Selection of potential antimicrobial producing fungi isolated from marine organisms

Abstract

Infections caused by drug resistant microorganisms are increasing worldwide. There is a need to find new sources of antibiotics to combat this problem. The aim of this study was to select marine-derived fungi isolated from marine organisms that produce antimicrobial metabolites. Five hundred and forty-seven fungi were isolated from 17 marine organisms and 123 isolates were selected based on their different colony morphologies for chemical extraction. Three extracts were obtained from each isolate including broth ethyl acetate (BE), cell ethyl acetate (CE) and cell hexane (CH) extracts. All extracts were preliminarily screened for their antimicrobial activity using colorimetric broth microdilution methods at 200 μg/ml against nine human pathogens. The results demonstrated that 219 extracts (59.35%) from 109 isolates (88.62%) showed inhibitory activity against at least one test strain. Most of active extracts were effective against Cryptococcus neoformans ATCC 90112 (CN) (35.77%) followed by Staphylococcus aureus ATCC 25923 (SA) (35.50%), methicillin-resistant S. aureus (MRSA) SK-1 (27.10%), Microsporum gypseum SH- MU4 (MG) (18.42%), Candida albicans ATCC90028 (CA) (10.02%), Talaromyces marneffei PSU-SKH1 (TM) (3.25%), Acinetobacter baumannii NPRC AB005 (AB005) (2.71%), Pseudomonas aeruginosa ATCC 27853 (PA) (1.89%) and Escherichia coli ATCC25922 (EC) (1.08%), respectively. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) values were varied in the range of 2-200 and 16->200 μg/ml., respectively. Thirty-six fungi presenting moderate to strong antimicrobial activity were identified based on morphological and/or molecular methods into two phyla, Ascomycota (35 isolates) and Basidiomycota (1 isolate). The active ascomycetous genera were Trichoderma (10 isolates), Aspergillus (8 isolates), Penicillium (8 isolates), Letendraea (2 isolates), Cladosporium (1 isolate), Fusarium (1 isolate), Pestalotiopsis (1 isolate), Phaeosphaeriopsis (1 isolate) and Trichothecium (1 isolate). Only one isolate, Schizophyllum commune AMF238 belonged to the phylum Basidiomycota and two isolates (AMF177 and AMF235) were unidentified. Broth ethyl acetate extracts from Aspergillus clavatonanicus AMF277 (AMF277BE) showed the broadest inhibitory activity against SA, MRSA, EC, PA, AB005, CN and TM with MIC values of 32, 64, 64, 32, 200, 200 and 200 ug/ml, respectively. AMF231CH from Aspergillus unguis AMF231 showed the strongest inhibitory activity against SA (MIC 4 μg/ml) and MG (MIC 16 μg/ml). AMF198CH from Phaeosphaeriopsis musae AMF198 was most active against MRSA (MIC 16 μg/ml). The mycelium and broth extracts of Trichothecium sp. AMF192 (AMF192CH and AMF192BE) displayed the most potential antifungal activity against CA and CN with MIC 8 μg/ml and 2 μg/ml, respectively. Electron microscopic observation of the treated cells with these active extracts showed morphological changes with deformation, collapsed, shrinkage and broken cell with holes. Furthermore, AMF222CE and AMF409BE from of Trichoderma spp. AMF222 and AMF409 presenting weak anti-AB (MIC 200 μg/ml) exhibited the synergistic effects with colistin against AB005. The checkerboard results showed fractional inhibitory concentration index (FICI) ranging from 0.25-0.5 and the time-kill assay revealed bactericidal activity (>3log10 CFU/ml reduction) of these two extracts in combination with colistin after 2 h of incubation. In addition, the extracts were preliminarily determined for their ability to inhibit quorum sensing (QS) using violacein inhibition assay with Chromobacterium violaceum DMST21761 by a disk diffusion methods. Only four extracts (AMF177BE, AMF199BE, AMF231BE and AMF408BE) exhibited anti-QS activity. The results from this study can indicate that the marine- derived fungi are a potential source of antimicrobial active metabolites.