Discovery Of Antibacterial-Producing Actinobacteria From Selected Malaysian Mangrove Sediments
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Date
2018-09
Authors
Mohd Syafiq Awang
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
The rapid emergence of multi-drug resistant pathogenic bacteria has called for
intensive research on the discovery of new and potent antibacterial metabolites.
Actinobacteria is one of the major producers of antibacterial secondary metabolites.
Among the most reliable sources for the isolation of actinobacteria are forest soil and
marine sediments. Mangrove is a rich marine environment, but it is seldom explored
for actinobacterial isolation. Accordingly, the present study was carried out with the
main aim to isolate and screen for potential antibacterial-producing actinobacteria
from mangrove sediments. The mangrove sediments were sampled from the
mangrove environment located at Balik Pulau, Pulau Pinang, Malaysia. A total of
131 actinobacterial isolates were successfully isolated by using a combination of
selective pre-treatment and isolation media. Preliminary screening of these isolates
against five species of Gram-positive and Gram-negative bacteria using cross streak
plate method showed that 54 % of the isolates were capable of producing
antibacterial metabolites. Among these active isolates, 70 % have broad-spectrum
antibacterial activity against both Gram-positive and Gram-negative test bacteria,
24 % have narrow-spectrum antibacterial activity against only Gram-positive
bacteria and 6 % against only Gram-negative test bacteria. Five actinobacterial
isolates with potent broad-spectrum antibacterial activity were selected for
cultivation in three litres submerged culture fermentation using a bubble-column
bioreactor. The crude fermentation broth extract was obtained through solvent
extraction using ethyl acetate at 1:1 ratio and tested for minimum inhibitory
concentration (MIC) and minimum bactericidal concentration (MBC) against 17 test bacteria. The lowest MIC value detected was 9.77 g/mL against the clinical
isolates Pseudomonas aeruginosa, Enterococcus faecalis and Listeria
monocytogenes ATCC 19114. No MBC value was recorded even at the highest test
concentration of 5000 g/mL of the ethyl acetate extract. Preliminary purification of
the most active actinobacterial ethyl acetate extract through column chromatography
produced a total of 11 fractions. Only Fractions 1 to 4 showed antibacterial activity
against clinical isolates P. aeruginosa, E. faecalis, Staphylococcus aureus, Methicilin
resistant Staphylococcus aureus, Shigella boydii ATCC 9207, and
Staphylococcus aureus ATCC 12600. Fraction 1 exhibited the lowest MIC and
MBC values of 0.15 g/mL and 2500 g/mL, respectively against P. aeruginosa and
E. faecalis. Fraction 1 was further separated using thin layer chromatography, which
produced three spots. Bioautography of these spots against S. aureus ATCC 12600
showed that only one spot with Rf value of 0.88 was active. Ultra-performance
liquid chromatography analysis also detected only one major peak in the
chromatogram, which might be the possible metabolite of interest. One
actinobacterial isolate with the most active ethyl acetate extract, namely isolate
PBD-310J was selected for isolate characterization and identification.
Characterization through morphological and physiological characteristics, and the
16S rRNA gene sequence showed that the isolate was a species of Streptomyces and
closely related to Streptomyces praecox. Thus, the finding from this study revealed
that the Malaysian mangrove sediments showed a good reservoir of antibacterialproducing
actinobacteria which could be candidates for future antibacterial agent.
Description
Keywords
Actinobacteria , Mangrove plants