A Non-Toxic Small Molecule Approach to Combating Multi-Drug Resistant Bacteria

Antibiotic resistance has significantly increased since the beginning of the 21^st century. Currently, the polymyxin colistin is typically viewed as the antibiotic of last resort for the treatment of multidrug resistant Gram-negative bacterial infections.However, even with the clinician’s knowledge of nephrotoxicity to the patient, colistin usage has increased. Consequently, it has also resulted in colistin resistant bacterial isolates becoming more common. The recent dissemination of plasmid-borne colistin resistance genes (mcr 1-8) into the human pathogen pool is further threatening to render colistin therapy ineffective. New methods to combat antibiotic resistant pathogens are needed. Herein the utilization of a colistin-adjuvant combination, where the adjuvant is non-toxic to the bacteria alone, is effective in potentiating colistin against Gram-negative bacteria in hopes of lowering toxic effects to the patient. Against colistin sensitive bacteria, a 2-aminoimidazole adjuvant enabled a 1000-fold reduction in the minimum inhibitory concentration (MIC) of colistin in vitro when tested at a concentration of 30 µM and at 15 µM, it reduced the colistin MIC against three colistin sensitive, multidrug resistant Acinteobacter baumannii strains from 1000 to 7.8, 3.9, and 3.9 ng/mL. In an additional study, an indole derived lead adjuvant increased colistin efficacy 32-fold against a colistin resistant bacteria strain containing the mcr-1 gene at 5 µM, and effects a 1024-fold increase in colistin efficacy against bacteria harboring chromosomally encoded colistin resistance determinants; these combinations lowered the colistin MIC to levels at or below clinical breakpoint levels for colistin susceptibility (≤2 µg/mL).

In addition to in vitro studies, the wax worm Galleria mellonella provides an in vivo model that provides an alternative to expensive and ethically challenged murine models. These studies provided here forth show the process from start to finish in developing such an assay, starting with bacterial virulence within the worms. Furthermore, a narrow-spectrum antibiotic synthesized within our lab showed increased worm survival against a multidrug resistant A. baumannii strain; tested at 50 mg/kg and 100 mg/kg, worm survival was 22% and 32% after a six-day study, respectively. This model has expanded to include antibiotic-adjuvant combinations to battle multidrug resistant bacteria. Antibiotic-adjuvant concentrations, where the adjuvant is tested at 50 mg/kg or 100 mg/kg in combination with an antibiotic, have increased survival of inoculated worms up to 50% over the course of several days. Two studies demonstrated a Gram-positive selective antibiotic was able to be used to combat Gram-negative bacteria when in combination with an adjuvant at 50 mg/kg or 100 mg/kg. Lastly, a chromosomally colistin resistant A. baumannii strain was resensitized to colistin with an adjuvant, where the combinatorial injection of colistin-adjuvant at 50 mg/kg afforded over 40% worm survival over the course of four days versus no survival for untreated, inoculated worms.