Abstract
The emergence of multi-drug resistant pathogenic bacteria represents a serious and growing threat to national healthcare systems. Most pressing is an immediate need for the development of novel antibacterial agents to treat Gram-negative multi-drug resistant infections, including the opportunistic, hospital-derived pathogen, Acinetobacter baumannii. Herein we report a naturally occurring 1,2-benzisoxazole with minimum inhibitory concentrations as low as 6.25 μg ml−1 against clinical strains of multi-drug resistant A. baumannii and investigate its possible mechanisms of action. This molecule represents a new chemotype for antibacterial agents against A. baumannii and is easily accessed in two steps via de novo synthesis. In vitro testing of structural analogs suggest that the natural compound may already be optimized for activity against this pathogen. Our results demonstrate that supplementation of 4-hydroxybenzoate in minimal media was able to reverse 1,2-benzisoxazole’s antibacterial effects in A. baumannii. A search of metabolic pathways involving 4-hydroxybenzoate coupled with molecular modeling studies implicates two enzymes, chorismate pyruvate-lyase and 4-hydroxybenzoate octaprenyltransferase, as promising leads for the target of 3,6-dihydroxy-1,2-benzisoxazole.
Highlights
Acinetobacter baumannii is a non-fermenting, Gramnegative member of the Gammaproteobacteria commonly implicated in nosocomial sepsis, wound infections, and ventilator-associated pneumonia [1]
We further provide evidence suggesting the mechanism of action (MOA) of 3,6-dihydroxy-1,2-benzisoxazole
Crude organic extract from the exudate of B. denitrificans (Isolate B158) was generated and initially screened in the p-iodonitrotetrazolium chloride (INT) assay to assess multidrug resistant (MDR) reversal potential as described [34]. This initial screening demonstrated the extract from isolate B158 potentiated the activity of erythromycin when tested against E. coli MDR strains MG1655 ΔBC/pABM, and MG1655 ΔBC/pXYM
Summary
Acinetobacter baumannii is a non-fermenting, Gramnegative member of the Gammaproteobacteria commonly implicated in nosocomial sepsis, wound infections, and ventilator-associated pneumonia [1]. A. baumannii tenacity in hospitals is enhanced both by its ability to develop antibiotic resistance and capacity to survive on surfaces, including skin, for several days [3]. Carbapenems, typically in combination with other antimicrobial agents (e.g., the polypeptide colistin) are a commonly utilized treatment option for multidrug resistant (MDR) A. baumannii, but it is estimated that more than half of MDR strains are carbapenem-resistant and colistin has significant toxicological and dosing concerns [1, 11, 12]. With the lack of new antibiotics in the drug discovery pipeline to treat Gramnegative infections, coupled with accelerated evolution of antibiotic resistance, it is imperative that new antibacterial drugs for treating A. baumannii infections be developed [13]. We report the identification of 3,6-dihydroxy-1,2benzisoxazole (1), a potent antibiotic against A. baumannii produced by a marine bacterium identified as a Bradyrhizobium denitrificans. In discovering 4-hydroxybenzoate (4-HB)’s ability to reverse the antibacterial property of 1, we investigate two possible 4-HButilizing target enzymes, chorismate pyruvate-lyase (CPL) and 4-HB octaprenyltransferase
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