Production and characterization of some antifungal metabolites produced by Alcaligenes and Lysinibacilli isolates

Abstract

Mycotic infections have emerged as a major threat to human health, particularly in the immunocompromised patients. Therefore, searching for new antifungal agents are urgently required for combating these infections especially with the rapid emergence of fungal resistance to the currently limited therapeutics. Resistance to antifungal agents represents a major clinical challenge leading to high morbidity and mortality rates especially in immunocompromised patients. Drugs from natural sources are believed to be safer and have fewer side effects than synthetic ones. In this study, we screened soil bacterial isolates for the capability of producing metabolites with antifungal activities via the cross streak and agar well diffusion methods. Two isolates, coded S6 and F2, showed significant antifungal activity against Candida (C.) albicans ATCC 10231 and Aspergillus (A.) niger clinical isolate. These strains were identified using a combined approach of phenotypic and molecular techniques as Lysinibacillus sp MK212927 and Alcaligenes (A.) faecalis MT332429, respectively. Extraction of the antifungal metabolite using ethyl acetate generated a dry weight yield of 1.07 g and gave inhibition zones of 26.6 and 24.3 mm against C. albicans and A. niger, respectively for Lysinibacillus sp. MK212927 isolate while for Alcaligenes (A.) faecalis MT332429 a dry weight yield of 0.91 g of the antifungal metabolite was extracted using ethyl acetate and gave inhibition zones of 20.6 and 19.6 mm against C. albicans and A. niger, respectively. The Lysinibacillus sp. metabolite displayed fungicidal activity, reserved its activity in a relatively wide range of temperatures (up to 60°C), pHs (6-7.8) and was stable in the presence of various enzymes and detergents .The fungicidal extracellular metabolite of Alcaligenes (A.) faecalis was relatively stable in the pH range from 7-8 and up to a temperature of 50 °C. It also showed stability in the presence of many detergents and enzymes. Using advanced spectroscopic techniques including, IR, UV absorbance, 1H and 13C and 2D NMR (COSY, HSQC and HMBC) the purified Lysinibacillus sp metabolite was identified as terbinafine and the metabolite of A. faecalis was identified as octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate. We employed a central composite design using Response Surface Methodology (RSM) to optimize the most five important variables influencing the production of the antifungal metabolites terbinafine and octadecyl 3-(3, 5-di-tert-butyl-4-hydroxyphenyl) propanoate. The optimum conditions for Lysinibacillus sp. MK212927 were found to be starch 5 g/l, ammonium chloride 5 g/l, temperature 32°C, agitation 150 rpm ,pH 7 while for A. faecalis MT332429 were found to be glucose 1 g/l , peptone 2 g/l , temperature 30°C, agitation 150 rpm and pH 8. The accuracy of the optimization procedure was evaluated by carrying out a confirmatory experiment in which, an increase in the antifungal metabolite terbinafine and octadecyl 3-(3, 5-di-tert-butyl-4-hydroxyphenyl) propanoate production by about 1.6 folds and 2.48 folds were obtained, respectively. To the best of our knowledge, this is the first report of terbinafine production and optimization by Lysinibacillus sp. MK212927 a fast-growing microbial source, with relatively high yield and subject to potential optimization for industrial production. Similarly, this is the first report for octadecyl 3-(3, 5-di-tert-butyl-4-hydroxyphenyl) propanoate production and optimization from the culture broth of A. faecalis MT332429 with a promising antifungal activity.