Live eels and processed fish products from Malaysia are routinely checked for microbial pathogens before export to Japan. The eels and water from the ponds are screened for Vibrio cholerae and Salmonella spp, whereas the processed fish products are tested for microbial contamination (aerobic plate count), coliforms, E. coil and Vibrio cholerae. Results showed that live eels and water samples were negative for Vibrio cholerae but Salmonella spp were isolated occasionally. Various types of processed fish products had counts below 1.0 x 10(5) whilst coliforms, E. coli and Vibrio cholerae were absent. Records available showed that procedures involved in the production and transportation of live eel, preparation and processing of fish products have resulted in relatively safe food products.
Bacterial isolates were made from the intestinal tracts ofcarious species of cockroaches (Periplaneta americana, Periplaneta brunnea, Periplaneta australasiae, Neostylopyga rhombifolia, Nauphoeta cinerea) trapped from kitchens and stores (houses and hospital), Shigello, flexneri, Salmonella typhi, Escherichia coli and Salmonella sp. were some of the bacteria isolated and identified.
Six independent isolates of Klebsiella from hospital environmental sources in Malaysia were found to be resistant to at least ampicillin, carbenicillin, cefoperazone, chloramphenicol, gentamicin and tetracycline. On the basis of their antibiograms, they were divided into four antibiogroups. They transferred all or part of their multiple antibiotic resistance traits to E. coli by conjugation. The results suggest that these Klebsiella strains harbour self-transmissible R plasmids. The significance of these findings are discussed.
Pharyngeal micro-organisms of 131 Australian and Malaysian children and adults were compared by analysis of aerobic culture of throat swab specimens. Enteric Gram-negative bacilli were commonly isolated in small numbers from Malaysian adults whether they had sore throats (28%) or not (36%), but were detected in only 9% of Australian adults without sore throats and in only 12% and 4% of Malaysian children with and without sore throats respectively. In other respects microbiological findings were similar in the different groups of subjects studied. It is concluded that the pharyngeal carriage rate of enteric Gram-negative bacilli may differ substantially between different groups of normal individuals. Our findings also suggest that these micro-organisms do not have a pathogenic role in pharyngitis.
Matched MeSH terms: Escherichia coli/growth & development
Hendra virus (HeV) and Nipah virus (NiV) are members of a new genus, Henipavirus, in the family paramyxoviridae. Each virus encodes a phosphoprotein (P) that is significantly larger than its counterparts in other known paramyxoviruses. The interaction of this unusually large P with its nucleocapsid protein (N) was investigated in this study by using recombinant full-length and truncated proteins expressed in bacteria and a modified protein-blotting protein-overlay assay. Results from our group demonstrated that the N and P of both viruses were able to form not only homologous, but also heterologous, N-P complexes, i.e. HeV N was able to interact with NiV P and vice versa. Deletion analysis of the N and P revealed that there were at least two independent N-binding sites on P and they resided at the N and C termini, respectively. Similarly, more than one P-binding site was present on N and one of these was mapped to a 29 amino acid (aa) C-terminal region, which on its own was sufficient to interact with the extreme C-terminal 165 aa region of P.
The gene encoding the excretory-secretory antigen TES-120 of dog ascarid worm Toxocara canis was cloned into the bacterium Escherichia coli. The specificity of the recombinant TES-120 antigen produced by the bacterium was investigated. A total of 45 human serum samples from patients infected with differenthelminthes and protozoa, including 8 cases of toxocariasis, were tested against the recombinant antigens in immunoblot assays. The results from the assays revealed that the recombinant TES-120 antigen reacted with sera from toxocariasis patients only. This highly specific recombinant TES-120 antigen can potentially be used for the development of an inexpensive serodiagnostic assay for human toxocariasis.
Infectious diseases remain a significant threat to human health, contributing to more than 17 million deaths, annually. With the worsening trends of drug resistance, there is a need for newer and more powerful antimicrobial agents. We hypothesized that animals living in polluted environments are potential sources of antimicrobials. Under polluted milieus, organisms such as cockroaches encounter different types of microbes, including superbugs. Such creatures survive the onslaught of superbugs and are able to ward off disease by producing antimicrobial substances. Here, we characterized antibacterial properties in extracts of various body organs of cockroaches (Periplaneta americana) and showed potent antibacterial activity in crude brain extract against methicillin-resistant Staphylococcus aureus and neuropathogenic Escherichia coli K1. The size-exclusion spin columns revealed that the active compound(s) are less than 10 kDa in molecular mass. Using cytotoxicity assays, it was observed that pre-treatment of bacteria with lysates inhibited bacteria-mediated host cell cytotoxicity. Using spectra obtained with LC-MS on Agilent 1290 infinity liquid chromatograph, coupled with an Agilent 6460 triple quadruple mass spectrometer, tissues lysates were analysed. Among hundreds of compounds, only a few homologous compounds were identified that contained the isoquinoline group, chromene derivatives, thiazine groups, imidazoles, pyrrole-containing analogs, sulfonamides, furanones, and flavanones and known to possess broad-spectrum antimicrobial properties and anti-inflammatory, anti-tumour, and analgesic properties. Further identification, characterization, and functional studies using individual compounds can act as a breakthrough in developing novel therapeutics against various pathogens including superbugs.
In this work, the potential of titanium dioxide nanoparticles incorporated gellan gum (GG + TiO2-NPs) biofilm as wound dressing material was investigated. The GG + TiO2-NPs biofilm was prepared via evaporative casting technique and was characterized using FTIR, XRD, and SEM to study their physiochemical properties. The mechanical properties, swelling and water vapor transmission rate (WVTR) of biofilm was determined to comply with an ideal wound dressing material. In vitro and in vivo wound healing studies was carried out to evaluate the performance of GG + TiO2-NPs biofilm. In vitro wound healing was studied on 3 T3 mouse fibroblast cells for cell viability, cell proliferation, and scratch assay. The acridine orange/propidium iodide (AO/PI) staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were used to evaluate the viability of cell and cell proliferation. Cell migration assay was analyzed using Essen BioScience IncuCyteTM Zoom system. In vivo wound healing via open excision wounds model on Sprague Dawley rat was studied within 14 days. The FT-IR spectra of GG + TiO2-NPs biofilm show main bands assigned to OH stretching, OH deformation, and TiO stretching modes. XRD pattern of GG + TiO2-NPs biofilm suggesting that TiO2-NPs was successfully incorporated in biofilm and well distributed on the surface as proved by SEM analysis. The GG + TiO2-NPs biofilm shows higher mechanical strength and swelling (3.76 ± 0.11 MPa and 1061 ± 6%) as compared to pure GG film (3.32 ± 0.08 Mpa and 902 ± 6%), respectively. GG + TiO2-NPs biofilm shows good antibacterial properties as 9 ± 0.25 mm and 11 ± 0.06 mm exhibition zone was observed against Staphylococcus aureus and Escherichia coli bacteria, respectively. While no exhibition zone was obtained for pure GG biofilm. GG + TiO2-NPs biofilm also demonstrated better cell-to-cell interaction properties, as it's promoted cell proliferation and cell migration to accelerate open excision wound healing on Sprague Dawley rat. The wound treated with GG + TiO2-NPs biofilm was healed within 14 days, on the other hand, the wound is still can be seen when it was treated with GG. However, GG and GG + TiO2-NPs biofilm show no cytotoxicity effects on mouse fibroblast cells.
Curculin isolated from Curculigo latifolia, a plant grown in Malaysia, has an intriguing property of modifying sour taste into sweet taste. In addition to this taste-modifying activity, curculin itself elicits a sweet taste. Although these activities have been attributed to the heterodimeric isoform and not homodimers of curculin, the underlying mechanisms for the dual action of this protein have been largely unknown. To identify critical sites for these activities, we performed a mutational and structural study of recombinant curculin. Based on the comparison of crystal structures of curculin homo- and heterodimers, a series of mutants was designed and subjected to tasting assays. Mapping of amino acid residues on the three-dimensional structure according to their mutational effects revealed that the curculin heterodimer exhibits sweet-tasting and taste-modifying activities through its partially overlapping but distinct molecular surfaces. These findings suggest that the two activities of the curculin heterodimer are expressed through its two different modes of interactions with the T1R2-T1R3 heterodimeric sweet taste receptor.
Response surface methodology (RSM) and artificial neural network (ANN) were used to optimize the effect of four independent variables, viz. glucose, sodium chloride (NaCl), temperature and induction time, on lipase production by a recombinant Escherichia coli BL21. The optimization and prediction capabilities of RSM and ANN were then compared. RSM predicted the dependent variable with a good coefficient of correlation determination (R² and adjusted R² values for the model. Although the R (2) value showed a good fit, absolute average deviation (AAD) and root mean square error (RMSE) values did not support the accuracy of the model and this was due to the inferiority in predicting the values towards the edges of the design points. On the other hand, ANN-predicted values were closer to the observed values with better R², adjusted R², AAD and RMSE values and this was due to the capability of predicting the values throughout the selected range of the design points. Similar to RSM, ANN could also be used to rank the effect of variables. However, ANN could not predict the interactive effect between the variables as performed by RSM. The optimum levels for glucose, NaCl, temperature and induction time predicted by RSM are 32 g/L, 5 g/L, 32°C and 2.12 h, and those by ANN are 25 g/L, 3 g/L, 30°C and 2 h, respectively. The ANN-predicted optimal levels gave higher lipase activity (55.8 IU/mL) as compared to RSM-predicted levels (50.2 IU/mL) and the predicted lipase activity was also closer to the observed data at these levels, suggesting that ANN is a better optimization method than RSM for lipase production by the recombinant strain.
The growing global awareness for environmental protection has inspired the exploration on producing active packaging films from bio-based materials. In present work, three types of active agents were studied by incorporating thymol(T), kesum(K), and curry(C) (10% wt.) into polylactic acid (PLA) to produce PLA-10T, PLA-10K, and PLA10-C packaging films via solvent casting method. The morphology, functional chemistry, thermal stability, permeability, and antimicrobial properties were evaluated for PLA films. Functional chemical analysis confirmed the presence of interfacial bonding between aromatic groups of active agents and PLA carbonyl group. PLA-10K exhibited the highest thermal resistance comparing to PLA-10T and PLA-10C while water vapor barrier was enhanced after incorporation of active agents. Qualitative observation had indicated that chicken meat could be preserved in the active films until 15 days, while odourless and firm texture properties retained in food sample. For disc diffusion assay (in vitro), it showed positive results against Gram-positive bacteria (Staphylococcus aureus) whereas with negative results against Gram-negative bacteria (Escherichia coli) and Aspergillus Brasiliensis due to embedded active agents within PLA matrix. We concluded that produced active agents filled PLA films potential to use in food packaging application to enhance the shelf life of meats, fruits and vegetables product.
Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of Bacillus sp. NR5 UPM β-cyclodextrin glycosyltransferase (CGT-BS) in recombinant Escherichia coli. Several rare codons were eliminated and replaced with the ones favored by E. coli cells, resulting in an increased codon adaptation index (CAI) from 0.67 to 0.78. The cultivation of the codon modified recombinant E. coli following optimization of glycine supplementation enhanced the secretion of β-CGTase activity up to 2.2-fold at 12 h of cultivation as compared to the control. β-CGTase secreted into the culture medium by the transformant reached 65.524 U/mL at post-induction temperature of 37 °C with addition of 1.2 mM glycine and induced at 2 h of cultivation. A 20.1-fold purity of the recombinant β-CGTase was obtained when purified through a combination of diafiltration and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. This combined strategy doubled the extracellular β-CGTase production when compared to the single approach, hence offering the potential of enhancing the expression of extracellular enzymes, particularly β-CGTase by the recombinant E. coli.
A stirred fluidized bed (SFB) ion exchange chromatography was successfully applied in the direct recovery of recombinant enhanced green fluorescent protein (EGFP) from the unclarified Escherichia coli homogenate. Optimal conditions for both adsorption and elution processes were determined from the packed-bed adsorption systems conducted at a small scale using the clarified cell homogenate. The maximal adsorption capacity and dissociation constant for EGFP-adsorbent complex were found to be 6.3 mg/mL and 1.3 × 10-3 mg/mL, respectively. In an optimal elution of EGFP with 0.2 M of NaCl solution (pH 9) and at 200 cm/h, the recovery percent of the EGFP was approximately 93%. The performances of SFB chromatography for direct recovery of EGFP was also evaluated under different loading volumes (50-200 mL) of crude cell homogenate. The single-step purification of EGFP by SFB recorded in a high yield (95-98%) and a satisfactory purification factor (~3 folds) of EGFP from the cell homogenate at 200 rpm of rotating speed.
One of the major environmental issues of textile industries is the discharge of large quantities of textile effluents, which are source of contamination of water bodies on surface of earth and quality of groundwater. The effluents are toxic, non-biodegradable, carcinogenic and prodigious threats to human and aquatic creatures. Since textile effluents can be treated efficiently and effectively by various advanced oxidation processes (AOPs). Among the various AOPs, cold atmospheric pressure plasma is a promising method among many prominent techniques available to treat the effluents. In this paper, we report about the degradation of simulated effluent, namely Direct Orange-S (DO-S) aqueous solution, using nonthermal atmospheric pressure plasma jet. The plasma treatment of DO-S aqueous solution was carried out as a function of various operating parameters such as potential and treatment time. The change in properties of treated DO-S dye was investigated by means of various analytical techniques such as high-performance liquid chromatography, UV-visible (UV-Vis) spectroscopy and determination of total organic content (TOC). The reactive species present in the samples were identified using optical emission spectrometry (OES). OES results confirmed that the formation of reactive oxygen and nitrogen species during the plasma treatment in the liquid surface was responsible for dye oxidation and degradation. Degradation efficiency, as monitored by color removal efficiency, of 96% could be achieved after 1 h of treatment. Concurrently, the TOC values were found to decrease with plasma treatment, implying that the plasma treatment process enhanced the non-toxicity nature of DO-S aqueous solution. Toxicity of the untreated and plasma-treated dye solution samples was studied using Escherichia coli (E. coli) and Staphylococcus (S. aureus) organisms, which demonstrated that the plasma-treated dye solution was non-toxic in nature compared with untreated one.
Mechanical and non-mechanical breakages of bacterial cells are usually the preliminary steps in intracellular protein purification. In this study, the recombinant green fluorescent protein (GFP) was purified from intact Escherichia coli cells using preparative PAGE. In this purification process, cells disruption step is not needed. The cellular content of E. coli was drifted out electrically from cells and the negatively charged GFP was further electroeluted from polyacrylamide gel column. SEM investigation of the electrophoresed cells revealed substantial structural damage at the cellular level. This integrated purification technique has successfully recovered the intracellular GFP with a yield of 82% and purity of 95%.
In this study, the effects of thermosonication and thermal treatment on Escherichia coli O157:H7 and Salmonella Enteritidis in mango juice were investigated at 50 and 60°C. Besides, nonlethal injury of Salm. Enteritidis after both treatments was also examined. The highest inactivation was attained with thermosonication at 60°C. The inactivation rate was different for both pathogens, and Salm. Enteritidis was found to be more sensitive to thermosonication than E. coli O157:H7. Salmonella Enteritidis was recovered in all treated samples, except those subjected to more than 5-min thermosonication at 60°C. It was found that the introduction of high-intensity ultrasound enhanced the inactivation of pathogens compared to thermal treatment alone. On the other hand, Salm. Enteritidis was detected in a number of samples following incubation in universal pre-enrichment broth, but no growth was detected after incubation in mango juice.
The application of nanomaterials has gained considerable momentum in various fields in recent years due to their high reactivity, excellent surface properties and quantum effects in the nanometer range. The properties of zinc oxide (ZnO) vary with its crystallite size or particle size and often nanocrystalline ZnO is seen to exhibit superior physical and chemical properties due to their higher surface area and modified electronic structure. ZnO nanoparticles are reported to exhibit strong bacterial inhibiting activity and silver (Ag) has been extensively used for its antimicrobial properties since ages. In this study, Ag doped ZnO nanoparticles were synthesized by mechanochemical processing in a high energy ball mill and investigated for antimicrobial activity. The nanocrystalline nature of zinc oxide was established by X-ray diffraction (XRD) studies. It is seen from the XRD data obtained from the samples, that crystallite size of the zinc oxide nanoparticles is seen to decrease with increasing Ag addition. Field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) data also supported the nanoparticle formation during the synthesis. The doped nanoparticles were subjected to antimicrobial investigation and found that both increase in Ag content and decrease in particle size contributed significantly towards antimicrobial efficiency. It was also observed that Ag doped ZnO nanoparticles possess enhanced antimicrobial potential than that of virgin ZnO against the studied microorganisms of Escherichia coli and Staphylococcus aureus.
The non-stereospecific α-haloalkanoic acid dehalogenase DehE from Rhizobium sp. RC1 catalyzes the removal of the halide from α-haloalkanoic acid D,L-stereoisomers and, by doing so, converts them into hydroxyalkanoic acid L,D-stereoisomers, respectively. DehE has been extensively studied to determine its potential to act as a bioremediation agent, but its structure/function relationship has not been characterized. For this study, we explored the functional relevance of several putative active-site amino acids by site-specific mutagenesis. Ten active-site residues were mutated individually, and the dehalogenase activity of each of the 10 resulting mutants in soluble cell lysates against D- and L-2-chloropropionic acid was assessed. Interestingly, the mutants W34→A,F37→A, and S188→A had diminished activity, suggesting that these residues are functionally relevant. Notably, the D189→N mutant had no activity, which strongly implies that it is a catalytically important residue. Given our data, we propose a dehalogenation mechanism for DehE, which is the same as that suggested for other non-stereospecific α-haloalkanoic acid dehalogenases. To the best of our knowledge, this is the first report detailing a functional aspect for DehE, and our results could help pave the way for the bioengineering of haloalkanoic acid dehalogenases with improved catalytic properties.
Cytochrome P450 (CYP) 2C19 is essential for the metabolism of clinically used drugs including omeprazole, proguanil, and S-mephenytoin. This hepatic enzyme exhibits genetic polymorphism with inter-individual variability in catalytic activity. This study aimed to characterise the functional consequences of CYP2C19*23 (271 G>C, 991 A>G) and CYP2C19*24 (991 A>G, 1004 G>A) in vitro. Mutations in CYP2C19 cDNA were introduced by site-directed mutagenesis, and the CYP2C19 wild type (WT) as well as variants proteins were subsequently expressed using Escherichia coli cells. Catalytic activities of CYP2C19 WT and those of variants were determined by high performance liquid chromatography-based essay employing S-mephenytoin and omeprazole as probe substrates. Results showed that the level of S-mephenytoin 4'-hydroxylation activity of CYP2C19*23 (V max 111.5 ± 16.0 pmol/min/mg, K m 158.3 ± 88.0 μM) protein relative to CYP2C19 WT (V max 101.6 + 12.4 pmol/min/mg, K m 123.0 ± 19.2 μM) protein had no significant difference. In contrast, the K m of CYP2C19*24 (270.1 ± 57.2 μM) increased significantly as compared to CYP2C19 WT (123.0 ± 19.2 μM) and V max of CYP2C19*24 (23.6 ± 2.6 pmol/min/mg) protein was significantly lower than that of the WT protein (101.6 ± 12.4 pmol/min/mg). In vitro intrinsic clearance (CLint = V max/K m) for CYP2C19*23 protein was 85.4 % of that of CYP2C19 WT protein. The corresponding CLint value for CYP2C19*24 protein reduced to 11.0 % of that of WT protein. These findings suggested that catalytic activity of CYP2C19 was not affected by the corresponding amino acid substitutions in CYP2C19*23 protein; and the reverse was true for CYP2C19*24 protein. When omeprazole was employed as the substrate, K m of CYP2C19*23 (1911 ± 244.73 μM) was at least 100 times higher than that of CYP2C19 WT (18.37 ± 1.64 μM) and V max of CYP2C19*23 (3.87 ± 0.74 pmol/min/mg) dropped to 13.4 % of the CYP2C19 WT (28.84 ± 0.61 pmol/min/mg) level. Derived from V max/K m, the CLint value of CYP2C19 WT was 785 folds of CYP2C19*23. K m and V max values could not be determined for CYP2C19*24 due to its low catalytic activity towards omeprazole 5'-hydroxylation. Therefore, both CYP2C19*23 and CYP2C19*24 showed marked reduced activities of metabolising omeprazole to 5-hydroxyomeprazole. Hence, carriers of CYP2C19*23 and CYP2C19*24 allele are potentially poor metabolisers of CYP2C19-mediated substrates.