Screening, purification and characterisation of anti-pseudomonas aeruginosa compounds produced by endophytic fungi from Kigelia Africana.

Abstract

The emergence of new diseases and drug resistant pathogens coupled with the side effects presented by conventional or synthetic drug use calls for the discovery of new antibiotics and chemotherapeutics. Pseudomonas aeruginosa is a multifaceted Gram-negative opportunistic pathogen which is responsible for ten percent of all hospital infections. This, therefore, directed the search for novel bioactive molecules with new targets from previously understudied sources. Plants have bioactive compounds that have been used for traditional healthcare for thousands of years. In the interest of plant preservation, the focus has shifted to include the plant microbiome; interestingly, not only were the plants themselves producing the bioactive metabolites but also their associated microbiome. The focus of this study was to screen and determine the optimum time to produce anti-Pseudomonas aeruginosa metabolites, to purify the compounds of interest and characterise them. Forty-five endophytic fungi were grown in solid substrate fermentation on rice to produce extracts of varying ages (one week to four weeks). Thin layer chromatography (TLC) coupled with bio-autography revealed that the anti-P. aeruginosa compound was produced after three weeks. Average zones of inhibition of 40.33, 20.33 and 22 mm were obtained using the Kirby-Bauer disc diffusion assay. All rows A, B, C and D show very strong activity as the MICs of the extracts were 156.5 μg/mL, 39.06 μg/mL, 78.73 μg/mL and 19.53 μg/mL for T1, T2, T3 and T4, respectively. Thin layer chromatography was conducted, and optimum separation was observed using hexane: ethyl acetate (60:40, v/v). Fractionation was carried out using a silica gel column with six different ratios of a hexane: ethyl acetate solvent system. The first round of purification resulted in twenty-seven fractions with five fractions having similar TLC profiles. These fractions were combined and subjected iii to a second round of purification that gave three fractions. One fraction was observed to have good anti-P. aeruginosa activity and acceptable purity levels after nuclear magnetic resonance spectroscopy. Compound one, a dilactone (3a,10b-dimethyl-1,2,3,3a,5a,7,10b,10c-octahydro-5,8-dioxa-acephenanthrylene-4,9-dione, molecular formula C16H18O4) was isolated as a white solid from the extract of the fungus Neofusicoccum luteum. This compound was previously isolated from the fungus Oidiodedron griseum. The relative configuration of the compound was confirmed by X-ray crystallography. Although the isolated compound is not novel, its ability to inhibit the growth of P. aeruginosa is new. This suggests that known compounds need to be screened across a wide range of pathogens and organisms to determine potential activity.