Metabolic Vulnerability of ESKAPE Pathogens to Exogenous L-Cysteine in M9 Media: Restoring Meropenem Susceptibility

The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) represent a critical threat to global health due to their multidrug resistance, virulence, and limited therapeutic options. The escalating failure of conventional antibiotics necessitates the identification of novel metabolic vulnerabilities that can be exploited for therapeutic intervention. Among metabolic targets, sulphur and redox metabolisms have gained increasing attention, as intracellular redox balance is essential for bacterial survival under stress and nutrient limitation. L-cysteine, a sulphur-containing amino acid, occupies a central position in bacterial metabolism as a precursor of glutathione and a regulator of cellular redox homeostasis. However, excess cysteine can induce oxidative stress through thiol autooxidation and the generation of reactive oxygen species, suggesting that cysteine availability may differentially impact bacterial viability. This study provides a comparative analysis of dosedependent, species-specific growth responses to exogenous L-cysteine across ESKAPE pathogens under nutritionally defined 0.5X M9 minimal medium conditions. Experiments were conducted over 24 hours with technical triplicates. Results demonstrate that cysteine exerts divergent effects across species. In Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterobacter spp., exogenous cysteine significantly inhibits growth in a dose-dependent manner, while in Acinetobacter baumannii and Staphylococcus aureus, it enhances growth. The inhibitory effect of L-Cysteine on susceptible ESKAPE pathogens prompted further investigation into its potential as an antibiotic adjuvant. Meropenem (Carbapenem class antibiotic) resistant Klebsiella pneumoniae JIIT/NS72/I4 strain present in the lab was used for validating the above hypothesis. Disc diffusion assays demonstrated that while meropenem-resistant JIIT/NS72/I4 showed no zones of inhibition with meropenem alone, co-supplementation with 10 mM L-Cysteine exhibited some growth inhibition across all tested concentrations of Meropenem. Notably, Meropenem susceptibility could be restored with 10 mM L-Cysteine under nutrient-limited 0.5X M9 conditions, suggesting cysteine's adjuvant potential with antibiotic under stress conditions. These findings reveal a previously unrecognised metabolic bifurcation within the ESKAPE group, highlighting species-specific differences in sulphur assimilation, redox buffering capacity, and metabolic wiring under nutrient-restricted conditions. The study advances understanding of ESKAPE pathogen physiology and identifies cysteine-induced redox imbalance as a tractable, species-selective vulnerability with potential for pathogen-selective therapeutic intervention.