Marine-Derived Actinobacteria and Their Ability to Produce Antimicrobial Substances against Human Pathogens

Abstract

The aims of this study were to isolate marine-derived actinomycetes from sediments and marine organisms from the South of Thailand and screen for their antimicrobial activity against human pathogens. A total of 525 isolates were obtained with members of the genus Streptomyces as the dominant population. Among the 274 representative actinomycetes, 57.7% exhibited antimicrobial activity against at least one test strain by a cross streak and hyphal inhibition tests. Two hundred and eighty- seven extracts from the 104 fast-growing active isolates were preliminary tested at 200 μg/ml by colorimetric broth microdilution methods. Among them, 160 extracts from 81 isolates showed varying spectrum of activity and their minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC) and minimum fungicidal concentrations (MFC) were further determined. The MIC values of active extracts ranged from ≤0.03 to 200 μg/ml and the MBCs and MFCs from 0.25 to >200 μg/ml and 1 to >200 μg/ml, respectively. Thirty-nine extracts from 19 isolates exhibited strong inhibitory activity (MICs ≤8 μg/ml), 33 extracts against bacteria and six extracts against fungi. The cell ethyl acetate extract from AMA11 (AMA11CE) exhibited the broadest activity against both Gram-positive bacteria, Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) (MIC 0.5 μg/ml) and Gram- negative bacteria, Acinetobacter baumannii (MICs 8 to 64 μg/ml) and Escherichia coli (64 μg/ml). In addition, AMA11CE inhibited biofilm formation of Staphylococcus epidermidis and quorum sensing of Chromobacterium violaceum. Gas chromatography-Mass spectrometry (GC-MS) analysis revealed that AMA11CE contained two compounds namely 3-nitro-1,2-benzenedicarboxylic acid and quinoxaline-2-carboxamide that may be associated with antibacterial activity. Six extracts displayed strong antifungal activity. The cell ethyl acetate extract from Streptomyces sp. AMR71 (AMR71CE) had the broadest antifungal activity against Candida albicans, Cryptococcus neoformans and Talaromyces marneffei with MICS of 4, 1 and 4 μg/ml, respectively. The cell hexane extract from Streptomyces sp. AMA50 (AMA50CH) exhibited the strongest activity against T. marneffei (MIC 0.5 μg/ml). Furthermore, in vivo study showed that AMA50CH protected the survival of T. marneffei infected Caenorhabditis elegans 60 to 70% as compared to the infected control group. GC-MS analysis revealed that AMA50CH contained seven compounds comprising n-hexadecanoic acid, tetradecanoic acid, pentadecanoic acid, heptadecenoic acid, palmitoleic acid, hexadecanoic acid 2-hydroxyl-1- (hydroxymethyl) ethyl ester and hexadecanoic acidmethyl ester that may be responsible for the antifungal activity. Scanning electron microscopic studies revealed that these active extracts may act on cell wall and cell membrane of pathogens. Finally, 63 selected isolates of marine-derived actinomycetes were identified by 16S IDNA sequence analysis. They could be divided into six orders and eight genera comprising the Streptomycetales (Streptomyces and Uncertain species), Corynebacteriales (Gordonia, Nocardia and Mycobacterium), Pseudonocardiales (Pseudonocardia), Streptosporangiales (Actinomadura), Jiangellales (Jiangella) and Micromonosporales (Micromonospora). In addition, strain AMA120 was classified as a novel species in the genus Gordonia based on a polyphasic study consisting of phenotypic, chemotaxonomic and phylogenetic properties. Therefore, the strain AMA120 was proposed as the type strain of a novel species with the name Gordonia sediminis sp. nov. The results from this study revealed that marine environments in the South of Thailand would be a good source of novel and antimicrobial producing marine-derived actinomycetes.