Antimicrobial activities of some marine actinonycetes isolated from Malaysians waters

Marine actinobacteria were isolated from sediment and water samples from Malaysian waters using starch-yeast extract agar with seawater and marine agar (Difco) as a selective medium to isolate the actinomycetes. Ten isolates were selected from a collection of 300 isolates for further investigations based on colony morphology and pigment production. Genomic DNA from these isolates were extracted and subjected to polymerase chain reaction (PCR) to detect the presence of polyketide synthase type I (PKS-I) and non-ribosomal peptide syntethases (NRPS) [1]. Molecular identification was also performed using 16S rRNA gene partial sequencing. A phylogenetic tree based on 16S rRNA gene sequences was constructed using MEGA version 4 [2]. Scanning electron microscopy (SEM) was also performed on these isolates. Evaluation of antimicrobial activity was performed for isolates that exhibited the presence of both PKS-I and NRPS genes, and NRPS gene only. These isolates were challenged against of 5 Gram negative bacteria - E.coli, P. vulgaris, P. aeruginosa, P. mirabilis and S. marcescens; 3 Gram positive bacteria – Methicillin-resistant S. aureus (MRSA), B.subtilis and S. aureus; and 3 fungi - Candida parapsilosis, A. niger and Candida albican. The antimicrobial test was conducted by placing the isolates on trypticase soy agar (TSA) which had been inoculated with the test organisms. The antimicrobial activity was determined as a clear zone of inhibition surrounding the isolate. Eight isolates exhibited the presence of both PKS-I and NRPS genes of which 5 isolates belong to the genus Streptomyces, while another 2 isolates displayed only NRPS gene. Five isolates, mostly Streptomyces species demonstrated high antimicrobial activity against most of the test organisms which could be related to the presence of PKS-I and NRPS genes. Streptomycetes produce approximately 75% of commercially and medically useful antibiotics worldwide [3] therefore, it was anticipated that the Streptomycetes isolates in this study would display high antimicrobial activities. However, 2 isolates displayed no antimicrobial activity at all even though both genes were detected in them. Based on 16S rRNA gene sequencing, these isolates were presumed to belong to the genus Rhodococcus and Pseudonocardia. The presence PKS-I and NRPS genes might not necessarily result in antimicrobial activities since the function of polyketides is not confined to antimicrobial activities but may have other useful properties such as antihelmintics, anticancer or immunosuppressive agents [4].Nevertheless, early evaluation of these isolates based on the presence of both genes would allow to focus the screening of isolates that possessed high metabolic potential. In conclusion, Malaysian waters have the potential as important natural resources for exploration of marine actinomycetes that possess the ability to produce a relatively high rate of new antimicrobial bioactive agents

Marine streptomyces SP. UKMCC_PT15 producing undecylprodigiosin with algicidal activity

Marine actinomycetes are now in demand as they are capable of producing unique and novel compounds with wide biological activities. Marine Streptomyces sp. UKMCC_PT15 previously isolated from seawater collected in Pulau Tinggi, Johor was used in this study. Molecular identification showed high similarity of Streptomyces sp. UKMCC_PT15 with Streptomyces fradiae and S. diastaticus subsp. ardesiacus based on partial sequence of 16S rRNA gene. Further characterisations of this bacterium include spore morphology using SEM, growth on various media, salt tolerance test and carbon utilisation profile. This bacterium had straight spore chain with smooth-surfaced spores and was able to tolerate up to 11% NaCl with capabilities of utilising >40 carbon sources. Undecylprodigiosin (C25H35N3O) was successfully purified through succession of column chromatography and finally using HPLC. Structure elucidation was confirmed through NMR spectroscopy, MS and comparison with established data. This compound demonstrated strong antibacterial activities against S. aureus, B. subtilis and C. albicans but weak antibacterial activities against E. coli, P. aeruginosa and Methicillin-resistant S. aureus (MRSA), Interestingly, undecylprodigiosin also demonstrated algicidal activity when tested against toxic dinoflagellates, A. minutum and P. bahamense, both of which are responsible for harmful algal blooms (HABs). Undecylprodiogiosin with concentration of 10-100 μg/ml gave ~100% algicidal activity against both dinoflagellates. Further testing with undecylprodigiosin concentration < 10 µg/ml showed that undecylprodigision was capable of killing significantly high numbers of both dinoflagellates, giving a high algicidal activity. Findings from this study suggested the potential use of undecylprodigiosin as algicidal agent which could be used for the mitigation of HABs

Study of Glucose-6-Phosphate dehydrogenase activity assay in mangrove streptomyces for actinohordin and undercylprodigiosin production

This study evaluates the potential of using glucose-6-phosphate dehydrogenase activity assay for Actinohordin and Undecylprodigiosin productions from mangrove Streptomyces. Previously, there were several methods used to screen antimicrobial activities such as agar spot test and disc diffusion assay, but those are lengthy screening methods and time consuming. Thus, to overcome the limitations plate-based assay is suggested to enable rapid screening on secondary metabolite production of numerous samples at one time. The development of plate-based assay was performed by optimizing glucose-6-phosphate dehydrogenase activity assay. This coupled assay was based on the production of dihydronicotinamideadenine dinucleotide phosphate (NADPH) whereby a right combination of nicotinamide adenine dinucleotide phosphate (NADP) and glucose-6-phosphate (G6P) were refined. The production of NADPH was measured at absorbance of 340 nm where reduced cofactor NADPH are absorbed readily at this wavelength. Sample with different concentrations of crude lysate was subjected to various substrates concentration to obtain the best activity curve. Even though elucidating clear patterns is speculative, it is believed that some improvements or optimizations of this study could offer promising knowledge which can serve as useful reference in futur

Verrucosispora sp. K2-04, Potential Xylanase Producer from Kuantan Mangrove Forest Sediment

Xylanase is the key enzyme that involves in the hydrolysis of xylan, the main constituent of the complex hemicellulose of the plant cell wall. In this study, forty actinomycetes that were isolated from the sediment of Kuantan Mangrove Forest, Malaysia, were tested for their ability to produce extracellular xylanase. At least 15 isolates were able to degrade xylan in the primary agar-based screening on marine agar containing 0.1% (v/v) azo-xylan (Birchwood). The degradation of xylan was indicated by the formation of halo zone around the colonies and the clear zone index (CZI) was calculated as a ratio of the clearing zones to the colony size. Isolate K2-04 with CZI 3.35 ± 1.91 was identified through 16S rRNA study as Verrucosispora sp. This isolate was further grown in marine broth and incubated at 30 °C, 200 rpm for 20 days. The growth of K2-04 and the xylanase activity was measured at day 2, 4, 6,12 and 18 respectively. The highest enzyme activity of the crude enzyme was recorded at day 18 (1.836 U/mL) and exhibited stability after 20 days storage at 4°C. This study serves as a preliminary study to characterize the properties of Verrucosispora sp. K2-04, rare actinomycete of Kuantan Mangrove Forest, Malaysi

Isolation of bacteria from the acidic peat swamp forest soil and their lignin degradation potential

The tropical peat swamp forest in Malaysia has reduced significantly due to increasing pressure for development and demand for agricultural land. Pekan peat swamp forest is part of the 200,000 hectares of peat swamp forest located in Pahang, Peninsular Malaysia. While more extensive studies were done on flora and fauna, the study on microbial diversity in this habitat is very limited. The highly acidic environment, low concentrations of nutrients and anoxic condition of the peat are among challenges that hampered the cultivation of microorganism from this environment. In this study two types of agar-based medium, M1 minimal medium (M1) and peat water medium (PW) supplemented with glucose, methanol and lignin were used to isolate bacteria from the peat sediment. In comparison to M1, the use of PW has resulted with higher number of isolates with different morphologies. The PW mainly contains the acidic peat water that was collected from the sampling location. Based on the growth on medium supplemented with lignin, selected isolates were identified using 16s rDNA sequencing. At least three of the isolates showed sequence similarity to Burkholderia sp., which is one of the common species, studied on their ligninase-producing abilities. The results from this study serve as the preliminary data for further work on growth characteristics and enzymatic potential of isolates from acidic peat swamp soil

Comparison of various culture media effectiveness in the isolation of bacteria from Pekan peat swamp forest soil

Aims: Previously described as non-favorable-microbial habitat, peat swamp forest has its own features, which are extremely acidic, poor in nutrient, water-logged and anoxic environment where rate of decomposition of plant litters is quiet slow. Interestingly, current research has proven that there is diversity of microbial communities in this ecosystem. The main objective of this study is to isolate bacteria from Pekan peat swamp forest soil that play a role in the decomposition of plant litters through cultivation on different agar-based medium. The success of isolation of bacteria from this neglected habitat could open the opportunity in unleashing the specific role of bacteria in peat swamp plant litter degradation as well as potential biotechnological application of these bacteria in lignocellulose-related industry. Methodology and results: To mimic the peat condition that is low in nutrient and comprised of plant debris, M1 and peat agar supplemented with cellulose, glucose, lignin and xylan were used. Specifically, for the isolation of actinomycetes, dry and wet heat pre-treatments were applied to the soil samples. Then, the samples were cultivated on three different agars which were oatmeal agar as well as M1 and peat agar supplemented with glucose. Enrichment method was applied in the isolation of cellulase-producing bacteria. It was found that higher number of bacteria and actinomycetes were successfully isolated from peat agar, followed by oatmeal agar and M1. In fact, more actinomycetes were isolated from soil that was treated with wet heat pre-treatment compared to dry heat pre-treatment and on peat agar compared to M1 and oatmeal agar. This finding is promising, indicating that the application of peat water in the agar-based medium is useful to mimic the actual environment of peat swamp and increase the possibility to isolate indigenous bacteria. Primary screening of isolates from samples enriched with carboxymethyl cellulose (CMC) showed positive result of decolourisation zone on Azo-CM-Cellulose agar indicating the ability of isolates to degrade cellulose compound. Conclusions, significance and impacts of study: The study indicates the effectiveness of different culture media in successful isolation of bacteria including actinomycetes. Using the enrichment method, bacteria that are able to degrade cellulose compound was successfully isolated even though it is well known that plant litter degradation in the peat swamp environment happens at very slow rates