Comparison of N-acetyl-L-cysteine-sodium hydroxide and Modified Petroff’s Decontamination Methods for Mycobacterium tuberculosis Culture

Indonesia is one of 22 countries with a high incidence of TB in the world, particularly related to TB-HIV and MDR-TB cases. Contamination of normal flora from nasopharyngeal tract is the main problem to isolate Mycobacterium tuberculosis (MTB) from sputum. It is needed a safe solution to decontaminate without killing MTB bacilli. N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) is compared with modified Petroff’s which is widely used in laboratories, to get better outcome. Of the 110 sputum samples were collected from suspected cases of Pulmonary TB. Decontamination using 2% NALC-NaOH and Modifed Petroff (4% NaOH) was performed before AFB smear and culture on LJ medium. Each group was assessed for contamination and culture positive rate. The positive culture was validated using chromatography test for detected antigen MPT-64 and PNB. Result of this study showed that NALC-NaOH and modified Petroff’s methods did not significantly affect positivity rate of Acid-Fast Bacillus (AFB) smear, 71% and 66% respectively. Contamination on culture was significantly (p=0,034) higher in samples treated with NALC-NaOH (21%) compared to Modified Petroff methods(13%). Proportion of positive culture on samples treated with NALC-NaOH was lower than Modified Petroff, 65% and 70% respectively with p value=1. Conclusion of this study showed that sputum decontamination using Modified Petroff methods is still more effective than NALC-NaOH to increase positivity rate of MTB culture. However, the two methods were not significantly different to get positivity result on the microscopic examination of AFB.

Are Trichoderma atroviride and Trichoderma harzianum Effective to Control Fusarium Associated With Tomato Wilt?

The pathogenic fungi, such as Fusarium in the rhizosphere of tomato (Solanum lycopersicum) negatively affects the yield and quality of the plant. A number of biological control agents have been used for protecting tomato plants against wilt diseases including various fungal species. The objective of this study was to evaluate the antagonism effects of Trichoderma atroviride and T. harzianum against the pathogen Fusarium sp. associated with tomato wilt. In this study, the antagonism of these Trichoderma spp. against the Fusarium sp. was tested in vitro by the dual culture technique, and the percentage inhibition of radial growth (PIRG) and the antagonism reaction (scale 1-5) were evaluated. The results showed that T. atroviride and T. harzianum led to 70.8% PIRG and scale 1 antagonism reaction, and 40.6% PIRG and scale 3 antagonism reaction against Fusarium sp. associated with tomato wilt after 7 days of incubation, respectively. These results indicate that application of T. atroviride and T. harzianum may be promising approach for biological control of Fusarium wilt of tomato and may play an important role in sustainable agriculture.

Analysis of The Phytochemical Characteristics of Jasmine Flower Against Escherichia coli and Staphylococcus aureus

Indonesia is a famous country had the diversity of plants, especially agricultural product and herbs, they had a potential of antimicrobial as a like Jasmine Flower (Jasminum sambac Ait). This study Indonesia is a famous country had the diversity of plants, especially agricultural product and herbs, they had a potential of antimicrobial as a like Jasmine Flower (Jasminum sambac Ait). This study aimed to determine the phytochemical and antimicrobial activity of jasmine flower. Extraction was done by maceration, such as using water solvent, methanol, ethyl acetate, and hexane as the solvent. The sample bacteria used were Escherichia coli and Staphylococcus aureus. This study used disc diffusion method, with a complete 2-factor random design and 4 replications. Processing of the data is using ANOVA. The results showed that the jasmine extracts with a concentration of 25%, 50%, 75% and 100% had an effect to inhibit of the growth of the bacteria. Jasmine extract with ethyl acetate solvent was the most effective to inhibit the growth of bacteria. It was known that jasmine flower extract is one of the good natural antimicrobials gave an inhibitory effect to the growth of Escherichia coli and Staphylococcus aureus bacteria. Keywords: jasmine, Escherichia coli, Sthaphylococcus aureus, antimicrobials

Production and Characterization of Thermoalkaliphilic Xylanase from Bacillus halodurans CM1 on Degumming Process of Ramie (Boehmeria nivea L.Gaud)Fiber as Textile Raw Material

Ramie fiber is a potential raw material to substitute imported raw materials such as cotton. Due to its higher hemicellulose content, ramie fiber required hydrolysis in a process called degumming. Enzymatic degumming is environmentally friendly compared to traditional process which using chemicals. Alkalithermophilic xylanase have high ability in hemicellulose hydrolysis. The production of xylanase was conducted by submerged fermentation of Bacillus halodurans CM1 in 20L bioreactor using Mamo and corncob medium with optimum conditions at 50°C, pH 9, 150 RPM and 1 vvm. The optimum specific activity of xylanase measured by Bailey method at 70°C and pH 9 is 475.41 U/mg. Xylanase was stable at 50°C, pH 9 and relatively stable to K+, Na2+, Co2+ and Ca2+ metal ions and Triton-X, Saba dan Tween-80 surfactants. Degumming process was carried out by immersing ramie fibers in formulated degumming solution with vlot 1:20 at 50°C, 150 RPM and 180 minutes. The enzymatic degumming process may substitute or reduce the use of chemicals due to its significant effect on ramie fiber quality. Enzymatic and chemical degumming process reduce the weight of Ramie Fiber to 7.23 %, and 7.72 %, slightly higher than enzymatic degumming 7.15%. Enzymatic degumming maintains tensile strength at 27.51 %. Whiteness index enhanced to 2.99% enzymatically and 3.49% chemically. Keywords: Bacillus halodurans CM1, enzymatic degumming, ramie fiber, textile industry, thermoalkaliphilic xylanase

DOMESTICATION AND FORMULATION OF RHIZOPODOPSIS JAVENSIS AS A TEMPEH STARTER

ABSTRACT Domestication of wild fungal strains involved in the manufacture of traditional fermented foods often occurs spontaneously. Rhizopodopsis javensis (Rh. javensis) is taxonomically close to Rhizopus. The wild strain Rhizopodopsis javensis has found in cool climates can be developed as a starter in tempeh production in temperate regions. Before formulating it as a tempeh starter, a wild strain of Rh. javensis needs to be domesticated in human-made niches. A wild strain of Rh. javensis was domesticated by subculture using rice flour media at optimum growth temperature and carried out every five days. The spore's density and viability and the starter's water content were used to determine its quality. The results showed that Rh. javensis grew optimally at 22 ℃. With seven-time subcultures using rice flour media, the domestication process did not change the Rh. javensis growth rate and colony appearance. The growth rate of Rh. javensis is relatively the same as that of commercial tempeh starter and pure R. microsporus var. oligosporus, at each optimal growth temperature. In the rice flour media as a carrier, Rh. javensis produces spore's density that is relatively the same as that of commercial tempeh starter but with lower spore's viability and higher water content. Therefore, Rh. javensis cannot be used as a starter to produce tempeh in the temperate region. The carrier material and drying processes still need to be modified to increase spore viability and improve the overall quality, including the starter's lifespan. Keywords: food fermentation, Rhizopus microsporus var. oligosporus, spore's viability, starter quality, wild strain ABSTRAK Domestikasi galur liar kapang yang terlibat dalam dalam pembuatan makanan fermentasi tradisional, sering terjadi secara spontan. Rhizopodopsis javensis (Rh. javensis) merupakan salah satu galur liar kapang yang memiliki hubungan taksonomi dekat dengan Rhizopus. Strain liar ini ditemukan di daerah beriklim sejuk, sehingga berpotensi untuk dikembangkan sebagai starter tempe untuk produksi di daerah beriklim sedang. Untuk mendapatkan kultur yang tumbuh subur di relung (niches) buatan manusia, strain liar Rh. javensis perlu didomestikasi terlebih dahulu. Penelitian ini bertujuan untuk mendomestikasi strain Rh. javensis liar yang dilanjutkan dengan memformulasikannya sebagai starter tempe. Domestikasi dilakukan dengan menumbuhkan strain liar Rh. javensis pada media tepung beras pada suhu pertumbuhan optimum dan diulangi setiap lima hari. Kerapatan dan viabilitas spora, serta kadar air starter digunakan sebagai penilaian keberhasilan starter. Hasil penelitian menemukan Rh. javensis tumbuh optimal pada suhu 22 ℃. Domestikasi dengan cara subkultur koloni Rh. javensis pada media tepung beras selama 7 kali tidak mengubah kecepatan pertumbuhan Rh. javensis dan penampakan koloni. Laju pertumbuhan Rh. javensis relatif sama dengan laju pertumbuhan starter tempe komersial dan R. microsporus var. oligosporus murni, pada suhu optimum pertumbuhan masing-masing. Formulasi tepung beras sebagai media pembawa starter Rh. javensis, menghasilkan kerapatan spora yang relatif sama dengan starter tempe komersial, namun viabilitas sporanya rendah dan kadar airnya tinggi. Starter Rh. javensis belum dapat digunakan untuk membuat tempe. Substrat dan proses pengeringan masih perlu dimodifikasi untuk meningkatkan viabilitas spora dan kualitas starter tempe secara keseluruhan, termasuk umur simpan starter. Keywords: fermentasi makanan, kualitas starter, Rhizopus microsporus var. oligosporus, strain liar, viabilitas spora

Immunogenicity of Recombinant DNA Vaccine Encoding Non-Structural Protein-1 Dengue Virus Serotype-2 in Balb/c Mice

Background: Dengue Hemorrhagic Fever (DHF) is an infectious disease caused by the dengue virus (DENV) which spread widely in tropical and subtropical regions of the world. DENV is a single-positive strand RNA virus with a genome size of ± 11kb which encodes three structural proteins, seven non-structural proteins, and two untranslated regions (UTR). The non-structural protein-1 (NS1) of DENV is known to have important role in dengue pathogenesis also promising to be developed as dengue vaccine. Lately, novel vaccine approach by DNA immunization have given new perspective for a safe, stable, and immunogenic vaccine platform. Previously, we have successfully construct DNA vaccine encoding NS1 protein of DENV2 (pUNS1) which express recombinant NS1 protein in-vitro. Thus, in this current study the ability of pUNS1 to induce humoral immune response will be further analyzed by in mice immunization. Methods: Sixteen BALB/c mice aged of 4 weeks were immunized 3 times with 100 µg of pUNS1 or pUMVC4a on 2 week time interval. Blood sampling was carried out just before immunization and termination was done 2 week after last immunization. Titer from individual mice sera against DENV-2 were measure with in-house ELISA. Results: IgG against NS1 protein of DENV2 titer from mice group immunized with recombinant pUNS1 shown high ELISA absorbancies, 5 times higher than pUMVC4a group. This result suggest the ability of pUNS1 to induce humoral immune response against NS1 DENV-2 in-vivo. Conclusion: Recombinant pUNS1 can induce humoral immune response in mice.

Molecular Identification of Hg-Resistant Bacteria and Their Potential in Reducing Mercury Contamination

The aim of this research was to obtain and determine the identity of Hg-resistant bacteria in soil contaminatedwith gold processing waste and test its ability to reduce mercury contamination. Soil samples as a source of Hgresistant bacterial isolates were obtained from the gold processing location in Ilangata Village, Anggrek District,North Gorontalo Regency. The research was conducted at the Microbiology Laboratory, Department of Biology,Faculty of Mathematics and Natural Sciences. Mercury analysis was carried out at the Laboratory of FisheriesProduct Quality Development and Testing (LPPMHP), Gorontalo Province, and bacterial identification wascarried out at the Hasanuddin University Medical Research Center Research Unit. The parameters observed werethe types of Hg resistant bacteria and the ability of the bacteria to reduce mercury contamination. The resultsshowed that there were four bacterial isolates on the soil contaminated with 4.5 ppm mercury, which were namedILb01, ILB02, ILb03, and ILb04. Molecular identification showed that ILb01 was closely related toStenotrophomonas sp. SB67 and ILb02 close to Enterobacter cloacae strain CM 1, these strains were not resistantto mercury contamination; while ILb03 which is similar to strain BS0591 and ILb04 which is similar to BacteriumBacillus albus strain SQ30 16S could be resistant and was able to reduce mercury contamination by 99% at 10ppm levels. Key words: heavy metals, Hg-resistant bacteria, molecular identification, mercury

Bacterial Population Dynamics of Natural Fermentation of Sumbawa Mare’s Milk Using Metagenomic Approach

This study aimed to assess the changing of bacterial density and the physicochemical aspects during natural fermentation of Sumbawa mare’s milk, and to evaluate the dynamics of bacterial population during the natural fermentation using metagenomic approach. Mare’s milk sample obtained from Regency of Dompu were fermented for 60 days. On the day 0, 7, 15, 30 and 60 mare milk sample were collected for further analysis, such as bacterial density enumeration, nutrition content, physical properties of the milk, and total DNA isolation. The total DNA samples obtained were analyzed using next generation sequencing. The density of lactic acid bacteria was decreased along with fermentation periods. Meanwhile, the density of aerobic bacteria on was relatively fluctuated. The physicochemical content of mare’s milk also changed during fermentation periods. Carbohydrate content and total sugar was decrease along with the decreasing of pH value. Moreover, the lipid content increase, and the protein content was fluctuated. The changing in physical properties such as whey color, acidity and gas was observed until the end of mare’s milk natural fermentation process. Using metagenomics analysis, the bacterial diversity from each sample periods categorized as low because of the dominance of Lactobacillus helveticus until the end of the fermentation. Lactobacillus helveticus as a member of LAB did not grow on isolation media on the late stage of fermentation periods (day-60). The presence of uncultivable bacteria can be detected with metagenomic approach, fulfilling the limited information on the bacterial composition of fermented Sumbawa mare’s milk products.

Single nucleotide polymorphism in the rpoB gene Mycobacterium tuberculosis from Papua-Indonesia and its impact on rifampicin resistance: A whole genome sequencing analysis

In the antibiotic era, Tuberculosis (TB) drugs resistance especially Rifampicin (RIF) is highly reported around the world. Resistance of RIF is caused by the mutation of genes that associated with RIF receptor. The aims of this study are detecting the Single Nucleotide Polymorphism of Rifampicin resistant genes using Whole Genome Sequencing (WGS) and analysing the profile of protein changing caused by SNP. Twenty Mycobacterium tuberculosis culture samples were passed on WGS procedure and 19 samples were adequate to further bioinformatics analysis. Single Nucleotide Polymorphisms Analysis was done using TBprofiler. Based on TBProfiler, seventeen samples were resistant to rifampicin. The mutations that cause the resistance are S450L, D435Y, H445Y, 430P, Q432K. Other Single Nucleotide Polymorphisms H835R, V534M and R224C were also found. The H835R mutants are present together with the S450L, V534M with S450L mutants, and R224C with Q432K mutants. Native protein for RNA Polymerase Subunit β used was the result of separation from the crystal structure of Mycobacterium tuberculosis H37Rv RNA polymerase (PDB: 5UHB). Binding affinity RIF to RNA Polymerase Subunit β calculated using AutoDock vina. Construction of mutant 3D structures using FoldX5. From the analysis, it was found that seventeen samples were resistant to rifampicin and two samples did not contain SNP which could cause resistance to rifampicin.

Growth Kinetic Study of Blue-green Microalgae Arthrospira platensis Using Buffalo Manure as Alternative Media

North Sumatera is one of the provinces in Indonesia with the highest buffalo population, which is responsible for the high accumulation of buffalo manure that can cause environmental and aesthetic problems if left untreated. One of the possible alternatives for solving this issue is by implementing buffalo manure as growth media for microorganisms, e.g. microalgae. In this research, buffalo manure was used as alternative media for Arthospira platensis cultivation. Buffalo manure was taken from Sitoluama village, Laguboti, Toba Regency of North Sumatra Province. Research steps included media and culture preparation, cultivation, sampling, sample analysis and verification of constructed models and validation. Buffalo manure concentration in media is varied from 1 g.L-1 to 8 g.L-1 which is analogous to nitrogen content of 0.002 mg.L-1 to 0.018 mg.L-1. Growth data was used for growth kinetic modelling, which was most satisfactory for Monod model (µmax = 0.5915 day-1, Ks = 0.421 g.L-1).