antibiotic stress, growth phase, specimen type, and staphylococcal species among clinical isolates from Nepal.

**Methods:** A total of 375 clinical staphylococcal isolates (161 S. aureus, 214 CNS) were collected from two tertiary care hospitals in Nepal. Biofilm formation was detected using the quantitative tissue culture plate (TCP) method and by PCR detection of icaAD genes. Antibiotic susceptibility was determined by Kirby-Bauer disc diffusion, and minimum inhibitory concentrations (MIC) were measured by agar dilution. Persister assays were performed on 60 antibiotic-susceptible isolates (10 isolates susceptible to each of six antibiotics: oxacillin, vancomycin, penicillin, gentamycin, ciprofloxacin, cotrimoxazole). Cells were grown to exponential phase (OD600 0.5–0.6) or stationary phase (16 h), then challenged with 20× MIC (exponential) or 100× MIC (stationary) of antibiotics. Colony-forming units (CFUs) were counted at designated time points up to 72 hours post-exposure.

**Key Results:** Among 375 isolates, 161 (43%) were S. aureus and 214 (57%) were CNS. TCP detected biofilm in 84 (52.2%) S. aureus and 90 (42.1%) CNS isolates; icaAD genes were detected in 86 (22.9%) isolates overall. Most biofilm producers and non-producers were sensitive to chloramphenicol (94.8% and 93.0%) and tetracycline (92.9% and 89.1%), but resistant to penicillin (92.0% and 94.0%). All isolates were sensitive to vancomycin. High persister frequency was observed in stationary phase across all isolates regardless of antibiotic used. In exponential phase, persister frequency was significantly higher among icaAD-positive isolates compared to icaAD-negative isolates (p < 0.01). Persister formation was observed in more than half of isolates exposed to vancomycin, oxacillin, ciprofloxacin, and penicillin, but no persisters formed with cotrimoxazole, chloramphenicol, or erythromycin. No significant difference in persister frequency was found between device-associated and other specimen types (p = 0.8), or between S. aureus and CNS (p = 0.54). Among biofilm producers, 51% formed persisters; 60% of icaAD-positive isolates formed persisters versus fewer icaAD-negative isolates (p < 0.01). Biofilm strength (strong vs weak) did not significantly affect persister formation (p = 0.78).

**Clinical Implications:** This study provides strong evidence that clinical staphylococcal isolates exhibit high persister cell frequency, particularly in stationary growth phase, which may explain treatment failures even in the absence of genetic antibiotic resistance. The significant association between icaAD genes and persister formation suggests that biofilm gene detection could help predict treatment failure risk. The finding that persister formation varies by antibiotic class—with cotrimoxazole, chloramphenicol, and erythromycin showing no persister survival—suggests that antibiotic choice may be critical in managing recurrent staphylococcal infections. The similar persister frequency between S. aureus and CNS highlights the clinical relevance of CNS in treatment failures, which is often underappreciated. These findings underscore the need for persister-directed therapeutic strategies and further research into the genetic determinants of persister formation in clinical settings.