AME-M290 confers aminoglycoside resistance in clinically identified P. aeruginosa KGH presenting efficacious treatment with Tobramycin and sulfonamide inhibitor combination

The study identified and characterized an unknown bacterial strain (UBS) from a sputum sample (120,000 CFU/ml) of a suspected bacterial pneumonia patient at Kingston General Hospital. Gram staining, biochemical tests, VITEK-2 AST, and 16S rRNA sequencing confirmed identity of P. aeruginosa KGH with multi-drug resistance (MDR) to common antibiotics. Plasmid analysis identified an aminoglycoside modifying acetyltransferase encoded by the AME-290 gene utilizing BLASTp. AME-M290 was cloned into pET-28a, expressed by transformed E. Coli BL21 (DE3). AME-M290 proteins are purified via size exclusion chromatography and Ni-NTA. Acetyltransferase assay displays AME-M290 acetylation of gentamicin at Vi of 0.1672 μM/s. S-02 is more potent with an IC-50 of 19.95 μM, compared to S-03 (IC50 = 80 μM). S-02 is more efficacious where percent inhibition (PI) is 90.43%, relative to S-01 (PI=3.89%). Broth microdilution displayed that tobramycin/S-03 had the lowest MIC (0.06 μg/mL) making it the best treatment for MDR P. aeruginosa KGH. This study supports dual antibiotic therapies as a viable treatment for AME P. aeruginosa resistance.