Beyond the Standard Antibiogram: Unveiling the Roles of pH and Salinity in Modulating Gentamicin Efficacy Against...

Urinary tract infections are highly prevalent diseases with a broad spectrum of clinical manifestations. This study investigates the influence of two key abiotic factors, pH and salinity, on the growth and antibiotic susceptibility of Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and a clinical E. coli isolate obtained from a human urine sample. Growth kinetics and gentamicin susceptibility were evaluated across a pH range (4.5-11.0) and NaCl concentrations (0-1000 mM) using Luria-Bertani and Mueller-Hinton (MH) media. In parallel, real urine was analyzed, and urine-mimicking MH media were prepared by adjusting pH and salinity independently and in combination. Gentamicin susceptibility was strongly influenced by environmental conditions. Salinity pre-exposure increased E. coli and P. aeruginosa resistance from 23.0 to 19.0 mm and from 25.5 to 18.5 mm at 800 mM, respectively. Direct pH modification enhanced gentamicin activity at extreme pH (up to 34.33 mm in E. coli and 33.5 mm in P. aeruginosa). In standard MH medium, the clinical E. coli isolate exhibited a 32.5-mm inhibition zone and a gentamicin minimum inhibitory concentration (MIC) of 4 µg/mL. Under urine-mimicking conditions, the inhibition zone decreased to 22.5 mm, while the MIC increased eightfold to 32 µg/mL, indicating a marked reduction in gentamicin efficacy under physiologically relevant urinary conditions. These findings demonstrate that environmental stress profoundly influences gentamicin efficacy and suggest that this effect may involve physiological adaptations, including RpoS-mediated stress responses, membrane remodeling, and osmoprotective mechanisms previously described in the literature.