Aims: This study was aimed to characterise nine clinical isolates in our culture collection that were categorized as Diutina species based on their molecular genetic profiles. D. rugosa is a species complex comprising four taxa., i.e., D. rugosa sensu stricto, D. pseudorugosa, D. neorugosa and D. mesorugosa. The most commonly used phenotypic identification methods for yeasts often lead to the misidentification of this species complex. Methodology and results: The Diutina isolates were received from two local referral hospitals as pure cultures. Species confirmation was performed using conventional phenotypic methods; CHROMagar and RapID Yeast Plus Kit. To study the inter- and intraspecific relationships among the clinical isolates, ITS region, D1/D2 domain and random amplified polymorphic DNA (RAPD) analyses were performed. The results were further validated using the housekeeping gene sequence similarity technique coupled with pairwise sequence alignment. The results from phenotypic methods results were ambiguous and inconclusive. The sequence analyses of ITS regions and D1/D2 domains revealed that the samples consisted of three yeast species; D. rugosa complex: D. rugosa (n=1), D. mesorugosa (n=6), Candida pararugosa (n=1) and Meyerozyma guilliermondii (n=1). The RAPD analysis with random primers, OPG4, OPG11 and OPA18, demonstrated good banding patterns that could distinguish between the Diutina isolates. The pairwise sequence alignment revealed that the Diutina isolates were genetically similar to D. rugosa ATCC 10571. Conclusion, significance and impact of study The molecular methods, D1/D2 domain, ITS1 and ITS4 region, and RAPD analyses have proven helpful for accurately identifying the yeasts, especially closely related species; D. rugosa and D. mesorugosa.

Tumour protein 53 (p53) plays an important role in the instruction of the cell cycle. In a variety of transformed cell lines, tumour protein is expressed in high amounts, and it is believed to contribute to transformation and malignancy. This research aimed to detect the anti-p53 antibodies in sera of patients with various malignant tumours and to evaluate the sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA). A case-control study was conducted on samples from 49 patients with various types of malignant tumours at Sultanah Bahiyah Hospital, Alor Setar, Kedah, Malaysia, and 32 healthy control cases with non‐malignant disease collected from Universiti Sains Malaysia clinic, Penang, Malaysia. The antibodies against p53 protein in the serum samples were analysed using the commercial ELISA kit, Calbiochem® p53- ELISAPLUS. The results showed that the rate of anti-p53 antibodies in patients with various malignant tumours was 13 out of 49 (26.5 %), compared with only 2 out of 32 (6.25%) in healthy controls (p < 0.001). The sensitivity of this kit reached 28.6% and the specificity was 93.8%. In conclusion, these results suggest that the anti-p53 antibodies can be detected in different sera of malignant tumour patients and the ELISA kit is highly specific; nevertheless, its discrimination power is not perfect because of its low sensitivity to determine the anti-p53 antibodies.

Aims: The exploration of natural products with innovative uses is dynamic and expanding rapidly. Medicinal plants have fascinated many researchers that subsequently lead to research publications highlighting plant extracts with wide range of secondary metabolites such as flavonoids, alkaloids, glycosides, quinones, terpenoids, tannins and saponins that exhibit antimicrobial activities and disease control. The concentration of these bioactive compounds in each plant species varies based on the pathosystem and environmental conditions. This study aims to uncover the various types of phytochemicals with antifungal properties. Methodology and results: Seven categories of plant-based antifungal compounds were reviewed, which are terpenoids, saponins, phenolic compounds, coumarins, alkaloids, essential oils and peptides, with examples and structures of some available compounds. The mechanism of action of each category of phytochemical was discussed. Also, the impact of some compounds was explained and elaborated. Conclusion, significance and impact of study It is of a great importance to explore natural plant fighters against fungal infection. Those active plant components do not only have antifungal properties, but they also help in the healing process and some even exhibit anticancer activities. The development and knowledge of antifungal activities from plant extracts have the potential for applications in antifungal therapy. Since the exact description of how antifungal compounds function in the human body is still unclear more studies are required to unveil phytochemicals’ properties and to elucidate their effects on living cells.
Phytochemicals--therapeutic use ; Antifungal Agents

Aim: A novel endophyte, Streptomyces kebangsaanensis was isolated from the stem of a Malaysian ethnomedicinal plant, Portulaca oleracea in 2013. Studies on S. kebangsaanensis crude extract showed that it had antifungal activities and further work led to isolation of a novel compound, phenazine-1-carboxylic acid (PCA). This study investigated the combinatorial effect of PCA isolated from S. kebangsaanensis with amphotericin B on the growth of four clinical Fusarium solani isolates. Methodology and results: Disk diffusion assay showed that the crude extract of S. kebangsaaneesis inhibited growth of all four F. solani isolates. Whereas, the compound PCA from this extract inhibited two of the tested F. solani isolates, UZ541/12, and UZ667/13 at minimum inhibitory concentration of 18.00 µg/mL Combinations of this compound with amphotericin B, reduced the minimum inhibitory concentration of amphotericin B for these two isolates from 8 to 0.13 µg/mL and 4 to 0.03 µg/mL respectively. Analysis of fractional inhibitory concentration index showed that a borderline synergism is present between the compound and amphotericin B. Conclusion, significance and impact of the study These results indicate PCA may be useful in improving actions of available drugs against antimicrobial resistant microorganisms.
Streptomyces

In the search for universal vaccine candidates for the prevention of avian influenza, the non-structural (NS)-1 protein of avian influenza virus (AIV) H5N1 has shown promising potential for its ability to effectively stimulate the host immunity. This study was aimed to produce a bacterial expression plasmid using pRSET B vector to harbour the NS1 gene of AIV H5N1 (A/Chicken/Malaysia/5858/2004 (H5N1)) for protein expression in Escherichia coli (E. coli). The NS1 gene (687 bp) was initially amplified by polymerase chain reaction (PCR) and then cloned into a pGEM-T Easy TA vector. The NS1 gene was released from pGEM-T-NS1 using EcoRI and XhoI restriction enzymes (RE). The pRSET B vector was also linearized using the same RE. The digested NS1 gene and linearized pRSET B were ligated using T4 DNA ligase to form the expression plasmid, pRSET B-NS1. The NS1 gene sequence in pRSET B-NS1 was confirmed by DNA sequencing. To prepare recombinant bacterial cells for protein expression in the future, pRSET B-NS1 was transformed into E. coli strain BL21 (DE3) by heat-shock. Colonies bearing the recombinant plasmid were screened using PCR. The DNA sequencing analysis revealed that the NS1 gene sequence was 97% homologous to that of AIV H5N1 A/Chicken/Malaysia/5858/2004 (H5N1). These results indicated that the NS1 gene of influenza A/Chicken/Malaysia/5858/2004 (H5N1) was successfully amplified and cloned into a pRSET B vector. Bacterial colonies carrying pRSET B-NS1 can be used for the synthesis of NS1-based influenza vaccine in the future and thereby aid in the prevention of avian influenza.

Ergosterol, a component of fungal cell membrane, has been frequently detected as an indicator of fungal presence and massin environmental samples like soil. However, its detection in major pathogenic fungal species has not been investigated.In this study, the ergosterol contents of ten pathogenic fungal species were determined. Liquid chromatography was usedfor the detection and quantification of ergosterol extracted from fungal broth cultures. Results showed that ergosteroleluted as a single, well resolved peak in the chromatogram profiles of all tested fungi. Based upon relative amounts ofergosterol produced per fungal mycelial dry weight, three groups of fungal pathogens were identified, namely low ergosterol(Aspergillus niger, Candida albicans and Cryptococcus neoformans at 4.62, 6.29 and 7.08 μg/mg, respectively), mediumergosterol (Fusarium solani, Aspergillus fumigatus, Mucor sp., Penicillium sp., Cryptococcus gattii and Rhizopus sp.at 9.40, 10.79, 10.82, 11.38, 12.60 and 13.40 μg/mg, respectively), and high ergosterol (Candida tropicalis at 22.84 μg/mg), producers. Ergosterol was not detectable in bacterial samples, which were included as controls. This first report onergosterol detection in major pathogenic fungal species indicates that ergosterol may be used as a biomarker to diagnoseinvasive fungal infections in clinical sampl

Aims: Endophytic fungi are microorganisms that live asymptomatically within plant tissues, producing a wide range of metabolites, including compounds potentially useful for drug development. We investigated endophytic fungi from Maliau Basin, Sabah to identify strains producing bioactive compounds, notably with antimicrobial activity. Methodology and results: A total of 23 plants were sampled yielding 345 endophytic fungal isolates. Of these, 44 isolates were screened for antimicrobial activity against nine species of bacteria and fungi, revealing 14 endophytes producing bioactive metabolites. Crude fungal extracts were obtained from broth cultures of endophytic isolates with promising activity while the fungal strains were identified using molecular methods. The crude extract of endophyte MB4 WA10, isolated from Callophyllum sp. (bintangor) showed IC50 of 2.6 mg/mL against S. aureus and 0.6 mg/mL against B. subtilis while the extract of MB22 WA16, an isolate identified as Valsaceae sp., was also active against S. aureus with an IC50 of 1.37 mg/mL. Another isolate, namely MB5 L4 (WA), was identified as a Phomopsis sp. and its extract was the most active against S. aureus with an IC50 of 1 mg/mL. The HPLC fraction of this fungal extract with the highest inhibition (92.37%) of S. aureus was purified for compound isolation and identification. A polyketide compound, 2,3-dihydro-2- hydroxy-2,4-dimethyl-5-trans-propenylfuran-3-one (C9H12O3), with molecular weight of 168.192 was identified based on mass spectral and NMR data analysis. This previously identified compound is known to have other antimicrobial properties. Conclusion, significance and impact of study Rainforests in Malaysia, especially Maliau Basin, harbour many species of fungal endophytes, producing useful bioactive compounds that may be explored for further potential uses, including antimicrobial activity.

Herbal-based slimming products which are consumed orally may be contaminated with heavy metals as well as microorganisms. This study aimed to evaluate the safety level of these slimming products by determining heavy metals and microbial contamination in different batch production. Six different brands of herbal-based slimming products (A, B, C, G, H and I) with three different batch productions (1, 2 and 3) were investigated (n =18). Five heavy metals Arsenic, Cadmium, Chromium, Copper and Zinc were determined using an Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). The presence of microorganisms was determined by total aerobic count and the bacteria were identified. The samples’ moisture content was determined by calculating the percentage of water loss after drying process. All batches of samples A and B had high content of zinc, over the permissible level of 5ppm while, 6 samples contained Chromium above the permissible level (1.5 ppm). All 3 batches of sample A presented with the highest total daily intake of heavy metals. Bacteria were present in all the samples tested with the highest numbers in samples G, H and A followed by B, I and C. The highest number of fungi was found in product A while product I was free from fungal contamination. Aspergillus spp. was the predominant fungus present in the samples. There was a weak correlation between moisture content and bacteria (r = 0.087) and fungal (r = 0.253) presence in the samples. As some herbal slimming products contain heavy metals as well as microorganisms, consumers need to be more vigilant and discerning when selecting products to be consumed.
Metals, Heavy

Background: This study aimed to determine the minimum inhibitory concentrations (MICs) of various antifungal agents against moulds isolated from dermatological specimens. Methods: We identified 29 moulds from dermatological specimens between October 2012 and March 2013 by conventional methods. We performed antifungal susceptibility testing on six antifungal agents, amphotericin B, clotrimazole, itraconazole, ketoconazole, miconazole and terbinafine, according to the Clinical and Laboratory Standards Institute guidelines contained in the M38-A2 document. Results: Most antifungal agents were active against the dermatophytes, except for terbinafine against Trichophyton rubrum (geometric mean MIC, MICGM 3.17 µg/mL). The dematiaceous moulds were relatively susceptible to amphotericin B and azoles (MICGM 0.17-0.34 µg/mL), but not to terbinafine (MICGM 3.62 µg/mL). Septate hyaline moulds showed variable results between the relatively more susceptible Aspergillus spp. (MICGM 0.25-4 µg/mL) and the more resistant Fusarium spp. (MICGM 5.66-32 µg/mL). The zygomycetes were susceptible to amphotericin B (MICGM 0.5 µg/mL) and clotrimazole (MICGM 0.08 µg/mL), but not to other azoles (MICGM 2.52-4 µg/mL). Conclusion: Amphotericin B and clotrimazole were the most effective antifungal agents against all moulds excepting Fusarium spp., while terbinafine was useful against dermatophytes (except T. rubrum) and Aspergillus spp. However, a larger study is required to draw more solid conclusions.

OBJECTIVETo study bioactivity and compounds produced by an endophytic Phoma sp. fungus isolated from the medicinal plant Cinnamomum mollissimum.

METHODSCompounds produced by the fungus were extracted from fungal broth culture with ethyl acetate. This was followed by bioactivity profiling of the crude extract fractions obtained via high performance liquid chromatography. The fractions were tested for cytotoxicity to P388 murine leukemic cells and antimicrobial activity against bacteria and pathogenic fungi. Compounds purified from active fractions which showed antibacterial, antifungal and cytotoxic activities were identified using capillary nuclear magnetic resonance analysis, mass spectrometry and admission to AntiMarin database.

RESULTSThree known compounds, namely 4-hydroxymellein, 4,8-dihydroxy-6-methoxy-3-methyl-3,4-dihydro-1H-isochromen-1-one and 1-(2,6-dihydroxyphenyl) ethanone, were isolated from the fungus. The polyketide compound 4-hydroxymellein showed high inhibitory activity against P388 murine leukemic cells (94.6%) and the bacteria Bacillus subtilis (97.3%). Meanwhile, 4,8-dihydroxy-6-methoxy-3-methyl-3,4-dihydro-1H-isochromen-1-one, a benzopyran compound, demonstrated moderate inhibitory activity against P388 murine leukemic cells (48.8%) and the fungus Aspergillus niger (56.1%). The second polyketide compound, 1 (2,6-dihydroxyphenyl) ethanone was inactive against the tested targets.

CONCLUSIONSThese findings demonstrate the potential of endophytes as producers of pharmacologically important compounds, including polyketides which are major secondary metabolites in fungi.