In the course of our chemotaxonomic study of the liverworts growing in Madagascar, mastigophoric acid methyl ester, along with eleven known compounds were isolated from Mastigophora diclados. Isolated metabolites showed that the Malagasy Mastigophora is more related to the samples from Borneo and Japan than to the Taiwanese or Malaysian ones. The biosynthesis of the herbertane type sesquiterpenoids from Mastigophora diclados is suggested to be similar to those found in the genus Herbertus. The herbertane-type sesquiterpenoids were screened for Staphylococcus aureus strain inhibition.
Matched MeSH terms: Staphylococcus aureus/growth & development
Otitis externa (OE) is a frequently reported disorder in dogs associated with secondary infections by Staphylococcus, Pseudomonas and yeast pathogens. The presence of biofilms may play an important role in the resistance of otic pathogens to antimicrobial agents. Biofilm production of twenty Staphylococcus pseudintermedius and twenty Pseudomonas aeruginosa canine otic isolates was determined quantitatively using a microtiter plate assay, and each isolate was classified as a strong, moderate, weak or nonbiofilm producer. Minimum biofilm eradication concentration (MBEC) of two ionophores (narasin and monensin) and three adjuvants (N-acetylcysteine (NAC), Tris-EDTA and disodium EDTA) were investigated spectrophotometrically (OD570nm ) and quantitatively (CFU/ml) against selected Staphylococcus and Pseudomonas biofilm cultures. Concurrently, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of planktonic cultures were assessed. 16/20 of the S. pseudintermedius clinical isolates were weak biofilm producers. 19/20 P. aeruginosa clinical isolates produced biofilms and were distributed almost equally as weak, moderate and strong biofilm producers. While significant antibiofilm activity was observed, no MBEC was achieved with narasin or monensin. The MBEC for NAC ranged from 5,000-10,000 µg/ml and from 20,000-80,000 µg/ml against S. pseudintermedius and P. aeruginosa, respectively. Tris-EDTA eradicated P. aeruginosa biofilms at concentrations ranging from 6,000/1,900 to 12,000/3,800 µg/ml. The MBEC was up to 16-fold and eightfold higher than the MIC/MBC of NAC and Tris-EDTA, respectively. Disodium EDTA reduced biofilm growth of both strains at concentrations of 470 µg/ml and higher. It can be concluded that biofilm production is common in pathogens associated with canine OE. NAC and Tris-EDTA are effective antibiofilm agents in vitro that could be considered for the treatment of biofilm-associated OE in dogs.
Brucella melitensis is one of the leading zoonotic pathogens with significant economic implications in animal industry worldwide. Lipopolysaccharide, however, remains by far the major virulence with substantial role in diseases pathogenesis. Nonetheless, the effect of B. melitensis and its lipopolysaccharide on immunopathophysiological aspects largely remains an enigma. This study examines the effect of B.melitensis and its lipopolysaccharide on immunopathophysiological parameters following experimental infection using mouse model. Eighty four (n = 84) mice, BALB/c, both sexes with equal gender distribution and 6-8 weeks-old were randomly assigned into three groups. Group 1-2 (n = 72) were orally inoculated with 0.4 mL containing 109 CFU/mL of B. melitensis and its LPS, respectively. Group 3 (n = 12) was challenged orally with phosphate buffered saline and served as a control group. Animals were observed for clinical signs, haematological and histopathological analysis for a period of 24 days post-infection. We hereby report that B.melitensis infected group demonstrated significant clinical signs and histopathological changes than LPS infected group. However, both infected groups showed elevated levels of interleukins (IL-1β and IL-6) and antibody levels (IgM and IgG) with varying degrees of predominance in LPS infected group than B. melitensis infected group. For hormone analysis, low levels of progesterone, estradiol and testosterone were observed in both B. melitensis and LPS groups throughout the study period. Moreover, in B. melitensis infected group, the organism was re-isolated from the organs and tissues of gastrointestinal, respiratory and reproductive systems thereby confirming the infection and transmission dynamics. This report is the first detailed investigation comparing the infection progression and host responses in relation to the immunopathophysiological aspects in a mouse model after oral inoculation with B. melitensis and its lipopolysaccharide.
Chilli (Capsicum annum L.) plant is a high economic value vegetable in Malaysia, cultivated in soilless culture containers. In soilless culture, the adoption of small container sizes to optimize the volume of the growing substrate could potentially reduce the production cost, but will lead to a reduction of plant growth and yield. By understanding the physiological mechanism of the growth reduction, several potential measures could be adopted to improve yield under restricted root conditions. The mechanism of growth reduction of plants subjected to root restriction remains unclear. This study was conducted to determine the physiological mechanism of growth reduction of root-restricted chilli plants grown in polyvinyl-chloride (PVC) column of two different volumes, 2392 cm3(root-restricted) and 9570 cm3(control) in soilless culture. Root restriction affected plant growth, physiological process, and yield of chilli plants. Root restriction reduced the photosynthesis rate and photochemical activity of PSII, and increased relative chlorophyll content. Limited root growth in root restriction caused an accumulation of high levels of sucrose in the stem and suggested a transition of the stem as a major sink organ for photoassimilate. Growth reduction in root restriction was not related to limited carbohydrate production, but due to the low sink demand from the roots. Reduction of the total yield per plant about, 23% in root restriction was concomitant, with a slightly increased harvest index which reflected an increased photoassimilate partitioning to the fruit production and suggested more efficient fruits production in the given small plant size of root restriction.
In this study, the effects of limited and excess nitrate on biomass, lipid production, and fatty acid profile in Messastrum gracile SE-MC4 were determined. The expression of fatty acid desaturase genes, namely stearoyl-ACP desaturase (SAD), omega-6 fatty acid desaturase (ω-6 FAD), omega-3 fatty acid desaturase isoform 1 (ω-3 FADi1), and omega-3 fatty acid desaturase isoform 2 (ω-3 FADi2) was also assessed. It was found that nitrate limitation generally increased the total oil, α-linolenic acid (C18:3n3) and total polyunsaturated fatty acid (PUFA) contents in M. gracile. The reduction of nitrate concentration from 1.76 to 0.11 mM increased the total oil content significantly from 32.5 to 41.85% (dry weight). Palmitic (C16:0) and oleic (C18:1) acids as the predominant fatty acids in this microalgae constituted between 82 and 87% of the total oil content and were relatively consistent throughout all nitrate concentrations tested. The expression of SAD, ω-6 FAD, and ω-3 FADi2 genes increased under nitrate limitation, especially at 0.11 mM nitrate. The ω-3 FADi1 demonstrated a binary up-regulation pattern of expression under both nitrate-deficient (0.11 mM) and -excess (3.55 mM) conditions. Thus, findings from this study suggested that limited or excess nitrate could be used as part of a cultivation strategy to increase oil and PUFA content following media optimisation and more efficient culture methodology. Data obtained from the expression of desaturase genes would provide valuable insights into their roles under excess and limited nitrate conditions in M. gracile, potentially paving the way for future genetic modifications.
Candida albicans is a commensal yeast commonly found on the skin and in the body. However, in immunocompromised individuals, the fungi could cause local and systemic infections. The carbon source available plays an important role in the establishment of C. albicans infections. The fungi's ability to assimilate a variety of carbon sources plays a vital role in its colonization, and by extension, its fitness and pathogenicity, as it often inhabits niches that are glucose-limited but rich in alternative carbon sources. A difference in carbon sources affect the growth and mating of C. albicans, which contributes to its pathogenicity as proliferation helps the fungi colonize its environment. The carbon source also affects its metabolism and signaling pathways, which are integral parts of the fungi's fitness and pathogenicity. As a big percentage of the carbon assimilated by C. albicans goes to cell wall biogenesis, the availability of different carbon sources will result in cell walls with variations in rigidity, adhesion, and surface hydrophobicity. In addition to the biofilm formation of the fungi, the carbon source also influences whether the fungi grow in yeast- or mycelial-form. Both forms play different roles in C. albicans's infection process. A better understanding of the role of the carbon sources in C. albicans's pathogenicity would contribute to more effective treatment solutions for fungal infections.
Matched MeSH terms: Candida albicans/growth & development
Acanthamoeba castellanii is a free-living amoeba which can cause a blinding keratitis and fatal granulomatous amoebic encephalitis. The treatment of Acanthamoeba infections is challenging due to formation of cyst. Quinazolinones are medicinally important scaffold against parasitic diseases. A library of nineteen new 3-aryl-6,7-dimethoxyquinazolin-4(3H)-one derivatives was synthesized to evaluate their antiamoebic activity against Acanthamoeba castellanii. One-pot synthesis of 3-aryl-6,7-dimethoxyquinazolin-4(3H)-ones (1-19) was achieved by reaction of 2-amino-4,5-dimethoxybenzoic acid, trimethoxymethane, and different substituted anilines. These compounds were purified and characterized by standard chromatographic and spectroscopic techniques. Antiacanthamoebic activity of these compounds was determined by amoebicidal, encystation, excystation and host cell cytopathogenicity in vitro assays at concentrations of 50 and 100 μg/mL. The IC50 was found to be between 100 and 50 μg/mL for all the compounds except compound 5 which did not exhibit amoebicidal effects at these concentrations. Furthermore, lactate dehydrogenase assay was also performed to evaluate the in vitro cytotoxicity of these compounds against human keratinocyte (HaCaT) cells. The results revealed that eighteen out of nineteen derivatives of quinazolinones significantly decreased the viability of A. castellanii. Furthermore, eighteen out of nineteen tested compounds inhibited the encystation and excystation, as well as significantly reduced the A. castellanii-mediated cytopathogenicity against human cells. Interestingly, while tested against human normal cell line HaCaT keratinocytes, all compounds did not exhibit any overt cytotoxicity. Furthermore, a detailed structure-activity relationship is also studied to optimize the most potent hit from these synthetic compounds. This report presents several potential lead compounds belonging to 3-aryl-6,7-dimethoxyquinazolin-4(3H)-one derivatives for drug discovery against infections caused by Acanthamoeba castellanii.
Matched MeSH terms: Acanthamoeba castellanii/growth & development
Anthracnose is a fungal disease causing major losses in crop production. Chemical fungicides widely used in crop plantations to combat fungal infections can be a threat to the environment and humans in the long term. Recently, biofungicides have gained much interest as an alternative to chemical fungicides due to their environmentally friendly nature. Biofungicide products in powder form can be formulated using the freeze-drying technique to provide convenient storage. Protective agent formulation is needed in maintaining the optimal viable cells of biofungicide products. In this study, 8.10 log colony-forming unit (CFU)/mL was the highest cell viability of Paenibacillus polymyxa Kp10 at 22 h during incubation. The effects of several selected protective agents on the viability of P. polymyxa Kp10 after freeze-drying were studied. Response surface methodology (RSM) was used for optimizing formulation for the protective agents. The combination of lactose (10% w/v), skim milk (20% w/v), and sucrose (27.5% w/v) was found to be suitable for preserving P. polymyxa Kp10 during freeze-drying. Further, P. polymyxa Kp10 demonstrated the ability to inhibit fungal pathogens, Colletotrichum truncatum and C. gloeosporioides, at 60.18% and 66.52% of inhibition of radial growth, respectively.
Species of fungi belonging to the order Mucorales can be found everywhere in the environment. Gilbertella persicaria, which belongs to this order, have often been isolated from fruits and in water systems. However, there has been no report of isolation of this fungus from human samples. During a gut mycobiome study, from the Segamat community, Gilbertella persicaria was isolated from a human fecal sample and was characterized through a series of morphological assessment, biochemical tests, and molecular techniques. The isolate produced a white velvety surface that turned grayish after 24 h. Although no biofilm production was observed, the results indicated that the isolate could form calcium oxalate crystals, produced urease, and was resistant to low pH. The isolate was sensitive to amphotericin but resistant to voriconazole and itraconazole. The features of this fungus that could help in its survival in the human gut are also discussed.
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.
Matched MeSH terms: Methicillin-Resistant Staphylococcus aureus/growth & development
Paenibacillus durus strain ATCC 35681T is a Gram-positive diazotroph that displayed capability of fixing nitrogen even in the presence of nitrate or ammonium. However, the nitrogen fixation activity was detected only at day 1 of growth when cultured in liquid nitrogen-enriched medium. The transcripts of all the nifH homologues were present throughout the 9-day study. When grown in nitrogen-depleted medium, nitrogenase activities occurred from day 1 until day 6 and the nifH transcripts were also present during the course of the study albeit at different levels. In both studies, the absence of nitrogen fixation activity regardless of the presence of the nifH transcripts raised the possibility of a post-transcriptional or post-translational regulation of the system. A putative SigA box sequence was found upstream of the transcription start site of nifB1, the first gene in the major nitrogen fixation cluster. The upstream region of nifB2 showed a promoter recognizable by SigE, a sigma factor normally involved in sporulation.
Matched MeSH terms: Paenibacillus/growth & development
Mangrove-dwelling microalgae are well adapted to frequent encounters of salinity fluctuations across their various growth phases but are lesser studied. The current study explored the adaptive changes (in terms of biomass, oil content and fatty acid composition) of mangrove-isolated C. vulgaris UMT-M1 cultured under different salinity levels (5, 10, 15, 20, 30 ppt). The highest total oil content was recorded in cultures at 15 ppt salinity (63.5% of dry weight) with uncompromised biomass productivity, thus highlighting the 'trigger-threshold' for oil accumulation in C. vulgaris UMT-M1. Subsequently, C. vulgaris UMT-M1 was further assessed across different growth phases under 15 ppt. The various short, medium and long-chain fatty acids (particularly C20:0), coupled with a high level of C18:3n3 PUFA reported at early exponential phase represents their physiological importance during rapid cell growth. Accumulation of C18:1 and C18:2 at stationary growth phase across all salinities was seen as cells accumulating substrate for C18:3n3 should the cells anticipate a move from stationary phase into new growth phase. This study sheds some light on the possibility of 'triggered' oil accumulation with uninterrupted growth and the participation of various fatty acid types upon salinity mitigation in a mangrove-dwelling microalgae.
Matched MeSH terms: Chlorella vulgaris/growth & development
Bioprospecting for biodiesel potential in microalgae primarily involves a few model species of microalgae and rarely on non-model microalgae species. Therefore, the present study determined changes in physiology, oil accumulation, fatty acid composition and biodiesel properties of a non-model microalga Messastrum gracile SE-MC4 in response to 12 continuous days of nitrate-starve (NS) and nitrate-replete (NR) conditions respectively. Under NS, the highest oil content (57.9%) was achieved despite reductions in chlorophyll content, biomass productivity and lipid productivity. However, under both NS and NR, palmitic acid and oleic acid remained as dominant fatty acids thus suggesting high potential of M. gracile for biodiesel feedstock consideration. Biodiesel properties analysis returned high values of cetane number (CN 61.9-64.4) and degree of unsaturation (DU 45.3-57.4) in both treatments. The current findings show the possibility of a non-model microalga to inherit superior ability over model species in oil accumulation for biodiesel development.
Matched MeSH terms: Microalgae/growth & development
Rapid immune plaque assays have been developed to quantify biohazard level 4 agents Hendra and Nipah viruses and detect neutralising antibodies to both viruses. The methods rely on the fact that both viruses rapidly generate large syncytia in monolayers of Vero cells within 24 h and that monospecific antiserum to the Hendra virus phosphoprotein (P) detects that protein in both Hendra and Nipah virus-induced syncytia after methanol fixation of virus-infected cells. The P protein is a constituent of the ribonucleoprotein core of the viruses and a component of the viral RNA-dependent RNA polymerase and is made in significant amounts in infected cells. In the immune plaque assay, anti-P antibody is localised by an alkaline phosphatase-linked second antibody and the Western blot substrates 5-bromo-4-chloro-3-indolyl phosphate and p-nitro blue tetrazolium. A modification of the rapid immune plaque assay was also used to detect antibodies to Nipah virus in a panel of porcine field sera from Malaysia and the results showed good agreement between the immune plaque assay and a traditional serum neutralisation test. After methanol fixation, plates can be stored for up to 7 months and may be used in the immune plaque assay to complement the enzyme-linked immunosorbent assay screening of sera for antibodies to Nipah virus. At present, all enzyme-linked immunosorbent assay positive sera are subject to confirmatory serum neutralisation tests. Use of the immune plaque assay may reduce the number of sera requiring confirmatory neutralisation testing for Nipah virus antibodies under biohazard level 4 conditions by identifying those that generate false positive in the enzyme-linked immunosorbent assay.
Matched MeSH terms: Paramyxovirinae/growth & development
Four subpopulations of a Plutella xylostella (L.) strain from Malaysia (F(4) to F(8)) were selected with Bacillus thuringiensis subsp. kurstaki HD-1, Bacillus thuringiensis subsp. aizawai, Cry1Ab, and Cry1Ac, respectively, while a fifth subpopulation was left as unselected (UNSEL-MEL). Bioassays at F(9) found that selection with Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai gave resistance ratios of >95, 10, 7, and 3, respectively, compared with UNSEL-MEL (>10,500, 500, >100, and 26, respectively, compared with a susceptible population, ROTH). Resistance to Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai in UNSEL-MEL declined significantly by F(9). The Cry1Ac-selected population showed very little cross-resistance to Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai (5-, 1-, and 4-fold compared with UNSEL-MEL), whereas the Cry1Ab-, B. thuringiensis subsp. kurstaki-, and B. thuringiensis subsp. aizawai-selected populations showed high cross-resistance to Cry1Ac (60-, 100-, and 70-fold). The Cry1Ac-selected population was reselected (F(9) to F(13)) to give a resistance ratio of >2,400 compared with UNSEL-MEL. Binding studies with (125)I-labeled Cry1Ab and Cry1Ac revealed complete lack of binding to brush border membrane vesicles prepared from Cry1Ac-selected larvae (F(15)). Binding was also reduced, although less drastically, in the revertant population, which indicates that a modification in the common binding site of these two toxins was involved in the resistance mechanism in the original population. Reciprocal genetic crosses between Cry1Ac-reselected and ROTH insects indicated that resistance was autosomal and showed incomplete dominance. At the highest dose of Cry1Ac tested, resistance was recessive while at the lowest dose it was almost completely dominant. The F(2) progeny from a backcross of F(1) progeny with ROTH was tested with a concentration of Cry1Ac which would kill 100% of ROTH moths. Eight of the 12 families tested had 60 to 90% mortality, which indicated that more than one allele on separate loci was responsible for resistance to Cry1Ac.
Teeth are the most durable structures in the human body. The timing and sequence of their development, as contained in dental development charts, have been used as valid criteria for age determination. The third molars however are the last teeth to erupt and are regarded as the most variable in the dentition. Age estimation in a legal context, using developing third molars must be carefully applied otherwise justice may miscarry. A case of wrongful use of the technique is presented here.
Anthelmintic resistance in nematode parasites of sheep and goats on a government farm in north Malaysia was monitored over a 3-year period (1997-2000). The faecal egg count reduction test (FECRT) was conducted on young sheep at the beginning and end of this period. Changes in management, designed to reduce the selection pressure for the development of anthelmintic resistance, were also implemented during this time. By far the most important parasite problem was Haemonchus contortus. In 1997, this nematode was found to be resistant to levamisole, with suspected resistance to closantel and moxidectin. However, when the FECRT was repeated 3 years later, its resistance status had become much more severe, with resistance to benzimidazole, levamisole and ivermectin, and suspected resistance to moxidectin. This rapid evolution to multiple anthelmintic resistance is a major concern that needs to be arrested. There is an urgent need to evaluate other control strategies that incorporate livestock management, the 'smart' use of drugs and non-chemotherapeutic approaches, such as biological control agents.
Immunomagnetic beads-PCR (IM-PCR), positively-charged virosorb filters (F), or a combination of both methods (F-IM-PCR) were used to capture, concentrate and rapidly detect hepatitis A virus (HAV) in samples of lettuce and strawberries experimentally contaminated. Direct reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the collected HAV-beads complex showed a detection limit of 0.5 plaque forming units (PFU) of the virus present in 1-ml of wash solution from the produce, which was several hundred-fold more sensitive than that demonstrated by RT-PCR. In separate trials, virus-containing wash solutions from the produce were passed through the filters and the captured virus was eluted with 10 ml volumes of 1% beef extract. Of the 62% filter-captured HAV, an average of 34.8% was eluted by the 1% beef extract. PCR amplification of 2 microl from this eluate failed to produce a clear positive band signal. As little as 10 PFU, present on each piece of the lettuce or strawberry, was detectable by the F-IM-PCR, which was almost 20 times less sensitive than the detection limit of 0.5 PFU by the IM-PCR. However, considering the large volumes (< or =50 ml) used in the F-IM-PCR, the sensitivity of detection could be much greater than that of the IM-PCR, which was restricted to < or =20 ml volumes. These data indicate that the F-IM-PCR method provides the potential for a greater sensitivity of detection than the IM-PCR, since low levels of virus could be detected from large volumes of sample than possible by the IM-PCR method. Although positively-charged filters captured a greater amount of virus than both the IM-PCR and F-IM-PCR methods, direct PCR amplification from beef extract eluates was not successful in detecting HAV from produce.
Matched MeSH terms: Hepatovirus/growth & development
Enterotoxigenic Bacillus cereus was detected in cooked foods (17), rice noodles (3), wet wheat noodles (2), dry wheat noodles (10), spices (8), grains (4), legumes (11) and legume products (3). One hundred ninety-four (42.3%), 70 (15.3%) and 23 (5.2%) of the 459 presumptive B. cereus colonies isolated from PEMBA agar were identified as B. cereus, Bacillus thuringiensis and B. mycoides, respectively. B. cereus isolates were examined for growth temperature, pH profile and enterotoxin production using both TECRA-VIA and BCET-RPLA kits. One hundred seventy-eight (91.8%) and 164 (84%) of the strains were enterotoxigenic as determined using TECRA-VIA and BCET-RPLA, respectively. Eighty-two (50%) of the enterotoxigenic strains were capable of growing at 5 degrees C, and 142 (86.6%) grew at 7 degrees C within 7 days of incubation. The enterotoxigenic strains did not grow at pH 4.0 but 69 (42.0%) of the strains were able to grow at pH 4.5 within 7 days at 37 degrees C. The isolates were resistant to ampicillin (98.8%), cloxallin (100%) and tetracycline (61.0%), and susceptible to chloroamphenicol (87%), erythromycin (77.4%), gentamycin (100%) and streptomycin (98.7%).
Matched MeSH terms: Bacillus cereus/growth & development