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Whole genome analysis of Klebsiella: Unique genes associated with isolates from Indonesian tempeh

Mahaldika Cesrany ; Adi Yulandi ; Iman Rusmana ; Antonius Suwanto

Malaysian Journal of Microbiology.2017;13(4):273-279.

Aims: Our previous study demonstrated that Klebsiella IIEMP-3 associated with tempeh was genetically different from those of medical isolates. In addition to the whole genome sequence of Klebsiella IIEMP-3, the draft genome sequence of another isolate, i.e. IWJB-6 was employed for comparison. In this study, the details of the virulence genes and unique gene in both Klebsiella isolates were compared employing in silico and in vitro analysis. Methodology and results: Whole genome of Klebsiella IIEMP-3 and IWJB-6 were annotated to investigate the virulence factor. Klebsiella IIEMP-3 and IWJB-6 were obtained from tempeh producers in Bogor, West Java - Indonesia. Genome sequences were analyzed employing BLAST Ring Image Generator (BRIG) software. The results showed that all of the samples, including isolates IIEMP-3 and IWJB-6 did not harbor rmpA, i.e. DNA sequence for K. pneumoniae virulence factor. Conclusion, significance and impact of study: Klebsiella could be found in almost all tempeh samples from Indonesia and could be harmless for human due to the absence of rmpA and other virulence-associated genes. The significance of this study showed that IIEMP-3 and IWJB-6 isolates were more closely related to K. variicola. However, K. variicola At22 harbored sdsA gene which is lacking in those two tempeh isolates. Combined with PCR analysis for specific gene/s; our study suggested that isolates from Indonesian tempeh were closely related to K. variicola, and proposed to be designated as K. variicola subsp. tempehensis.

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Metagenome analysis of tempeh production: Where did the bacterial community in tempeh come from?

Rahmadina Radita ; Antonius Suwanto ; Norio Kurosawa ; Aris Tri Wahyudi ; Iman Rusmana

Malaysian Journal of Microbiology.2017;13(4):280-288.

Aims: Tempeh is a soy-based traditional food fermented by Rhizopus oligosporus. Although this mold is the main microorganism responsible for tempeh fermentation, various unknown bacteria presence in tempeh could enhance tempeh’s nutritional value. This study is aimed to examine the identity of bacteria in tempeh bacterial community by combining metagenomics analysis and culturable technique. Methodology and results: Samples were obtained from a tempeh producer which consists of raw soybeans, fresh water used to soak the beans, soaking water after the beans were soaked for 18 h, dehulled-soybean before inoculation, starter culture, and fresh tempeh. All samples were plated onto Enterobacteriaceae and Lactic Acid Bacteria agar media, and the total DNA was extracted for metagenomics analysis based on 16S rRNA gene cloning and High-Throughput Sequencing (HTS). Metagenomic analysis indicated that Firmicutes and Proteobacteria were the predominant and subdominant bacteria, respectively, while the culturable technique showed Proteobacteria were the predominant bacteria. Firmicutes species detected in tempeh were similar to the ones in the soaking water, which were populated by Lactobacillus. However, another predominant bacteria from tempeh, Enterococcus, was similar to minor population of Enterococcus detected in dehulled-soybean before inoculation. Based on the cloned 16S rRNA genes, we observed L. agilis, L. fermentum, and E. cecorum as the predominant bacteria in tempeh. The starter culture, which was dominated by Clostridium, did not alter bacterial community in tempeh, since its proportion was only 2.7% in tempeh clean reads. Conclusion, significance and impact of study: The dominant bacteria in tempeh was Lactobacillus from Firmicutes. The bacterial community in tempeh was not affected by the starter culture used, but mainly because of the soybean soaking process.

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Identification and prevention of microbial contaminants of potato culture in temporary immersion bioreactor (TIB) system

Md. Zamilur Rahman ; S. M. Shahinul Islam ; A. N. Chowdhury ; Sreeramanan Subramaniam

Malaysian Journal of Microbiology.2017;13(4):289-297.

Aims: Temporary Immersion Bioreactor (TIB) system is an advanced technology for commercial mass production of potato microtubers. Despite of several advantages, this system possess a great risk of culture loss at any stage of micropropagation due to microbial contamination. The aims of this study were to identify microbial contaminants isolated during potato shoot growth in the TIB system, evaluate the efficacy of antimicrobial agents to prevent them, to investigate the effect of those agents in vitro on growth and morphology of potato plantlets. Methodology and results: Six bacteria namely Pseudomonas, Staphylococcus, Klebsiella, Corynebacterium, Proteus, Bacillus and five fungi Aspergillus, Penicillium, Mucor, Fusarium and Rhizopus were isolated from the TIB system. We examined the effect of three antibacterial (Gentamycin, Vancomycin and Tetracycline) and four antifungal agents (Mencozeb, Propiconazole, Bavistin and Copper oxychloride) on the contaminants and on potato shoot growth. Results show that Gentamycin (50 mg/L) and Propiconazole (0.15%) were most effective against the isolated bacteria (35 mm inhibition zone) and fungi (100%) respectively, whereas Gentamycin in combination with Bavistin showed better performance on potato shoot and root development. Conclusion, significance and impact of study: Present study will provide useful guidelines to reduce or eliminate the risk of contamination during micropropagation.

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The potential of a novel β-specific dehalogenase from Bacillus cereus WH2 as a bioremediation agent for the removal of β-haloalkanoic acids

Wafaa Hassan Muslem ; Mohamed Faraj Edbeib ; Roswanira Abdul Wahab ; Elham Khalili ; Iffah Izzati Zakaria ; Fahrul Huyop

Malaysian Journal of Microbiology.2017;13(4):298-307.

Aims: This study aims to describe the biochemical and kinetic properties of a dehalogenase produced by a bacterium, Bacillus cereus WH2 (KU721999), that is uniquely adept at degrading a β-haloalkanoic acid, i.e., 3-chloropropionic acid (3-CP), and using it as the bacterium’s sole carbon source. The bacterium was isolated from abandoned agricultural land in Universiti Teknologi Malaysia that was previously exposed to herbicides and pesticides. Methodology and results: The B. cereus impressively removed 97% of 3-CP after 36 h of culturing. The intracellular WH2 dehalogenase of the bacterium was purified 2.5-fold and has an estimated molecular mass of 37 kDa. The highest activity of the dehalogenase was achieved under conditions of 30 °C and pH 7. The metal ions Hg2+ and Ag2+ substantially repressed the enzyme’s activity, but the enzyme’s activity was uninhibited by dithiothreitol (DTT) and EDTA. The WH2 dehalogenase showed a higher affinity for 3-CP (Km = 0.32 mM, kcat = 5.74 s-1 ) than for 3-chlorobutyric acid (3-CB) (Km = 0.52 mM; kcat = 5.60 s-1 ). The enzyme was ~1.6-fold more catalytically efficient (kcat/Km) in dehalogenating the three-carbon substrate 3-CP (17.8 mM-1 s -1 ) than the four-carbon 3-CB (11.2 mM-1 s -1 ). Conclusion, significance and impact of study: The novel B. cereus bacterium isolated in this study may prove applicable as a bioremediation agent to cleaning environments that are polluted with β-halogenated compounds. Furthermore, such an approach to treat polluted environments is more sustainable and potentially safer than chemical treatments.

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Genotypic and phenotypic characterization of methicillin resistance determinants and β-lactamase in Staphylococcus species

Abdul Rahim Abdul Rachman ; Norhidayah Mat Azis ; Pung Hui Ping ; Zarizal Suhaili ; Syafinaz Amin Nordin ; Zulkefley Othman ; Mohd Nasir Mohd Desa

Malaysian Journal of Microbiology.2017;13(4):308-317.

Aims: To characterize the genotypic distribution of mec complex, bla complex, methicillin-resistance level (cefoxitinMIC) and β-lactamase activity in carriage methicillin-resistant Staphylococcus species for a potential correlation. Methodology and results: Biochemical test, 30 µg cefoxitin diffusion disc test, cefoxitin E-test, mec and bla complexes distributions, Pbp2a and β-lactamase assays were conducted to characterize phenotypic and genotypic of MRSA and MRCoNS in our collection. Phylogenetic tree was constructed using MEGA6 software to trace the diversity of blaZ gene of MRSA and MRCoNS. Sixteen MRSA and nineteen MRCoNS were identified by biochemical tests followed by 30 µg cefoxitin antibiotic disc susceptibility test and mecA gene screening. Twenty nine isolates carry complete mecA genes (2.1 kb), incomplete mec regulator (negative or truncated) and positive Pbp2a assay for both MRSA and MRCoNS. Only MRCoNS SC177 isolate with cefoxitin MIC of 32 µg/mL carries complete mec complex. Thirty-one of thirty-five isolates carry complete bla complex (blaZ, blaRI, blaI) with 10 MRSA produce strong β-lactamase and cefoxitin MIC of ≥12 µg/mL. Only 4 MRCoNS with cefoxitin MIC of ≤8 µg/mL produce strong β-lactamase. The diversity of blaZ gene was demonstrated by phylogenetic analysis and unusual amino acid mutation at position 145 for MRSA SA60 isolate may compromise its β-lactamase activity with low cefoxitin MIC level (2 µg/mL). Conclusions, significance and impact of the study: Isolates that carry complete complete mecA gene were largely consistent with the expression of Pbp2a. Nevertheless, there is no clear correlation of mec regulator genes in relation to cefoxitin-MIC in both methicillin resistant (MR) Isolates that carry Staphylococcus species. On the other hand, various expression level of β-lactamase may correlate with cefoxitin-MIC level in MRSA as compared to MRCoNS.

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Characterization and comparison of phytase production by Bacillus and Paenibacillus strains from Thai soils

Saowapar Khianngam ; Yupa Pootaeng-On ; Apinya Sonloy ; Juthamat Kajorn-aroonkij ; Somboon Tanasupawat

Malaysian Journal of Microbiology.2017;13(4):318-325.

Aims: The objective of this research was to isolate, screen and identify phytase-producing bacteria from soils and a potent isolate was selected for its phytase production. Methodology and results: Eight spore-forming bacteria isolated from agricultural soils in Thailand were screened for their phytase production. They were identified as Bacillus and Paenibacillus strains based on their phenotypic characteristics and 16S rRNA gene sequence analyses. The phytase production by Bacillus amyloliquefaciens CH3-1 [Group I(a)] was 20.956 ± 0.099 U/mL, while Bacillus subtilis SR9-3 [Group I(b)] produced 20.588 ± 0.099 U/mL. Five isolates in Group I(c), identified as Bacillus aryabhattai, produced phytase at levels ranging from 2.436 ± 0.116 to 20.910 ± 0.000 U/mL, while Paenibacillus cineris CM5-3 (Group II) produced 1.261 ± 0.111 U/mL. A potent strain, CH3-1, produced the highest phytase when cultivated in Phytate Specific Medium (PSM) supplemented with 1% glucose, at pH 7.0 and incubated at 45 °C. Additionally, wheat bran and sorghum seed (0.5%) substrates were used to induce phytase production by replacing Na-phytate. Conclusion, significance and impact of study: Phytase producing bacteria were isolated from soils in Thailand. Gram-positive spore forming thermotolerant Bacillus strains displayed higher phytase activity than a Paenibacillus strain. A potent strain, CH3-1, could utilize agricultural waste as a substrate, which may be useful for animal feed supplementation.

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Effects of storage temperatures on survival and enterotoxin production of Staphylococcus aureus in Turkish white pickled cheese

Alper Baran ; Ahmet Erdoğan ; Mustafa Atasever

Malaysian Journal of Microbiology.2017;13(4):326-333.

Aims: Turkish white pickled cheese is the most consumed cheese type in Turkey and it is an important food to be evaluated in terms of food safety. In this study we investigated the behavior (survival and production of enterotoxin) of Staphylococcus aureus (S. aureus) NCTC 10654 in Turkish white pickled cheeses, which were ripened at 4 °C and 12 °C for 90 days. Methodology and results: Counting of microorganisms was carried out by conventional methods on appropriate media. Detection of enterotoxins was performed by double-sandwich ELISA technique and gene region responsible for enterotoxin production by reverse transcription-PCR (RT-PCR). The counts of S. aureus decreased (p < 0.05) in all of the cheese samples during ripening, where they decreased by 102 (CFU/g) at the end of the 90-day ripening period. The reduction in the S. aureus count was 2.5 times lower in cheeses ripened at the higher temperature, but the temperature was determined that had no significant effect on S. aureus survival (p > 0.05). Staphylococcal enterotoxin could not be detected in the cheeses during ripening. Staphylococcal enterotoxin (SE) B mRNA was detected in cheese samples on days 1, 15, and 30 of ripening by RT-PCR. The SEB mRNA expression levels had differed according to the storage temperature. Conclusion, significance and impact of study: This study showed that enterotoxin B producing S. aureus decreased in Turkish white pickled cheese stored at different temperatures and it could not produce enterotoxins, possibly due to factors such as type and nature of the cheese, and the conditions of production and activity of the starter culture.

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In vitro evaluation of caffeic acid derivatives as efflux pump inhibitor in Pseudomonas aeruginosa and Burkholderia pseudomallei

Noor Zawani Zakaria ; Norshima Abu Hasan ; Ahmad Fahim Mohd Dani ; Amirin Sadikun ; Pazilah Ibrahim ; Ezatul Ezleen Kamarulzaman ; Suriani Mohamad

Malaysian Journal of Microbiology.2017;13(4):334-342.

Aims: Bacterial pathogens such as Pseudomonas aeruginosa and Burkholderia pseudomallei are intrinsically resistant to many classes of antibiotics. This is not only due to the poor permeability of their outer membrane but also because of expression of multiple efflux pumps. A promising strategy to minimize the efflux of drugs by these pumps is the use of efflux pump inhibitors (EPIs). In this study, the potential of caffeic acid derivatives as EPIs in P. aeruginosa and B. pseudomallei were evaluated. Methodology and results: The potential of caffeic acid and its derivatives, i.e. chlorogenic acid, caffeic acid phenethyl ester (CAPE) and caffeic acid phenethyl amide (CAPA) to act as EPIs in P. aeruginosa and B. pseudomallei were assessed using the ethidium bromide (EtBr) accumulation and minimum inhibitory concentration (MIC) validation assays. Among the four test compounds, CAPE was found to significantly increased intracellular accumulation of EtBr in both P. aeruginosa and B. pseudomallei. An increase of 21.4% and 16.8% in cell fluorescence, over a 5-min time frame was observed in P. aeruginosa and B. pseudomallei respectively. Combination of CAPE with kanamycin significantly reduced MICs of this aminoglycoside by a factor of 8-fold in P. aeruginosa and 2-fold in B. pseudomallei. Combination of CAPE with gentamicin also led to a reduction of 4-fold MIC value of this antibiotic in B. pseudomallei. Conclusion, significance and impact of study: The in-vitro results suggest that CAPE has the potential to act as an EPI in P. aeruginosa and B. pseudomallei, thus improving the efficacy of aminoglycosides as antimicrobial agents.

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In vitro evaluation of cell adhesion and immunomodulatory properties of five Lactobacillus rhamnosus strains isolated from infants

Hlaing Hlaing Thaw ; Namfon Seubwongsa ; Rattiya Thongrung ; Marutpong Panya ; Viraphong Lulitanond

Malaysian Journal of Microbiology.2017;13(4):343-349.

Aims: Five Lactobacillus rhamnosus strains (UBU03, UBU06, UBU09, UBU34 and UBU37) with good in vitro probiotic properties, isolated from breast-fed infants, were evaluated for in vitro adhesion, competitive adhesion and immunomodulatory properties. Knowledge of such properties is important when considering specific circumstances when these strains might be used clinically. Methodology and results: The Caco-2 cell line was used for adhesion assays and for competitive adhesion assays against Escherichia coli O157:H7. Lactobacillus rhamnosus GG was used as the reference strain for adhesion assays. The immunomodulatory activities of the five strains were evaluated by determining the levels of the inflammatory cytokines IL-6, IL-12 and TNF-α, and of the immunoregulatory cytokine IL-10, produced by bacterial-activated THP-1 cells after 6, 12 and 24 h of stimulation. In the cell-adhesion assays, all five strains showed high adhesion properties. For UBU09, UBU34 and UBU37, adhesive capacity was higher than that of the reference strain. All strains except UBU03 showed the ability to inhibit adhesion of E. coli O157:H7 to Caco-2 cells. All strains induced IL-6 production but not IL-12 production. UBU03 and UBU09 could induce only one cytokine IL-6. UBU06 and UBU34 could each induce two (IL-6/IL-10 and IL-6/TNF-α, respectively). UBU37 could induce three cytokines (IL-6/TNF-α /IL-10). Conclusion, significance and impact of study: These five probiotic L. rhamnosus strains with high adhesion properties and with different in vitro cytokine induction profiles should be investigated further in different immunological conditions to identify appropriate circumstances for their clinical use.

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Shotgun metagenomic analysis of microbial communities in the surface waters of the Eastern South China Sea

Jessica Song ; Aazani Mujahid ; Po-Teen Lim ; Azizan Abu Samah ; Birgit Quack ; Klaus Pfeilsticker ; Sen-Lin Tang ; Elena Ivanova ; Moritz Müller

Malaysian Journal of Microbiology.2017;13(4):350-362.

Aims: The South China Sea (SCS) harbours a rich biodiversity. However, few studies have been published on its diverse communities, particularly its microbial counterparts. As key players behind many of the vital processes carried out in the ocean, microbes are the focus of this study, placing particular emphasis on community composition, structure, and function. Methodology and results: By employing next generation shotgun sequencing technologies (Illumina HiSeq2000), we assessed the taxonomic structure and functional diversity of the prokaryotic communities in surface waters collected from 3 representative sites in the Eastern SCS: Sarawak (Kuching), Sabah (Kota Kinabalu), and Philippines (Manila). Comparisons were undertaken to similar studies from coastal and open ocean environments. All 3 locations were dominated by members of the Proteobacteria (Alpha- and Gamma-) and Cyanobacteria (Synechococcus sp. and Prochlorococcus sp.). The highest proportion of Gammaproteobacteria was found in Sarawak, representing an approximate 20% of total sequences. Archaeal assemblages were made up largely of Euryarchaeota and unclassified sequences, while Crenarchaeota and Thaumarchaeota were present in much smaller proportions, except in the Philippines where Thaumarchaeota made up almost 40% of the entire taxa. Conclusion, significance and impact of study: The majority of the microbial communities adhered to a core set of functional genes across the different locations. However, differences existed particularly in Sarawak waters which are hypothesized to be due to local environmental parameters such as riverine influence. The results obtained from this study provide the first comparison of prokaryotic communities in the surface waters of the eastern SCS and will serve as a good platform for prospective studies in the field of environmental science.

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