Endophytic bacteria, such as Streptomyces, have the potential to act as a source for novel bioactive molecules with medicinal properties. The present study was aimed at assessing the antimalarial activity of crude extract isolated from various strains of actinobacteria living endophytically in some Malaysian medicinal plants. Using the four day suppression test method on male ICR strain mice, compounds produced from three strains of Streptomyces (SUK8, SUK10, and SUK27) were tested in vivo against Plasmodium berghei PZZ1/100 in an antimalarial screen using crude extracts at four different concentrations. One of these extracts, isolated from Streptomyces SUK10 obtained from the bark of Shorea ovalis tree, showed inhibition of the test organism and was further tested against P. berghei-infected mice for antimalarial activity at different concentrations. There was a positive relationship between the survival of the infected mouse group treated with 50 µg/kg body weight (bw) of ethyl acetate-SUK10 crude extract and the ability to inhibit the parasites growth. The parasite inhibition percentage for this group showed that 50% of the mice survived for more than 90 days after infection with the parasite. The nucleotide sequence and phylogenetic tree suggested that Streptomyces SUK10 may constitute a new species within the Streptomyces genus. As part of the drug discovery process, these promising finding may contribute to the medicinal and pharmaceutical field for malarial treatment.
The contamination of food and feed by Aspergillus has become a global issue with a significant worldwide economic impact. The growth of Aspergillus is unfavourable to the development of food and feed industries, where the problems happen mostly due to the presence of mycotoxins, which is a toxic metabolite secreted by most Aspergillus groups. Moreover, fungi can produce spores that cause diseases, such as allergies and asthma, especially to human beings. High temperature, high moisture, retarded crops, and poor food storage conditions encourage the growth of mold, as well as the development of mycotoxins. A variety of chemical, biological, and physical strategies have been developed to control the production of mycotoxins. A biological approach, using a mixed culture comprised of Saccharomyces cerevisiae and Lactobacillus rhamnosus resulted in the inhibition of the growth of fungi when inoculated into fermented food. The results reveal that the mixed culture has a higher potential (37.08%) to inhibit the growth of Aspergillus flavus (producer of Aflatoxin) compared to either single culture, L. rhamnosus NRRL B-442 and S. cerevisiae, which inhibit the growth by 63.07% and 64.24%, respectively.
This study was undertaken to investigate the influence of culture conditions and medium components on production of antibacterial compounds by Serratia sp. WPRA3 (JX020764) which was isolated from marine water of Port Dickson, Malaysia. Biochemical, morphological, and molecular characteristics suggested that the isolate is a new candidate of the Serratia sp. The isolate showed strong antimicrobial activity against fungi, Gram-negative and Gram-positive bacteria. This bacterium exhibited optimum antibacterial compounds production at 28°C, pH 7 and 200 rev/min aeration during 72 h of incubation period. Highest antibacterial activity was obtained when sodium chloride (2%), yeast extract (0.5%), and glucose concentration (0.75%) were used as salt, nitrogen, and carbon sources respectively. Different active fractions were obtained by Thin-Layer Chromatography (TLC) and Flash Column Chromatography (FCC) from ethyl acetate crude extracts namely OCE and RCE in different culture conditions, OCE (pH 5, 200 rev/min) and RCE (pH 7/without aeration). In conclusion, the results suggested different culture conditions have a significant impact on the types of secondary metabolites produced by the bacterium.
The effect of Leucaena leucocephala hybrid-Bahru (LLB), which contains a high concentration of condensed tannins, on cellulolytic rumen fungal population in goats was investigated using real-time PCR. The fungal population in goats fed LLB was inhibited during the first 10 days of feeding, but after 15 days of feeding, there was a tremendous increase of fungal population (157.0 μg/ml), which was about fourfold more than that in control goats (39.7 μg/ml). However, after this period, the fungal population decreased continuously, and at 30 days of feeding, the fungal population (50.6 μg/ml) was not significantly different from that in control goats (55.4 μg/ml).
This study examined the effect of host extract in the culture medium on anti-candidal activity of Phomopsis sp. ED2, previously isolated from the medicinal herb Orthosiphon stamineus Benth. Interestingly, upon addition of aqueous host extract to the culture medium, the ethyl acetate extract prepared from fermentative broth exhibited moderate anti-candidal activity in a disc diffusion assay. The minimal inhibitory concentration of this extract was 62.5 μg/ml and it only exhibited fungistatic activity against C. albicans. In the time-kill study, a 50% growth reduction of C. albicans was observed at 31.4 h for extract from the culture incorporating host extract. In the bioautography assay, only one single spot (Rf 0.59) developed from the extract exhibited anti-candidal activity. A spot with the a similar Rf was not detected for the crude extract from YES broth without host extract. This indicated that the terpenoid anti-candidal compound was only produced when the host extract was introduced into the medium. The study concluded that the incorporation of aqueous extract of the host plant into the culture medium significantly enhanced the anti-candidal activity of Phomopsis sp. ED2.
Endophytic bacteria (Pseudomonas aeruginosa UPMP3 and Burkholderia cepacia UMPB3), isolated from within roots of oil palm (Elaeis guineensis Jacq.) were tested for their presymbiotic effects on two arbuscular mcorrhizal fungi, Glomus intraradices UT126 and Glomus clarum BR152B). These endophytic bacteria were also tested for antagonistic effects on Ganoderma boninense PER 71, a white wood rot fungal pathogen that causes a serious disease in oil palm. Spore germination and hyphal length of each arbuscular mycorrhizal fungal (AMF) pairing with endophytic bacteria was found to be significantly higher than spores plated in the absence of bacteria. Scanning electron microscopy (SEM) showed that the endophytic bacteria were scattered, resting or embedded on the surface hyaline layer or on the degraded walls of AMF spores, possibly feeding on the outer hyaline spore wall. The antagonistic effect of the endophytic bacteria was expressed as severe morphological abnormalities in the hyphal structures of G. boninense PER 71. The effects of the endophytic bacteria on G. boninense PER 71 hyphal structures were observed clearly under SEM. Severe inter-twisting, distortion, lysis and shriveling of the hyphal structures were observed. This study found that the effect of endophytic bacteria on G. intraradices UT126 and G. clarum BR152B resembled that of a mycorrhiza helper bacteria (MHB) association because the association significantly promoted AMF spore germination and hyphal length. However, the endophytic bacteria were extremely damaging to G. boninense PER 71.
Molecular diversity of rumen archaeal populations from bovine rumen fluid incubated with or without condensed tannins was investigated using 16S rRNA gene libraries. The predominant order of rumen archaea in the 16S rRNA gene libraries of the control and condensed tannins treatment was found to belong to a novel group of rumen archaea that is distantly related to the order Thermoplasmatales, with 59.5% (15 phylotypes) and 81.43% (21 phylotypes) of the total clones from the control and treatment clone libraries, respectively. The 16S rRNA gene library of the control was found to have higher proportions of methanogens from the orders Methanomicrobiales (32%) and Methanobacteriales (8.5%) as compared to those found in the condensed tannins treatment clone library in both orders (16.88% and 1.68% respectively). The phylotype distributed in the order Methanosarcinales was only found in the control clone library. The study indicated that condensed tannins could alter the diversity of bovine rumen methanogens.
In this study, an organic solvent tolerant bacterial strain was isolated. This strain was identified as Pseudomonas sp. strain S5, and was shown to degrade BTEX (Benzene, Toluene, Ethyl-Benzene, and Xylene). Strain S5 generates an organic solvent-tolerant lipase in the late logarithmic phase of growth. Maximum lipase production was exhibited when peptone was utilized as the sole nitrogen source. Addition of any of the selected carbon sources to the medium resulted in a significant reduction of enzyme production. Lower lipase generation was noted when an inorganic nitrogen source was used as the sole nitrogen source. This bacterium hydrolyzed all tested triglycerides and the highest levels of production were observed when olive oil was used as a natural triglyceride. Basal medium containing Tween 60 enhanced lipase production to the most significant degree. The absence of magnesium ions (Mg2+) in the basal medium was also shown to stimulate lipase production. Meanwhile, an alkaline earth metal ion, Na+, was found to stimulate the production of S5 lipase.
In this study, the growth kinetics of Lactobacillus rhamnosus and lactic acid production in continuous culture were assessed at a range of dilution rates (0.05 h(-1) to 0.40 h(-1)) using a 2 L stirred tank fermenter with a working volume of 600 ml. Unstructured models, predicated on the Monod and Luedeking-Piret equations, were employed to simulate the growth of the bacterium, glucose consumption, and lactic acid production at different dilution rates in continuous cultures. The maximum specific growth rate of L. rhamnosus, mu-max, was estimated at 0.40 h(-1), and the Monod cell growth saturation constant, Ks, at approximately 0.25 g/L. Maximum cell viability (1.3 x 10(10) CFU/ml) was achieved in the dilution rate range of D = 0.28 h(-1) to 0.35 h(-1). Both maximum viable cell yield and productivity were achieved at D = 0.35 h(-1). The continuous cultivation of L. rhamnosus at D = 0.35 h(-1) resulted in substantial improvements in cell productivity, of 267% (viable cell count) that achieved via batch cultivation.
This study examined the capacity of immobilized bacteria to degrade petroleum hydrocarbons. A mixture of hydrocarbon-degrading bacterial strains was immobilized in alginate and incubated in crude oil-contaminated artificial seawater (ASW). Analysis of hydrocarbon residues following a 30-day incubation period demonstrated that the biodegradation capacity of the microorganisms was not compromised by the immobilization. Removal of n-alkanes was similar in immobilized cells and control cells. To test reusability, the immobilized bacteria were incubated for sequential increments of 30 days. No decline in biodegradation capacity of the immobilized consortium of bacterial cells was noted over its repeated use. We conclude that immobilized hydrocarbon-degrading bacteria represent a promising application in the bioremediation of hydrocarbon-contaminated areas.
In this paper, we investigated the development of a simplified and rapid primary capture step for the recovery of M13 bacteriophage from particulate-containing feedstock. M13 bacteriophage, carrying an insert, was propagated and subsequently purified by the application of both conventional multiple steps and expanded bed anion exchange chromatography. In the conventional method, precipitation was conducted with PEG/NaCl, and centrifugation was also performed. In the single step expanded bed anion exchange adsorption, UpFront FastLine 20 (20 mm i.d.) from UpFront Chromatography was used as the contactor, while 54 ml (Ho = 15 cm) of STREAMLINE DEAE (rho = 1.2 g/cm3) from Amersham Pharmacia Biotechnology was used as the anion exchanger. The performance of the two methods were evaluated, analysed, and compared. It was demonstrated that the purification of the M13 bacteriophage, using expanded bed anion exchange adsorption, yielded the higher recovery percentage, at 82.86%. The conventional multiple step method yielded the lower recovery percentage, 36.07%. The generic application of this integrated technique has also been assessed.
A single chain variable fragment (scFv) specific towards B. pseudomallei exotoxin had previously been generated from an existing hybridoma cell line (6E6AF83B) and cloned into the phage display vector pComb3H. In this study, the scFv was subcloned into the pComb3X vector to facilitate the detection and purification of expressed antibodies. Detection was facilitated by the presence of a hemagglutinin (HA) tag, and purification was facilitated by the presence of a histidine tag. The culture was grown at 30 degrees C until log phase was achieved and then induced with 1 mM IPTG in the absence of any additional carbon source. Induction was continued at 30 degrees C for five h. The scFv was discerned by dual processes-direct enzyme-linked immunosorbent assays (ELISA), and Western blotting. When compared to E. coli strains ER2537 and HB2151, scFv expression was observed to be highest in the E. coli strain Top10F'. The expressed scFv protein was purified via nickel-mediated affinity chromatography and results indicated that two proteins a 52 kDa protein, and a 30 kDa protein were co-purified. These antibodies, when blotted against immobilized exotoxin, exhibited significant specificity towards the exotoxin, compared to other B. pseudomallei antigens. Thus, these antibodies should serve as suitable reagents for future affinity purification of the exotoxin.
In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with Ni2+ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was 1.26% and 5.56%, respectively. It was demonstrated that EBA achieved the highest final protein yield of 9.6% with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.
In this study, we assessed the susceptibility of 12 Lactobacillus strains, all of which had been isolated from the gastrointestinal tracts of chicken, to three antibiotics (chloramphenicol, erythromycin and tetracycline) used commonly as selective markers in transformation studies of lactic acid bacteria. Among these strains, 17%, 58%, and 25% were found to exhibit a high degree of resistance to 200 microg/ml of tetracycline, erythromycin, and chloramphenicol, respectively. Seven of the 12 Lactobacillus strains exhibiting resistance to at least 50 microg/ml of chloramphenicol or erythromycin, and five strains exhibiting resistance to at least 50 microg/ml of tetracycline, were subsequently subjected to plasmid curing with chemical curing agents, such as novobiocin, acriflavin, SDS, and ethidium bromide. In no cases did the antibiotic resistance of these strains prove to be curable, with the exception of the erythromycin resistance exhibited by five Lactobacillus strains (L. acidophilus I16 and I26, L. fermentum I24 and C17, and L. brevis C10). Analysis of the plasmid profiles of these five cured derivatives revealed that all of the derivatives, except for L. acidophilus I16, possessed profiles similar to those of wild-type strains. The curing of L. acidophilus I16 was accompanied by the loss of 4.4 kb, 6.1 kb, and 11.5 kb plasmids.
Antiplasmodial nortriterpenes with 3,4-seco-27-norlanostane skeletons, almost entirely obtained from fruiting bodies, represent the main evidential source for bioactive secondary metabolites derived from a relatively unexplored phytopathogenic fungus, Ganoderma boninense. Currently lacking is convincing evidence for antimicrobial secondary metabolites in this pathogen, excluding that obtained from commonly observed phytochemicals in the plants. Herein, we aimed to demonstrate an efficient analytical approach for the production of antibacterial secondary metabolites using the mycelial extract of G. boninense. Three experimental cultures were prepared from fruiting bodies (GBFB), mycelium cultured on potato dextrose agar (PDA) media (GBMA), and liquid broth (GBMB). Through solvent extraction, culture type-dependent phytochemical distributions were diversely exhibited. Water-extracted GBMB produced the highest yield (31.21 ± 0.61%, p < 0.05), but both GBFB and GBMA elicited remarkably higher yields than GBMB when polar-organic solvent extraction was employed. Greater quantities of phytochemicals were also obtained from GBFB and GBMA, in sharp contrast to those gleaned from GBMB. However, the highest antibacterial activity was observed in chloroform-extracted GBMA against all tested bacteria. From liquid-liquid extractions (LLE), it was seen that mycelia extraction with combined chloroform-methanol-water at a ratio of 1:1:1 was superior at detecting antibacterial activities with the most significant quantities of antibacterial compounds. The data demonstrate a novel means of assessing antibacterial compounds with mycelia by LLE which avoids the shortcomings of standardized methodologies. Additionally, the antibacterial extract from the mycelia demonstrate that previously unknown bioactive secondary metabolites of the less studied subsets of Ganoderma may serve as active and potent antimicrobial compounds.
In this study, bacterial strains Ha5T, Ta1, and Jb2 were isolated from different colonies of weaver ant Oecophylla smaragdina. They were identified as bacterial symbionts of the ant belonging to family Acetobacteraceae and were distinguished as different strains based on distinctive random-amplified polymorphic DNA (RAPD) fingerprints. Cells of these bacterial strains were Gram-negative, rod-shaped, aerobic, non-motile, catalase-positive and oxidase-negative. They were able to grow at 15-37°C (optimum, 28-30°C) and in the presence of 0-1.5% (w/v) NaCl (optimum 0%). Their predominant cellular fatty acids were C18:1ω7c, C16:0, C19:0ω8c cyclo, C14:0, and C16:0 2-OH. Strains Ha5T, Ta1, and Jb2 shared highest 16S rRNA gene sequence similarity (94.56-94.63%) with Neokomagataea tanensis NBRC106556T of family Acetobacteraceae. Both 16S rRNA gene sequence-based phylogenetic analysis and core gene-based phylogenomic analysis placed them in a distinct lineage in family Acetobacteraceae. These bacterial strains shared higher than species level thresholds in multiple overall genome-relatedness indices which indicated that they belonged to the same species. In addition, they did not belong to any of the current taxa of Acetobacteraceae as they had low pairwise average nucleotide identity (< 71%), in silico DNA-DNA hybridization (< 38%) and average amino acid identity (< 67%) values with all the type members of the family. Based on these results, bacterial strains Ha5T, Ta1, and Jb2 represent a novel species of a novel genus in family Acetobacteaceae, for which we propose the name Oecophyllibacter saccharovorans gen. nov. sp. nov., and strain Ha5T as the type strain.
Lactobacillus plantarum is a non-gas-producing lactic acid bacterium that is generally regarded as safe (GRAS) with Qualified Presumption of Safety (QPS) status. Although traditionally used for dairy, meat and vegetable fermentation, L. plantarum is gaining increasing significance as a probiotic. With the newly acclaimed gut-heart-brain axis, strains of L. plantarum have proven to be a valuable species for the development of probiotics, with various beneficial effects on gut health, metabolic disorders and brain health. In this review, the classification and taxonomy, and the relation of these with safety aspects are introduced. Characteristics of L. plantarum to fulfill the criteria as a probiotic are discussed. Emphasis are also given to the beneficial functions of L. plantarum in gut disorders such as inflammatory bowel diseases, metabolic syndromes, dyslipidemia, hypercholesteromia, obesity, and diabetes, and brain health aspects involving psychological disorders.
Getah virus (GETV), which was first isolated in Malaysia in 1955, and Sagiyama virus (SAGV), isolated in Japan in 1956, are members of the genus Alphavirus in the family Togaviridae. It is a consensus view that SAGV is a variant of GETV. In the present study, we determined the complete sequences of the prototype GETV MM2021 and SAGV M6-Mag132 genomic RNA extracted from plaque-purified viruses. The MM2021 genome was 11,692 nucleotides (nt) in length in the absence of 3' poly(A) tail, and the length of M6-Mag132 genome was 11,698 nt. Through sequence alignment of MM2021 and M6-Mag132, we located all the amino acid differences between these two strains, which were scattered in all the encoded proteins. Subsequently, we validated the close evolutionary relationship between GETV and SAGV by constructing phylogenetic trees based on either complete genomes or structural genomes. We eventually analyzed the growth kinetics of GETV and SAGV as well as other representative alphaviruses in various mammalian and insect cell lines. It was shown that human-oriented cell lines such as HEK-293T and Hela cells were relatively resistant to GETV and SAGV infection due to absence of proviral factors or species-specific barrier. On the other hand, both GETV and SAGV replicated efficiently in non-human cell lines. Our results provide essential genetic information for future epidemiological surveillance on Alphaviruses and lay the foundation for developing effective interventions against GETV and SAGV.
Ganoderma boninense is the causal agent of a devastating disease affecting oil palm in Southeast Asian countries. Basal stem rot (BSR) disease slowly rots the base of palms, which radically reduces productive lifespan of this lucrative crop. Previous reports have indicated the successful use of Trichoderma as biological control agent (BCA) against G. boninense and isolate T. virens 7b was selected based on its initial screening. This study attempts to decipher the mechanisms responsible for the inhibition of G. boninense by identifying and characterizing the chemical compounds as well as the physical mechanisms by T. virens 7b. Hexane extract of the isolate gave 62.60% ± 6.41 inhibition against G. boninense and observation under scanning electron microscope (SEM) detected severe mycelial deformation of the pathogen at the region of inhibition. Similar mycelia deformation of G. boninense was observed with a fungicide treatment, Benlate(®) indicating comparable fungicidal effect by T. virens 7b. Fraction 4 and 5 of hexane active fractions through preparative thin layer chromatography (P-TLC) was identified giving the best inhibition of the pathogen. These fractions comprised of ketones, alcohols, aldehydes, lactones, sesquiterpenes, monoterpenes, sulphides, and free fatty acids profiled through gas chromatography mass spectrometry detector (GC/MSD). A novel antifungal compound discovery of phenylethyl alcohol (PEA) by T. virens 7b is reported through this study. T. virens 7b also proved to be an active siderophore producer through chrome azurol S (CAS) agar assay. The study demonstrated the possible mechanisms involved and responsible in the successful inhibition of G. boninense.
There have been relatively few studies which support a link between Ganoderma boninense, a phytopathogenic fungus that is particularly cytotoxic and pathogenic to plant tissues and roots, and antimicrobial compounds. We previously observed that liquid-liquid extraction (LLE) using chloroformmethanol-water at a ratio (1:1:1) was superior at detecting antibacterial activities and significant quantities of antibacterial compounds. Herein, we demonstrate that antibacterial secondary metabolites are produced from G. boninense mycelia. Antibacterial compounds were monitored in concurrent biochemical and biophysical experiments. The combined methods included high performance thin-layer chromatography (HPTLC), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR) spectroscopy. The antibacterial compounds derived from mycelia with chloroform-methanol extraction through LLE were isolated via a gradient solvent elution system using HPTLC. The antibacterial activity of the isolated compounds was observed to be the most potent against Staphylococcus aureus ATCC 25923 and multidrug-resistant S. aureus NCTC 11939. GC-MS, HPLC, and FTIR analysis confirmed two antibacterial compounds, which were identified as 4,4,14α-trimethylcholestane (m/z = 414.75; lanostane, C30H54) and ergosta-5,7,22-trien-3β-ol (m/z = 396.65; ergosterol, C28H44O). With the aid of spectroscopic evaluations, ganoboninketal (m/z = 498.66, C30H42O6), which belongs to the 3,4-seco-27-norlanostane triterpene family, was additionally characterized by 2D-NMR analysis. Despite the lack of antibacterial potential exhibited by lanostane; both ergosterol and ganoboninketal displayed significant antibacterial activities against bacterial pathogens. Results provide evidence for the existence of bioactive compounds in the mycelia of the relatively unexplored phytopathogenic G. boninense, together with a robust method for estimating the corresponding potent antibacterial secondary metabolites.