The mitogen-activated protein (MAP) kinase pathways has been implicated in the pathogenicity of various pathogenic fungi and plays important roles in regulating pathogenicity-related morphogenesis. This work describes the isolation and characterization of MAP kinase gene, Cgl-SLT2, from Colletotrichum gloeosporioides. A DNA sequence, including 1,633 bp of Cgl-SLT2 open-reading frame and its promoter and terminator regions, was isolated via DNA walking and cloned. To analyze gene function, a gene disruption cassette containing hygromycin-resistant gene was constructed, and Cgl-SLT2 was inactivated via gene deletion. Analysis on Cgl-slt2 mutant revealed a defect in vegetative growth and sporulation as compared to the wild-type strain. When grown under nutrient-limiting conditions, hyperbranched hyphal morphology was observed in the mutant. Conidia induction for germination on rubber wax-coated hard surfaces revealed no differences in the percentage of conidial germination between the wild-type and Cgl-slt2 mutant. However, the percentage of appressorium formation in the mutant was greatly reduced. Bipolar germination in the mutant was higher than in the wild-type at 8-h post-induction. A pathogenicity assay revealed that the mutant was unable to infect either wounded or unwounded mangoes. These results suggest that the Cgl-SLT2 MAP kinase is required for C. gloeosporioides conidiation, polarized growth, appressorium formation and pathogenicity.
Vitellibacter aquimaris D-24T (=KCTC 42708T=DSM 101732T), a halophilic marine bacterium, was isolated from seawater collected from Desaru beach, Malaysia. Here, we present the draft genome sequence of D-24T with a genome size of approximately 3.1Mbp and G+C content of 39.93%. The genome of D-24T contains genes involved in reducing a potent greenhouse gas (N2O) in the environment and the degradation of proteinaceous compounds. Genome availability will provide insights into potential biotechnological and environmental applications of this bacterium.
This study aimed to isolate and identify Lactobacillus in the honey stomach of honeybee Apis dorsata. Samples of honeybee were collected from A. dorsata colonies in different bee trees and Lactobacillus bacteria isolated from honey stomachs. Ninety two isolates were Gram-stained and tested for catalase reaction. By using bacterial universal primers, the 16S rDNA gene from DNA of bacterial colonies amplified with polymerase chain reaction (PCR). Forty-nine bacterial 16S rDNA gene were sequenced and entrusted in GenBank. Phylogenetic analysis showed they were different phylotypes of Lactobacillus. Two of them were most closely relevant to the previously described species Lactobacillus plantarum. Other two phylotypes were identified to be closely related to Lactobacillus pentosus. However, only one phylotype was found to be distantly linked to the Lactobacillus fermentum. The outcomes of the present study indicated that L. plantarum, L. pentosus, and L. fermentum were the dominant lactobacilli in the honey stomach of honeybee A. dorsata collected during the dry season from Malaysia forest area - specifically "Melaleuca in Terengganu".
Bioconverting glycerol into various valuable products is one of glycerol's promising applications due to its high availability at low cost and the existence of many glycerol-utilizing microorganisms. Bioethanol and biohydrogen, which are types of renewable fuels, are two examples of bioconverted products. The objectives of this study were to evaluate ethanol production from different media by local microorganism isolates and compare the ethanol fermentation profile of the selected strains to use of glucose or glycerol as sole carbon sources. The ethanol fermentations by six isolates were evaluated after a preliminary screening process. Strain named SS1 produced the highest ethanol yield of 1.0 mol: 1.0 mol glycerol and was identified as Escherichia coli SS1 Also, this isolated strain showed a higher affinity to glycerol than glucose for bioethanol production.
Both Gram-positive and Gram-negative bacteria can take up exogenous DNA when they are in a competent state either naturally or artificially. However, the thick peptidoglycan layer in Gram-positive bacteria's cell wall is considered as a possible barrier to DNA uptake. In the present work, two transformation techniques have been evaluated in assessing the protocol's ability to introduce foreign DNA, pBBRGFP-45 plasmid which harbors kanamycin resistance and green fluorescent protein (GFP) genes into a Gram-positive bacterium, Bacillus cereus EB2. B. cereus EB2 is an endophytic bacterium, isolated from oil palm roots. A Gram-negative bacterium, Pseudomonas aeruginosa EB35 was used as a control sample for both transformation protocols. The cells were made competent using respective chemical treatment to Gram-positive and Gram-negative bacteria, and kanamycin concentration in the selective medium was also optimized. Preliminary findings using qualitative analysis of colony polymerase chain reaction (PCR)-GFP indicated that the putative positive transformants for B. cereus EB2 were acquired using the second transformation protocol. The positive transformants were then verified using molecular techniques such as observation of putative colonies on specific media under UV light, plasmid extraction, and validation analyses, followed by fluorescence microscopy. Conversely, both transformation protocols were relatively effective for introduction of plasmid DNA into P. aeruginosa EB35. Therefore, this finding demonstrated the potential of chemically prepared competent cells and the crucial step of heat-shock in foreign DNA transformation process of Gram-positive bacterium namely B. cereus was required for successful transformation.
As the largest genus in Actinobacteria family, Streptomyces species have the ability to synthesize numerous compounds of diverse structures with bioactivities. Streptomyces mangrovisoli MUSC 149T was previously isolated as a novel streptomycete from mangrove forest in east coast of Peninsular Malaysia. The high quality draft genome of MUSC 149T comprises 9,165,825bp with G+C content of 72.5%. Through bioinformatics analysis, 21 gene clusters identified in the genome were associated with the production of bioactive secondary metabolites. The presence of these biosynthetic gene clusters in MUSC 149T suggests the potential exploitation of the strain for production of medically important compounds.
Streptomycetes remain as one of the important sources for bioactive products. Isolated from the mangrove forest, Streptomyces gilvigriseus MUSC 26T was previously characterised as a novel streptomycete. The high quality draft genome of MUSC 26T contained 5,213,277bp with G+C content of 73.0%. Through genome mining, several gene clusters associated with secondary metabolites production were revealed in the genome of MUSC 26T. These findings call for further investigations into the potential exploitation of the strain for production of pharmaceutically important compounds.
The time courses for production of fungal biomass, lipid, phenolic and arachidonic acid (ARA) as well as expression of the genes involved in biosynthesis of ARA and lipid were examined in Mortierella alpina CBS 754.68. A significant increase in the arachidonic acid content in lipids that coincided with reduced levels of lipid was obtained. Reduced gene expression occurred presumably due to the steady reduction of carbon and nitrogen resources. However, these energy resources were inefficiently compensated by the breakdown of the accumulated lipids that in turn, induced up-regulated expression of the candidate genes. The results further indicated that the expression of the GLELO encoding gene is a rate-limiting step in the biosynthesis of ARA in the early growth phase.
Multidrug-resistant (MDR) and extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae associated with nosocomial infections have caused serious problems in antibiotic management with limited therapeutic choices. This study aimed to determine the genotypic and phenotypic characteristics of K. pneumoniae strains isolated from a tertiary hospital in Malaysia. Ninety-seven clinical K. pneumoniae strains were analyzed for antimicrobial susceptibility, all of which were sensitive to amikacin and colistin (except one strain), while 31.9 % and 27.8 % were MDR and ESBL producers, respectively. PCR and DNA sequencing of the amplicons indicated that the majority of MDR strains (26/27) were positive for blaTEM, followed by blaSHV (24/27), blaCTX-M-1 group (23/27), blaCTX-M-9 group (2/27), and mcr-1 (1/27). Thirty-seven strains were hypervirulent and PCR detection of virulence genes showed 38.1 %, 22.7 %, and 16.5 % of the strains were positive for K1, wabG, and uge genes, respectively. Genotyping by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) showed that these strains were genetically diverse and heterogeneous. Sequence types, ST23, ST22, and ST412 were the predominant genotypes. This is the first report of colistin-resistant K. pneumoniae among clinical strains associated with mcr-1 plasmid in Malaysia. The findings in this study have contributed to the effort in combating the increase in antimicrobial resistance by providing better understanding of genotypic characteristics and resistance mechanisms of the organisms.
Biofilm formation can lead to various consequences in the food processing line such as contamination and equipment breakdowns. Since formation of biofilm can occur in various conditions; this study was carried out using L. monocytogenes ATCC 19112 and its biofilm formation ability tested under various concentrations of sodium chloride and temperatures. Cultures of L. monocytogenes ATCC 19112 were placed in 96-well microtitre plate containing concentration of sodium chloride from 1-10% (w/v) and incubated at different temperature of 4 °C, 30 °C and 45 °C for up to 60 h. Absorbance reading of crystal violet staining showed the density of biofilm formed in the 96-well microtitre plates was significantly higher when incubated in 4 °C. The formation of biofilm also occurs at a faster rate at 4 °C and higher optical density (OD 570 nm) was observed at 45 °C. This shows that storage under formation of biofilm that may lead to a higher contamination along the processing line in the food industry. Formation of biofilm was found to be more dependent on temperature compared to sodium chloride stress.
Antibacterial activity of cell-free supernatant from Escherichia coli E against selected pathogenic bacteria in food and aquaculture was the highest against Edwardsiella tarda 3, a significant aquaculture pathogen. Biochemical properties of the bacteriocins were studied and bacteriocin was found to be sensitive to proteinase K, demonstrating its proteinaceous nature. In addition, pH and temperature affected bacteriocin activity and stability. The bacteriocins were partially purified by ammonium sulfate precipitation. The antibacterial activity was only detected in 20% ammonium sulfate fraction and direct detection of its activity was performed by overlaying on the indicator strains. The inhibition zone associated with the antibacterial activity was detected in the sample overlaid by E. tarda 3 and Staphylococcus aureus DMST8840 with the relative molecular mass of about 27 kDa and 10 kDa, respectively. Bacteriocin showed no cytotoxic effect on NIH-3T3 cell line; however, two virulence genes, aer and sfa, were detected in the genome of E. coli E by PCR. The characteristics of bacteriocins produced by E. coli E exhibited the antibacterial activity against both Gram-positive and Gram-negative pathogenic bacteria and the safe use determined by cytotoxicity test which may have interesting biotechnological applications.
This study aimed to investigate the impact of nonionic surfactants on the efficacy of fluorine degradation by Polyporus sp. S133 in a liquid culture. Fluorene was observed to be degraded in its entirety by Polyporus sp. S133 subsequent to a 23-day incubation period. The fastest cell growth rate was observed in the initial 7 days in the culture that was supplemented with Tween 80. The degradation process was primarily modulated by the activity of two ligninolytic enzymes, laccase and MnP. The highest laccase activity was stimulated by the addition of Tween 80 (2443U/L) followed by mixed surfactant (1766U/L) and Brij 35 (1655U/L). UV-vis spectroscopy, TLC analysis and mass spectrum analysis of samples subsequent to the degradation process in the culture medium confirmed the biotransformation of fluorene. Two metabolites, 9-fluorenol (λmax 270, tR 8.0min and m/z 254) and protocatechuic acid (λmax 260, tR 11.3min and m/z 370), were identified in the treated medium.
Flagellar-mediated motility is a crucial virulence factor in many bacterial species. A dual flagellar system has been described in aeromonads; however, there is no flagella-related study in the emergent human pathogen Aeromonas dhakensis. Using 46 clinical A. dhakensis, phenotypic motility, genotypic characteristics (flagellar genes and sequence types), biochemical properties and their relationship were investigated in this study. All 46 strains showed swimming motility at 30 °C in 0.3% Bacto agar and carried the most prevalent 6 polar flagellar genes cheA, flgE, flgG, flgH, flgL, and flgN. On the contrary, only 18 strains (39%) demonstrated swarming motility on 0.5% Eiken agar at 30 °C and they harbored 11 lateral flagellar genes lafB, lafK, lafS, lafT, lafU, flgCL, flgGL, flgNL, fliEL, fliFL, and fliGL. No association was found between biochemical properties and motility phenotypes. Interestingly, a significant association between swarming and strains isolated from pus was observed (p = 0.0171). Three strains 187, 277, and 289 isolated from pus belonged to novel sequence types (ST522 and ST524) exhibited fast swimming and swarming profiles, and they harbored > 90% of the flagellar genes tested. Our findings provide a fundamental understanding of flagellar-mediated motility in A. dhakensis.
Aeromonas dhakensis is an emergent human pathogen with medical importance. This study was aimed to determine the sequence types (STs), genetic diversity, and phylogenetic relationships of different clinical sources of 47 A. dhakensis from Malaysia using multilocus sequence typing (MLST), goeBURST, and phylogenetic analyses. The analysis of a concatenated six-gene tree with a nucleotide length of 2994 bp based on six housekeeping genes (gyrB, groL, gltA, metG, ppsA, and recA) and independent analyses of single gene fragments was performed. MLST was able to group 47 A. dhakensis from our collection into 36 STs in which 34 STs are novel STs. The most abundant ST521 consisted of five strains from peritoneal fluid and two strains from stools. Comparison of 62 global A. dhakensis was carried out via goeBURST; 94.4% (34/36) of the identified STs are novel and unique in Malaysia. Two STs (111 and 541) were grouped into clonal complexes among our strains and 32 STs occurred as singletons. Single-gene phylogenetic trees showed varying topologies; groL and rpoD grouped all A. dhakensis into a tight-cluster with bootstrap values of 100% and 99%, respectively. A poor phylogenetic resolution encountered in single-gene analyses was buffered by the multilocus phylogenetic tree that offered high discriminatory power (bootstrap value = 100%) in resolving all A. dhakensis from A. hydrophila and delineating the relationship among other taxa. Genetic diversity analysis showed groL as the most conserved gene and ppsA as the most variable gene. This study revealed novel STs and high genetic diversity among clinical A. dhakensis from Malaysia.
The wide use of whole-genome sequencing approach in the modern genomic era has opened a great opportunity to reveal the prospective applications of halophilic bacteria. Robertkochia marina CC-AMO-30DT is one of the halophilic bacteria that was previously taxonomically identified without any inspection on its biotechnological potential from a genomic aspect. In this study, we present the whole-genome sequence of R. marina and demonstrated the ability of this bacterium in solubilizing phosphate by producing phosphatase. The genome of R. marina has 3.57 Mbp and contains 3107 predicted genes, from which 3044 are protein coding, 52 are non-coding RNAs, and 11 are pseudogenes. Several phosphatases such as alkaline phosphatases and pyrophosphatases were mined from the genome. Further genomic study (phylogenetics, sequence analysis, and functional mechanism) and experimental data suggested that the alkaline phosphatase produced by R. marina could potentially be utilized in promoting plant growth, particularly for plants on saline-based agricultural land.
Neoscytalidium dimidiatum and Bipolaris species are fungal plant pathogens that have been reported to cause human diseases. Recently, we have isolated numerous N. dimidiatum and Bipolaris species from the skin scrapings and nails of different patients. In this work, we have sequenced the genome of one strain of N. dimidiatum. The sequenced genome was compared to that of a previously reported Bipolaris papendorfii genome for a better understanding of their complex lifestyle and broad host-range pathogenicity. Both N. dimidiatum UM 880 (~ 43 Mb) and B. papendorfii UM 226 (~ 33 Mb) genomes include 11,015-12,320 putative coding DNA sequences, of which 0.51-2.49% are predicted transposable elements. Analysis of secondary metabolism gene clusters revealed several genes involved in melanin biosynthesis and iron uptake. The arsenal of CAZymes related to plants pathogenicity is comparable between the species, including genes involved in hemicellulose and pectin decomposition. Several important gene encoding keratinolytic peptidases were identified in N. dimidiatum and B. papendorfii, reflecting their potential pathogenic role in causing skin and nail infections. In this study, additional information on the metabolic features of these two species, such as nutritional profiling, pH tolerance, and osmotolerant, are revealed. The genomic characterization of N. dimidiatum and B. papendorfii provides the basis for the future functional studies to gain further insights as to what makes these fungi persist in plants and why they are pathogenic to humans.
A total of 63 beef offal samples (beef liver = 16; beef lung = 14; beef intestine = 9; beef tripe = 15; beef spleen = 9) from three wet markets (A, B, and C) in Selangor, Malaysia were examined for the prevalence and microbial load of Listeria monocytogenes. A combination of the most probable number and polymerase chain reaction (MPN-PCR) method was employed in this study. It was found that L. monocytogenes detected in 33.33% of the beef offal samples. The prevalence of L. monocytogenes in beef offal purchased from wet markets A, B, and C were 22.73%, 37.50% and 41.18% respectively. The density of L. monocytogenes in all the samples ranged from < 3 up to > 2,400 MPN/g. The findings in this study indicate that beef offal can be a potential vehicle of foodborne listeriosis.
The accumulation of nitrogen compounds in shrimp farming water and effluent presents a major challenge. Ammonia is a form of nitrogen that limits shrimp growth due to its potential toxicity and effects on shrimp health and water quality. This study is aimed at identifying promising bioremediators from shrimp pond sludge to mitigate ammonia levels in both culture water and wastewater and at determining major bacterial communities in sludge using metagenomic analysis. A sludge sample was collected from a shrimp pond in Selangor, Malaysia, to isolate potential ammonia-removing bacteria. Out of 64 isolated strains, Bacillus flexus SS2 showed the highest growth in synthetic basal media (SBM) containing ammonium sulfate at a concentration of 70 mg/L as the sole nitrogen source. The strain was then incubated in SBM with varying pH levels and showed optimal growth at pH 6.5-7. After 24 h of incubation, B. flexus SS2 reduced the ammonia concentration from an initial concentration of 5 to 0.01 mg/L, indicating a 99.61% reduction rate, which was highest in SBM at pH 7. Moreover, the strain showed ammonia removal ability at concentrations ranging from 5 to 70 mg/L. Metagenomic analysis revealed that Proteobacteria was the most abundant phylum in the sludge, followed by Cyanobacteria, Actinobacteria, Chloraflexi, Firmicutes, and Campilobacterota. Bacillus flexus SS2 belongs to the Bacillota phylum and has the potential to serve as a bioremediator for removing ammonia from shrimp culture water and wastewater.
This research evaluates the bioactivity of twelve endophytic fungi successfully isolated and characterised from Gynura procumbens. The fungal extracts displayed inhibitory activity against Staphylococcus aureus, Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli and Salmonella typhi with the MIC and MBC of 5000 µg/mL. High antioxidant activity using DPPH free radical scavenging assay with inhibition of 86.6% and IC50 value of 104.25 ± 18.51 µg/mL were exhibited by ethyl acetate extract of Macrophomina phaseolina SN6. In contrast, the highest scavenging activity percentage of methanolic extract was exhibited by Mycoleptodiscus indicus SN4 (50.0%). Besides that, the highest ferric reducing antioxidant power (FRAP) value of ethyl acetate and methanolic extract was recorded from M. phaseolina SN6 (239.9 mg Fe (II)/g) and M. indicus SN4 (44.7 mg Fe (II)/g), respectively. Total phenolic content (TPC) and total flavonoid content (TFC) of ethyl acetate and methanolic fungal extracts were determined using Folin-Ciocalteu and aluminium chloride, respectively. The highest TPC for ethyl acetate and methanolic extracts were exhibited by Colletotrichum gloeosporioides SN11 (87.0 mg GAE/g) and M. indicus SN4 (35.0 mg GAE/g), whereas the highest TFC of ethyl acetate and methanolic extracts were showed by M. phaseolina SN6 (122.8 mg QCE/g) and M. indicus SN4 (60.4 mg QCE/g), respectively. Bioactive metabolites of isoelemicin (50.8%), terpinen-4-ol (21.5%), eucalyptol (24.3%), oleic acid (19.8%) and β-pinene (10.9%) were detected. Owing to the higher content of phytochemicals represented in the ethyl acetate extract of M. phaseolina, SN6 is therefore identified to be a superior candidate in exhibiting strong antioxidant and antimicrobial properties be fit for further pharmaceutical studies.