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ERIC-PCR fingerprint profiling and genetic diversity of coagulase negative Staphylococcus in Malaysia

Malaysian Journal of Microbiology.2020;16(1):7-16. doi:10.21161/mjm.180149

Aims: The coagulase-negative staphylococci (CoNS) are a group of Staphylococcus that is gaining clinical significance as major agents of nosocomial infections, especially amongst neonates and immuno-compromised patients. The identification of CoNS remains problematic, and there has been little information on their molecular genotyping. The overall aim of this study was to evaluate Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) as a rapid and cost-effective tool for the genotyping of CoNS isolates from within a hospital setting. Methodology and results: A total of 200 isolates of CoNS were collected from Hospital Tuanku Ampuan Rahimah, Klang, Malaysia and identified via sodA gene sequence analysis. Genetic diversity among the isolates was evaluated using the ERIC-PCR. The most frequently isolated species was S. epidermidis (37%) followed by S. haemolyticus (30%), S. hominis (18%) and S. capitis (8.5%). ERIC-PCR was found to be efficient for the differentiation of S. hominis isolates with a discriminatory index (DI) of 0.949 and satisfactory for S. epidermidis isolates at DI of 0.808. Poor discriminatory power was observed in S. haemolyticus (0.377) and S. capitis (0.111). The majority of the S. haemolyticus and S. capitis isolates were found to be genetically homogenous which imply that the source of these infections are due to hospital-derived contaminants. In contrast, the S. epidermidis and S. hominis strains displayed high genetic diversity suggesting the presence of different endemic strains and inflow of exogenous strains brought in by nonlocal residents. Conclusion, significance and impact of study: ERIC-PCR is a useful tool to differentiate and track selected species of CoNS.

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Detection and distribution of putative pathogenicity-associated genes among serologically important Leptospira strains and post-flood environmental isolates in Malaysia

Nurul Najian Aminuddin Baki

Malaysian Journal of Microbiology.2020;16(1):17-28. doi:10.21161/mjm.180208

Aims: Leptospirosis is an infectious disease that is endemic to many tropical regions. Large epidemics usually happen after heavy rainfall and flooding. This potentially fatal zoonosis is caused by pathogenic bacteria belonging to the genus Leptospira. Leptospirosis can be diagnosed using specific biomarkers such as target genes and virulence indicators that are well preserved across various Leptospira spp., including those that are prevalent in clinical samples and in the environment. To date, several pathogenicity-determinant genes, including lipL32 and lipL41, have been described and used for diagnosing leptospirosis. However, prevalence of these genes in leptospiral strains is unclear. Methodology and results: In the present study, we assessed the distribution of eight pathogenicity-determinant genes in reference Leptospira strains and environmental isolates in Malaysia, by polymerase chain reaction (PCR). We found that only lipL32 and ligB were consistently expressed in all pathogenic Leptospira strains compared with the other tested genes. Moreover, our results suggested that the use of lipL41, lipL21, ompL1, lfb1, ligA, and ligC as biomarkers could incorrectly misdetect pathogenic Leptospira strains present in the environment. Conclusion: Thus, our results suggest that the pathogenicity-determinant genes lipL32 and ligB can be used as biomarkers for detection pathogenic Leptospira

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Detection methods of avian influenza - current and novel approaches

May Ye Yee ; Shaharum Shamsuddin ; Quaza Nizamuddin Hassan Nizam ; Muhammad Redzwan Sidik ; Faizul Fikri Mohd Yusop ; Faizah Hanim Mohd. Saeid ; Ismail Aziah

Malaysian Journal of Microbiology.2019;15(6):492-504. doi:10.21161/mjm.190380

Avian influenza (AI), caused by the avian strain of influenza A virus (AIV) is one of the significant health concerns globally. Human infections with AI viruses were reported sporadically and often exhibited high mortality and morbidity rate. AI outbreaks also influenced the safety of the food supply and caused significant economic losses. Immediate control measures are required during AI outbreaks in poultry to prevent further viruses spreading. Hence, accurate, sensitive, and rapid detection methods are pivotal for decision making. Traditional methods of detection, such as virus isolation in embryonated chicken eggs, immuno-based methods, and nucleic acid amplification method, pose different limitations. These always grab the attention of researchers to improve existing methods or invent novel diagnostic approaches to compensate for the shortcoming of current methods applied. However, the method of choice is highly dependent on the availability of facilities and resources. Among the detection methods, reverse transcription-polymerase chain reaction (RT-PCR) is the most favourable method used for detecting AIV. However, a constant review of the virus genome is crucial to maintain the assay’s sensitivity. More comprehensive research and evaluation study are needed for new diagnostic approaches.

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Biosorption and proteomic analysis of an encapsulated endophytic heavy-metal resistant Pestalotiopsis sp.

Jenny Choo ; Norasfaliza Rahmad ; Jameel R. Al-Obaidi ; Aazani Mujahid ; LikFong Ting ; Moritz Mü ; ller

Malaysian Journal of Microbiology.2019;15(7):505-517. doi:10.21161/mjm.180037

Aims: A study on biosorption ability using encapsulated endophytic fungi has been carried out to investigate its biosorption potential in removing heavy metals. Biosorption has emerged as an alternative bioremediation process to remove and sequester heavy metal ions from polluted water. An endophytic Pestalotiopsis sp. (isolated from Nypa fruticans) was found to be able to resist copper (Cu), chromium (Cr), lead (Pb) and zinc (Zn) up to 1,000 ppm and thus the aim of this study was to investigate the biosorption ability using encapsulated live and dead Pestalotiopsis sp. biomass (at pH 4-6) to remove heavy metals. Additionally, a proteomic study was conducted to investigate down- and up-regulation expression levels of proteins under the treatment of the heavy metals. Methodology and results: Encapsulated live fungal biomass displayed higher efficiency in removing chromium at pH 5 and 6, while both encapsulated live and dead biomass were able to remove lead at pH 4 and 5 and copper at pH 5. Five (5) proteins of interest were identified via MALDI-ToF analysis. Among the proteins identified, multidrug resistance protein (MRP homolog) was up-regulated in the presence of lead. Conclusion, significance and impact of study The data obtained in this study provides an initial understanding of the biosorptive and defensive mechanisms of Pestalotiopsis sp. under heavy metal stress.

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In vitro antifungal effect of eugenol in combination with fluconazole against Candida sp.

Anek Pootong ; Kanokwan Wongdech ; Thitaporn Changrua ; Narissara Mungkornkeaw

Malaysian Journal of Microbiology.2019;15(7):518-522. doi:10.21161/mjm.180256

Aims: A combination of the antimicrobial drug with the herbal derived antifungal agent was exploited as alternative therapeutic approaches for infectious diseases caused by drug resistant strains. In this study, we determine the antifungal effects of eugenol alone and in combination with fluconazole against Candida sp. Methodology and results: Candida strains including fluconazole resistant (C. parapsilosis ATCC 22019 and C. albicans U821/10) and susceptible strains (C. tropicalis U624/10 and C. glabrata U71/1) were used in this study. By broth microdilution technique, eugenol exhibited antifungal activity with MIC and MFC against Candida sp. tested ranging from 0.5-1 mg/mL. The interaction between eugenol and fluconazole against Candida sp. was determined by chequerboard microtiter technic. Eugenol decreased the MIC of fluconazole against Candida sp. tested. No antagonism was observed in strains test. Conclusion, significance and impact of study From these results, eugenol displayed a promising antifungal effect alone as well as combination with fluconazole against Candida sp.

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Volatile organic compounds profiles emitted by Cochliobolus miyabeanus, a causal agent of brown spot disease of rice

Faten Najihah Shamsir ; Sazlinda Kamaruzaman ; Nur Ain Izzati Mohd Zainudin

Malaysian Journal of Microbiology.2019;15(7):523-528. doi:10.21161/mjm.180302

Aims: Brown spot disease is among the important crop diseases of rice caused by the infection of a pathogenic fungus, Cochliobolus miyabeanus that results in yield losses. Nowadays, limited studies on volatile organic compounds (VOCs) have been carried out using pathogenic fungal isolate. Hence, this study was conducted to identify VOCs produced by C. miyabeanus wild-type isolate, WK1C, a causal agent of brown spot disease using gas chromatography-mass spectrometry (GC-MS). Methodology and results: Fungal isolate WK1C was cultured on potato dextrose agar (PDA) and in potato dextrose broth (PDB) for extraction. The extracts were analysed using GC-MS and the profiles of VOCs were obtained. Cochliobolus miyabeanus WK1C isolate showed a significant presence of various types of organic compound including ester, alcohol, phenol, alkane, alkene, ketone, carboxylic acid, amide and aldehyde. Conclusion, significance and impact of study This study important for a preliminary assessment of VOCs profiles of C. miyabeanus, a causal agent of brown spot disease. In order to identify the compounds contribute to pathogenicity, further study can be conducted to identify the virulence factor of brown spot disease using different approaches

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Characterization of Cd(II)/Pb(II) transcriptional regulator Alcaligenes faecalis SF-S1-60

Nik Marzuki Sidik ; Roslina Mat Yazid ; Nardiah Rizwana Jaafar ; Ainon Hamzah

Malaysian Journal of Microbiology.2019;15(7):529-535. doi:10.21161/mjm.190344

Aims: Metal transcriptional regulators controlled the regulation of metal ion homeostasis in bacteria genera. Cd(II)/Pb(II) transcriptional regulator is one of the member of MerR family found in Alcaligenes faecalis SF-S1-60 (PbrT-AF). Methodology and results: The PbrT-AF gene with 432 bp open reading frame was successfully isolated from genomic DNA of A. faecalis using polymerase chain reaction (PCR) analysis. This gene was phylogenetically grouped with A. alcaligenes species using PHYLIP version 3.69 by the neighbor-joining method with 1000 bootstrap replicates. Phylogeny analysis shows that these proteins have distinct amino acids compared to Cd(II)/Pb(II) regulators from different species. The structure of PbrT-AF shows similar conformation with other members of MerR family using MODELLERv9.17. We also demonstrated that the expression of Pbrt-AF in Escherichia coli BL21 were able to increase the bacteria tolerance towards Pb up to 1000 ppm. Conclusion, significance and impact of study This result suggests that PbrT-AF promotes cell adaptation and tolerance towards Pb toxicity.

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Effect of calcium carbonate nanoparticles on alkaline protease production from Streptomyces spororaveus

Waleed Mohamed Hussain Abdulkhair ; Mousa Abdullah Alghuthaymi

Malaysian Journal of Microbiology.2019;15(7):536-542. doi:10.21161/mjm.190350

Aims: This study aims to detect the effect of nano-sized calcium carbonate as an ingredient in growth medium on the production of alkaline protease by Streptomyces spororaveus. The proportional relationship between highly production of alkaline protease and calcium carbonate nanoparticles emphasizes the unique and super properties of nanotechnology that applied in all field. Methodology and results: The high production of protease from S. spororaveus accompanied with presence of calcium carbonate nanoparticles as one of growth medium's constituents. Both qualitative and quantitative tests for proteolytic activity proved this fact. Agar-well diffusion method revealed that, the proteolytic activity with calcium carbonate nanoparticles (45 mm) is higher than that with calcium carbonate (30 mm). Calcium carbonate nanoparticles led to 150 μg/mL of protease, while calcium carbonate led to 65 μg/mL only. The crude protease was purified by ammonium sulphate precipitation and gel filtration column chromatography using Sephadex G-100. The purified protease was separated by SDS-PAGE as a single band at 30 kDa. The highest proteolytic activity was obtained at pH 8.5 and 45 °C as optimum environmental conditions. The purified protease has inhibited the growth of Candida albicans ATCC-10231 and Aspergillus brasiliensis ATCC-16404 at 8 mm and 10 mm of inhibition zone respectively. Conclusion, significance and impact of study Calcium carbonate nanoparticles in the composition of starch nitrate broth is good stimulus for highly proteolytic activity of S. spororaveus. Shake-flask fermentation method proved that, the concerned protease is an alkaline and thermostable up to 70 °C. However, the best pH and temperature values are 8.5 and 45 °C, respectively. This study can be applied to manufacture a modified starch nitrate broth medium for highly production of proteases from Streptomyces bacteria.

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Bacterial endophytes from mangrove leaves with antibacterial and enzymatic activities

Heru Pramono ; Noer Tommy Irawan ; Moch. Rizal Arif Firdaus ; Sudarno ; Laksmi Sulmartiwi ; A. Shofy Mubarak

Malaysian Journal of Microbiology.2019;15(7):543-553. doi:10.21161/mjm.190352

Aims: The aim of this study was to screen bacterial endophytes with antibacterial and enzyme activity from mangrove leaves of Indonesia. Methodology and results: Bacterial endophytes were isolated and evaluated for antibacterial activity against five strains of pathogenic bacteria. Enzymatic Index (EI) was measured to evaluate the production of protease, amylase and cellulase. Hemolysin test was performed on Blood Agar and the sensitivity to antibiotic was performed. Bacterial endophyte Strain 1-1 isolated from Bruguiera gymnorrhiza showed strong inhibition against Escherichia coli, Salmonella sp., Staphylococcus aureus and Pseudomonas aeruginosa with inhibition zone of 12.6 ± 1.4, 8.8 ± 4.1, 12.5 ± 2.3 and 8.4 ± 0.9 mm respectively. Isolate 1-16 which isolated from B. gymnorrhiza exhibited antibacterial activity against E. coli and P. aeruginosa, while Isolate 6-10 isolated from Avicennia lanata exhibited strong inhibition on Salmonella sp. (13.1 ± 3.3 mm). All of those three isolates produced protease, non-haemolysin-producing strain and sensitive to gentamicin or kanamycin but resistant to ampicillin, tetracycline and chloramphenicol. Those three isolates were identified based on homology of 16S rDNA sequence. Strain 1-1 and 1-16 were identified as P. aeruginosa, while strain 6-10 identified as S. marcescens. Conclusion, significance and impact of study This finding was showed the potential endophytic bacteria from Indonesian mangrove plants with antibacterial and enzyme production.

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Characteristics of a red-pigmented thermotolerant bacteria (GSB-001) isolated from Gedong Songo hot spring, Bandungan, Semarang – Indonesia

Endang Kusdiyantini ; Iffan Alif ; Salma Fuadiyah ; Muhammad Fadhil Randa Putra ; Dyah Wulandari ; Anto Budiharjo

Malaysian Journal of Microbiology.2019;15(7):554-559. doi:10.21161/mjm.190382

Aims: Pigments used as food additives for a long time and people prefer natural pigment than synthetic ones because of their safety. Microorganisms are interesting for pigment production and many of them have been used in food industry. Many research have been conducted to find out for natural pigment sources. The objective of this research was to investigate the characteristics of a red-pigmented bacteria isolated from Gedong Songo hot spring, Bandungan- Indonesia. Methodology and results: Bacterial isolates were grown on Nutrient Agar for 24 h, and the morphology of the colonies and of the cells were identified. Biochemical tests included indole, methyl red, catalase, urease tests and carbohydrate fermentation. Molecular identification was based on 16S rRNA sequence. The isolate was a rod shape and Gram-negative. Biochemical tests showed that the isolate was indole negative, catalase positive, methyl red negative and urease negative. This isolate was glucose, maltose and sucrose positive and negative for lactose. 16S rRNA sequence was BLAST and it matched with Serratia marcescens strain S823. The red pigment antioxidant activity showed the highest DPPH radical scavenging of 49.11% obtained from 48 h of incubation. Functional group of S. marcescens pigment on Fourier Transform InfraRed Spectroscopy (FTIR) displayed specific peak at 1740 cm-1 represented of C═O (carbonyl) stretching group. Conclusion, significance and impact of study Based on morphological, biochemical and moleculer identifications, it showed that the bacteria isolated from Gedong Songo hot spring Bandungan-Indonesia had 94% homology with Serratia marcescens strain S823. Based on DPPH radical scavenging test, it demonstrated that S. marcescens had potency as an antioxidant.

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