The emergence of beta lactamase producing bacterial strains eliminated the use of beta lactam antibiotics as chemotherapeutic alternative. Beta lactam antibiotics can be coupled with non-antibiotic adjuvants to combat these multidrug resistant strains. We study the synergistic antibiotic effect of stigmasterol as adjuvant of ampicillin against clinical isolates. Ampicillin was used in this study as a beta lactam antibiotic model. All test bacteria were beta lactamase producing clinical isolates. The combination showed significantly better antibiotic activity on all bacteria tested. The two test substances have synergistic antibiotic activity, and the effect was observed in both Gram positive and Gram negative bacteria. The synergistic antibiotic effect of stigmasterol and ampicillin was evident by the low fractional inhibitory concentration (FIC) index on Checkerboard Assay. The results suggest that the combination of ampicillin and stigmasterol acts additively in the treatment of infections caused by beta-lactamase producing pathogens. In bacterial growth reduction assay, ampicillin and stigmasterol alone exhibited very weak inhibitory effect on the bacterial growth, relative to ethanol control. Comparatively, combination of stigmasterol-ampicillin greatly reduced the colony counts at least by 98.7%. In conclusion, we found synergistic effects of stigmasterol and ampicillin against beta lactamase producing clinical isolates. This finding is important as it shows potential application of stigmasterol as an antibiotic adjuvant.
Previously, a hypothetical protein (HP) termed Bleg1_2437 (currently named Bleg1_2478) from Bacillus lehensis G1 was discovered to be an evolutionary divergent B3 subclass metallo-β-lactamase (MBL). Due to the scarcity of clinical inhibitors for B3 MBLs and the divergent nature of Bleg1_2478, this study aimed to design and characterise peptides as inhibitors against Bleg1_2478. Through in silico docking, RSWPWH and SSWWDR peptides with comparable binding energy to ampicillin were obtained. In vitro assay results showed RSWPWH and SSWWDR inhibited the activity of Bleg1_2478 by 50% at concentrations as low as 0.90 µM and 0.50 µM, respectively. At 10 µM of RSWPWH and 20 µM of SSWWDR, the activity of Bleg1_2478 was almost completely inhibited. Isothermal titration calorimetry (ITC) analyses showed slightly improved binding properties of the peptides compared to ampicillin. Docked peptide-protein complexes revealed that RSWPWH bound near the vicinity of the Bleg1_2478 active site while SSWWDR bound at the center of the active site itself. We postulate that the peptides caused the inhibition of Bleg1_2478 by reducing or blocking the accessibility of its active site from ampicillin, thus hampering its catalytic function.