LV Prasad Eye Institute Microbiology Research

Research Question

Can pharmacological inhibition of CrtM-mediated staphyloxanthin production using celastrol weaken Staphylococcus aureus–Pseudomonas aeruginosa polymicrobial biofilms and improve their susceptibility to gentamicin?

Experimental Design

Clinical ocular keratitis isolates were screened to identify low-, medium-, and high-staphyloxanthin S. aureus phenotypes (SA-L, SA-M, SA-H) and a high-pyocyanin P. aeruginosa isolate (PA-H). S. aureus ATCC 29213 and P. aeruginosa ATCC 27853 were used for assay standardization.

Biofilm formation was compared across individual isolates and SA-L, SA-M, and SA-H co-cultured with PA-H at 48 and 72 hours under untreated and celastrol-treated conditions. The project integrates biofilm quantification, structural analysis, antibiotic susceptibility testing, and computational molecular docking to evaluate whether inhibiting the CrtM–staphyloxanthin pathway can weaken biofilm-associated protection.

Methods

Staphyloxanthin-associated pigmentation was quantified spectrophotometrically following methanol extraction. Biofilm biomass was measured using crystal-violet staining in 96-well plates across individual and dual-species cultures exposed to different celastrol concentrations.

Scanning electron microscopy was used to examine changes in bacterial aggregation, surface coverage, and biofilm architecture following celastrol treatment. Gentamicin susceptibility was assessed by comparing MIC values with and without celastrol. Molecular docking was also used to investigate celastrol’s predicted binding efficiency, binding location, and interactions with CrtM, the enzyme responsible for the first committed step of staphyloxanthin biosynthesis.

Current Work

H₂O₂ oxidative-stress assay and data compilation/processing.

Skills Developed

Aseptic technique, clinical isolate handling, 96-well plate biofilm assays, crystal-violet staining, spectrophotometric analysis, staphyloxanthin quantification, antimicrobial susceptibility testing, experimental controls, replicate-based data analysis, scientific data processing, primary-literature analysis, molecular-docking interpretation, and working within an active clinical microbiology research environment.