Molecular reprogramming of Streptococcus pyogenes virulence by sub-inhibitory antibiotic exposure

Bacteria frequently encounter sub-inhibitory concentrations of antibiotics within host tissues and natural environments, where these exposures can function as regulatory signals rather than growth inhibitors. Streptococcus pyogenes, an important human pathogen, transitions between asymptomatic colonization and invasive disease through complex regulation of virulence factors and stress responses. In this article we aim to show that there is alteration in virulence of S. pyogenes on exposure to CWA antibiotics. In clinical practice, cell wall-active antibiotics remain the cornerstone of therapy against S. pyogenes, therefore understanding their ability to modulate virulence and stress responses is critical.

S. pyogenes was exposed to sub-MIC levels of penicillin, bacitracin, and colistin. Capsule formation, biofilm development, and macrophage interactions were assessed phenotypically. Transcriptional analysis of various important virulence determinants (speB, slo, hasA) and oxidative stress response gene (sodA, ahpC, gpo) were analysed. Adhesion and invasion assays were performed using murine macrophages. The activity of the serine/threonine kinase (stk) and oxidative stress regulator (spx) was also examined. Change in morphology in presence of CWA antibiotics was assessed by staining and measuring cell size.

The results demonstrate that sub-MIC of CWA antibiotics significantly altered capsule formation, enhanced biofilm development, and increased adhesion and invasion of macrophages, indicating a potential role in immune evasion and persistence. Transcriptional analysis revealed upregulation of both virulence-associated and oxidative stress defense genes, alongside increased activity of stk and spx.

Sub-inhibitory concentrations of CWA antibiotics act as environmental cues, reprogramming molecular regulatory networks that contribute to the shift from colonization to invasive disease. Our results highlight that beyond their antimicrobial function, CWAs influence key molecular pathways that may inadvertently promote persistence and pathogenicity.