Displaying publications 161 - 180 of 830 in total

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  1. Process intensification of core streptavidin production through high-cell-density cultivation of recombinant E. coli and a temperature-based refolding method
    Chua LH, Tan SC, Liew MWO
    J Biotechnol, 2018 Jun 20;276-277:34-41.
    PMID: 29679607 DOI: 10.1016/j.jbiotec.2018.04.012
    An intensified process was developed that enables high level production of recombinant core streptavidin (cSAV), a non-glycosylated tetrameric protein utilised in a wide range of applications. A pH-stat fed-batch feeding strategy was employed to achieve high-cell-density and improve volumetric yield of cSAV which was expressed as inclusion bodies (IBs). The effect of induction at different cell densities (OD 20, 60 and 100) on volumetric and specific yield were then studied. Highest volumetric yield of cSAV (1550 mg L-1) was obtained from induction at OD 100 without significant reductions in specific yield. To recover active cSAV from IBs, the possibility of refolding using a temperature-based refolding method was investigated. Refolded cSAV obtained from temperature-based refolding were then compared against cSAV refolded with conventional dialysis and dilution methods using quantitative and qualitative metrics. The temperature-based refolding method was found to improve the yield of cSAV by 6-18% in comparison to conventional methods without compromising quality. Intensification was achieved by reductions in process volumes and a more concentrated product stream. Using the newly developed process, the volumetric yield of cSAV IBs was improved by thirty-six fold in comparison to low-cell-density shake flask cultivation, and 33% of cSAV can be recovered from IBs at 90% purity.
    Matched MeSH terms: Escherichia coli/genetics; Escherichia coli/metabolism*
  2. Stepwise optimization of recombinant protein production in Escherichia coli utilizing computational and experimental approaches
    Packiam KAR, Ramanan RN, Ooi CW, Krishnaswamy L, Tey BT
    Appl Microbiol Biotechnol, 2020 Apr;104(8):3253-3266.
    PMID: 32076772 DOI: 10.1007/s00253-020-10454-w
    Over the past few decades, Escherichia coli (E. coli) remains the most favorable host among the microbial cell factories for the production of soluble recombinant proteins. Recombinant protein production (RPP) via E. coli is optimized at the level of gene expression (expression level) and the process condition of fermentation (process level). Presently, the reported studies do not give a clear view on the selection of methods employed in the optimization of RPP. Here, we have reviewed various optimization methods and their preferences with respect to the factors at expression and process levels to achieve the optimal levels of soluble RPP. With a greater understanding of these optimization methods, we proposed a stepwise methodology linking the factors from both levels for optimizing the production of soluble recombinant protein in E. coli. The proposed methodology is further explained through five sets of examples demonstrating the optimization of RPP at both expression and process levels.Key Points• Stepwise methodology of optimizing recombinant protein production is proposed.• In silico tools can facilitate the optimization of gene- and protein-based factors.• Optimization of gene- and protein-based factors aids host-vector selection.• Statistical optimization is preferred for achieving optimal levels of process factors.
    Matched MeSH terms: Escherichia coli/genetics*; Escherichia coli/metabolism
  3. Fulltext Synthesis, characterisation and cytotoxicity of gold microwires for ultra-sensitive biosensor development
    Lah NAC, Gray R, Trigueros S
    Microb Cell Fact, 2021 Feb 17;20(1):46.
    PMID: 33596912 DOI: 10.1186/s12934-020-01478-y
    With the long-term goal of developing an ultra-sensitive microcantilever-based biosensor for versatile biomarker detection, new controlled bioreceptor-analytes systems are being explored to overcome the disadvantages of conventional ones. Gold (Au) microwires have been used as a probe to overcome the tolerance problem that occurs in response to changes in environmental conditions. However, the cytotoxicity of Au microwires is still unclear. Here, we examined the cytotoxicity of Au microwires systems using both commercial and as-synthesised Au microwires. In vitro experiments show that commercial Au microwires with an average quoted length of 5.6 µm are highly toxic against Gram-negative Escherichia coli (E. coli) at 50 µg/mL. However, this toxicity is due to the presence of CTAB surfactant not by the microwires. Conversely, the as-synthesised Au microwires show non-cytotoxicity even at the maximum viable concentration (330 µg/mL). These findings may lead to the development of potentially life-saving cytotoxicity-free biosensors for an early diagnostic of potential diseases.
    Matched MeSH terms: Escherichia coli/cytology; Escherichia coli/drug effects*
  4. Microbial water quality and the detection of multidrug resistant E. coli and antibiotic resistance genes in aquaculture sites of Singapore
    Ng C, Chen H, Goh SG, Haller L, Wu Z, Charles FR, et al.
    Mar Pollut Bull, 2018 Oct;135:475-480.
    PMID: 30301062 DOI: 10.1016/j.marpolbul.2018.07.055
    Poor microbial water quality jeopardizes the health and safety of food produced by aquaculture farms. Three fish farms and transect sites in Singapore were assessed for microbial water quality and antimicrobial resistance determinants. Of the 33 multidrug resistant E. coli isolated from surface waters of the Johor Straits, 81.8% were ESBL producers. The relative abundance of sul1, qnrA and intI1 genes were higher in sediments than surface waters. Among the surface water samples, higher concentrations (10-1-101) of beta-lactamases (blaSHV, blaOXA, blaCTX-M) were detected in the transect sites. This study highlights a potential antimicrobial resistance transmission chain from environmental waters, to animal carriers and humans.
    Matched MeSH terms: Escherichia coli/drug effects; Escherichia coli/isolation & purification*
  5. Site-directed mutagenesis: role of lid region for T1 lipase specificity
    Mohamed RA, Salleh AB, Leow TC, Yahaya NM, Abdul Rahman MB
    Protein Eng. Des. Sel., 2018 06 01;31(6):221-229.
    PMID: 30239965 DOI: 10.1093/protein/gzy023
    A broad substrate specificity enzyme that can act on a wide range of substrates would be an asset in industrial application. T1 lipase known to have broad substrate specificity in its native form apparently exhibits the same active sites as polyhydroxylalkanoate (PHA) depolymerase. PhaZ6Pl is one of the PHA depolymerases that can degrade semicrystalline P(3HB). The objective of this study is to enable T1 lipase to degrade semicrystalline P(3HB) similar to PhaZ6Pl while maintaining its native function. A structural study on PhaZ6Pl contains no lid in its structure and therefore T1 lipase was designed with removal of its lid region. BSLA lipase was chosen as the reference protein for T1 lipase modification since it contains no lid. Initially, structures of both enzymes were compared via protein-protein superimposition in 3D-space and the location of the lid region of T1 lipase was highlighted. A total of three variants of T1 lipase without lid were successfully designed by referring to BSLA lipase (a lipase without lid). The ability of T1 lipase without lid variants in degrading P(3HB) was investigated quantitatively. All the variants showed activity towards the substrate which confirmed that T1 lipase without lid is indeed able to degrade P(3HB). In addition, D2 was recorded to have the highest activity amongst other variants. Results obtained in this study highlighted the fact that native T1 lipase is a versatile hydrolase enzyme which does not only record triglyceride degradation but also P(3HB) by simply removing the lid region.
    Matched MeSH terms: Escherichia coli/enzymology; Escherichia coli/genetics
  6. Insensitivity of bacterial nucleic acid biosyntheses to a morphine-like narcotic
    Wolfe AD, Hahn FE
    Naturwissenschaften, 1975 Feb;62(2):99.
    PMID: 1683
    Matched MeSH terms: Escherichia coli/drug effects*; Escherichia coli/metabolism
  7. Characterization of a novel type I l-asparaginase from Acinetobacter soli and its ability to inhibit acrylamide formation in potato chips
    Jiao L, Chi H, Lu Z, Zhang C, Chia SR, Show PL, et al.
    J Biosci Bioeng, 2020 Jun;129(6):672-678.
    PMID: 32088137 DOI: 10.1016/j.jbiosc.2020.01.007
    l-Asparaginases have the potential to inhibit the formation of acrylamide, a harmful toxin formed during high temperature processing of food. A novel bacterium which produces l-asparaginase was screened. Type I l-asparaginase gene from Acinetobacter soli was cloned and expressed in Escherichia coli. The recombinant l-asparaginase had an activity of 42.0 IU mL-1 and showed no activity toward l-glutamine and d-asparagine. The recombinant l-asparaginase exhibited maximum catalytic activity at pH 8.0 and 40°C. The enzyme was stable in the pH ranging from 6.0 to 9.0. The activity of the recombinant enzyme was substantially enhanced by Ba2+, dithiothreitol, and β-mercaptoethanol. The Km and Vmax values of the l-asparaginase for the l-asparagine were 3.22 mmol L-1 and 1.55 IU μg-1, respectively. Moreover, the recombinant l-asparaginase had the ability to mitigate acrylamide formation in potato chips. Compared with the untreated group, the content of acrylamide in samples treated with the enzyme was effectively decreased by 55.9%. These results indicate that the novel type I l-asparaginase has the potential for application in the food processing industry.
    Matched MeSH terms: Escherichia coli/genetics; Escherichia coli/metabolism
  8. Fulltext Preparation of kenaf stem hemicellulosic hydrolysate and its fermentability in microbial production of xylitol by Escherichia coli BL21
    Shah SSM, Luthfi AAI, Low KO, Harun S, Manaf SFA, Illias RM, et al.
    Sci Rep, 2019 03 11;9(1):4080.
    PMID: 30858467 DOI: 10.1038/s41598-019-40807-z
    Kenaf (Hibiscus cannabinus L.), a potential fibre crop with a desirably high growth rate, could serve as a sustainable feedstock in the production of xylitol. In this work, the extraction of soluble products of kenaf through dilute nitric-acid hydrolysis was elucidated with respect to three parameters, namely temperature, residence time, and acid concentration. The study will assist in evaluating the performance in terms of xylose recovery. The result point out that the maximum xylose yield of 30.7 g per 100 g of dry kenaf was attained from 2% (v/v) HNO3 at 130 °C for 60 min. The detoxified hydrolysate was incorporated as the primary carbon source for subsequent fermentation by recombinant Escherichia coli and the performance of strain on five different semi-synthetic media on xylitol production were evaluated herein. Among these media, batch cultivation in a basal salt medium (BSM) afforded the highest xylitol yield of 0.35 g/g based on xylose consumption, which corresponded to 92.8% substrate utilization after 38 h. Subsequently, fermentation by E. coli in the xylose-based kenaf hydrolysate supplemented with BSM resulting in 6.8 g/L xylitol which corresponding to xylitol yield of 0.38 g/g. These findings suggested that the use of kenaf as the fermentation feedstock could be advantageous for the development of sustainable xylitol production.
    Matched MeSH terms: Escherichia coli/genetics; Escherichia coli/metabolism
  9. Preparation, characterization, immobilization, and molecular docking analysis of a novel detergent-stable subtilisin-like serine protease from Streptomyces mutabilis strain TN-X30
    Mechri S, Allala F, Bouacem K, Hasnaoui I, Gwaithan H, Chalbi TB, et al.
    Int J Biol Macromol, 2022 Dec 01;222(Pt A):1326-1342.
    PMID: 36242508 DOI: 10.1016/j.ijbiomac.2022.09.161
    We recently described the production of a detergent-biocompatible crude protease from Streptomyces mutabilis strain TN-X30. Here, we describe the purification, characterization, and immobilization of the serine alkaline protease (named SPSM), as well as the cloning, sequencing, and over-expression of its corresponding gene (spSM). Pure enzyme was obtained after ammonium sulphate precipitation followed by heat-treatment and Sephacryl® S-200 column purification. The sequence of the first 26 NH2-terminal residues of SPSM showed a high sequence identity to subtilisin-like serine proteases produced by actinobacteria. The spSM gene was heterologously expressed in Escherichia coli BL21(DE3)pLysS and E. coli BL21-AI™ strains using pTrc99A (rSPSM) and Gateway™ pDEST™ 17 [(His)6-tagged SPSM] vectors, respectively. Results obtained indicated that the (His)6-tagged SPSM showed the highest stability. The SPSM was immobilized using encapsulation and adsorption-encapsulation approaches and three different carriers. Features of SPSM in soluble and immobilized forms were analyzed by Fourier transform infrared (FTIR) spectroscopy in attenuated total reflection (ATR) mode, X-ray diffraction (XRD), zeta potential measurements, and field emission scanning electron microscopy (FE-SEM). The white clay and kaolin used in this study are eco-friendly binders to alginate-SPSM and show great potential for application of the immobilized SPSM in various industries. Molecular modeling and docking of N-succinyl-l-Phe-l-Ala-l-Ala-l-Phe-p-nitroanilide in the active site of SPSM revealed the involvement of 21 amino acids in substrate binding.
    Matched MeSH terms: Escherichia coli/genetics; Escherichia coli/metabolism
  10. Rapid preparation of adherent mammalian cells for basic scanning electron microscopy (SEM) analysis
    Osahor A, Deekonda K, Lee CW, Sim EU, Radu A, Narayanan K
    Anal Biochem, 2017 10 01;534:46-48.
    PMID: 28693990 DOI: 10.1016/j.ab.2017.07.008
    Sample preparation for scanning electron microscope analysis involves reagents and equipment that are expensive and often hazardous. Here we demonstrate a circumvention of Osmium tetroxide and critical point drying, greatly reducing the duration, complexity and cost of the process. We captured early stage interactions of invasive-bacteria and HeLa cells during the process of bacteria-mediated gene delivery and illustrate sufficient clarity can be obtained using this procedure to preserve and clearly visualize relevant cellular structures. This protocol is significantly cheaper and easier to adapt compared to conventional methods, and will allow routine preparation/viewing of eukaryotic or bacterial samples for basic morphological studies.
    Matched MeSH terms: Escherichia coli/genetics*; Escherichia coli/isolation & purification
  11. Controlled expression of cholera toxin B subunit from Vibrio cholerae in Escherichia coli
    Haryanti T, Mariana NS, Latifah SY, Yusoff K, Raha AR
    Pak J Biol Sci, 2008 Jul 01;11(13):1718-22.
    PMID: 18819625
    The ctxB gene, the causative agent of cholera epidemic was successfully cloned from V. cholerae in E. coli. The insertion of the gene was confirmed by PCR as well as restriction digestion analyses. The sequencing results for the gene confirmed that the insert was in the correct orientation and in-frame with the P(BAD) promoter and it showed that the gene was 99% homologous to the published ctxB sequence. The CTB protein was successfully expressed in E. coli using the pBAD/His vector system. The expected protein of approximately 14 kDa was detected by SDS-PAGE and Western blot. The use of pBAD/His vector to express the cholera toxin gene in E. coli would facilitate future study of toxin gene products.
    Matched MeSH terms: Escherichia coli/genetics; Escherichia coli/metabolism*
  12. Prevalence of AmpC beta-lactamase and extended spectrum beta-lactamase co-producer in Escherichia coli and Klebsiella species in a teaching hospital
    Ting SMV, Ismail Z, Hanafiah A
    Malays J Pathol, 2024 Apr;46(1):79-89.
    PMID: 38682847
    INTRODUCTION: Beta-lactamase producing bacterial infection has been on surge due to selection pressure and injudicious antibiotics usage. Organisms that co-produced more than one beta lactamase enzyme posed diagnostic challenges which may result in inadequate treatment. To date, there is no standardised guideline offering phenotypic detection of AmpC β-lactamase. The purpose of this study was to determine the prevalence of ESBLs, AmpC β-lactamase and co-producer organisms in a teaching hospital.

    MATERIALS AND METHODS: Three hundred and four isolates of E. coli and Klebsiella sp. had been selected via convenient sampling. These isolates were identified using conventional laboratory methods and their antimicrobial susceptibilities were determined using disc diffusion method. Those isolates were then proceeded with ESBL confirmatory test, cloxacillin-containing Muller Hinton confirmatory test, modified double disk synergy test and AmpC disk test.

    RESULTS: Out of 304 isolates, 159 isolates were E. coli and 145 were Klebsiella sp. The prevalence of organisms which co-produced AmpC β-lactamase and ESBL enzymes were 3.0%. Besides that, 39 cefoxitin resistant and three cefoxitin susceptible isolates (13.8%) were proven to produce AmpC β-lactamase through AmpC disk test. Through the CLSI confirmatory test, 252 (82.9%) isolates were identified as ESBLs producers and the prevalence increased slightly when cloxacillin-containing Muller Hinton were used. Only three ESBLs positive organisms were positive for modified double disk synergy test.

    CONCLUSION: Distinguishing between AmpC β-lactamase and ESBL-producing organisms has epidemiological significance as well as therapeutic importance. Moreover, AmpC β-lactamase and ESBLs co-producing organisms can lead to false negative ESBL confirmatory test. Therefore, knowing the local prevalence can guide the clinician in navigating the treatment.

    Matched MeSH terms: Escherichia coli Infections/microbiology; Escherichia coli Infections/epidemiology
  13. Production of refolded Toxoplasma gondii recombinant SAG1-related sequence 3 (SRS3) and its use for serodiagnosis of human toxoplasmosis
    Mirzadeh A, Saadatnia G, Golkar M, Babaie J, Noordin R
    Protein Expr Purif, 2017 05;133:66-74.
    PMID: 28263855 DOI: 10.1016/j.pep.2017.03.001
    SAG1-related sequence 3 (SRS3) is one of the major Toxoplasma gondii tachyzoite surface antigens and has been shown to be potentially useful for the detection of toxoplasmosis. This protein is highly conformational due to the presence of six disulfide bonds. To achieve solubility and antigenicity, SRS3 depends on proper disulfide bond formation. The aim of this study was to over-express the SRS3 protein with correct folding for use in serodiagnosis of the disease. To achieve this, a truncated SRS3 fusion protein (rtSRS3) was produced, containing six histidyl residues at both terminals and purified by immobilized metal affinity chromatography. The refolding process was performed through three methods, namely dialysis in the presence of chemical additives along with reduced/oxidized glutathione and drop-wise dilution methods with reduced/oxidized glutathione or reduced DTT/oxidized glutathione. Ellman's assay and ELISA showed that the protein folding obtained by the dialysis method was the most favorable, probably due to the correct folding. Subsequently, serum samples from individuals with chronic infection (n = 76), probable acute infection (n = 14), and healthy controls (n = 81) were used to determine the usefulness of the refolded rtSRS3 for Toxoplasma serodiagnosis. The results of the developed IgG-ELISA showed a diagnostic specificity of 91% and a sensitivity of 82.89% and 100% for chronic and acute serum samples, respectively. In conclusion, correctly folded rtSRS3 has the potential to be used as a soluble antigen for the detection of human toxoplasmosis.
    Matched MeSH terms: Escherichia coli/genetics; Escherichia coli/metabolism
  14. Heat treatment of unclarified Escherichia coli homogenate improved the recovery efficiency of recombinant hepatitis B core antigen
    Ng MY, Tan WS, Abdullah N, Ling TC, Tey BT
    J Virol Methods, 2006 Oct;137(1):134-9.
    PMID: 16860402
    Heat precipitation procedure has been regularly incorporated as a selective purification step in various thermostable proteins expressed in different hosts. This method is efficient in precipitation of most of the host proteins and also deactivates various host proteases that can be harmful to the desired gene products. In this study, introduction of heat treatment procedure in the purification of hepatitis B core antigen (HBcAg) produced in Escherichia coli has been investigated. Thermal treatment of the cell homogenate at 60 degrees C for 30 min prior to subsequent clarification steps has resulted in 1.4 times and 18% higher in purity and recovery yield, respectively, compared to the non-heat-treated cell homogenate. In direct capture of HBcAg by using anion-exchangers from unclarified feedstock, pre-conditioning the feedstock by heat treatment at 60 degrees C for 45 min has increased the recovery yield of HBcAg by 2.9-fold and 42% in purity compared to that treated for 10 min. Enzyme-linked immunosorbent assay (ELISA) analysis showed that the antigenicity of the core particles was not affected by the heat treatment process.
    Matched MeSH terms: Escherichia coli/genetics; Escherichia coli/metabolism*
  15. beta-Lactam resistance phenotype determination in Escherichia coli isolates from University Malaya Medical Centre
    Wong JS, Mohd Azri ZA, Subramaniam G, Ho SE, Palasubramaniam S, Navaratnam P
    Malays J Pathol, 2003 Dec;25(2):113-9.
    PMID: 16196367
    beta-Lactamases have been identified as the major cause of antimicrobial resistance to beta-lactam antibiotics in Escherichia coli. The activities of ampicillin-sulbactam and amoxicillin-clavulanate as well as a range of beta-lactam antibiotics were studied with 87 clinical E. coli isolates from patients of the University Malaya Medical Center using the disc diffusion technique. Susceptible, intermediate and resistant categories were established based on the diameter of zones of inhibition set by the National Committee for Clinical Laboratory Standards (NCCLS). The isolates were then classified into 6 phenotypes according to the criteria stated in the methodology: S (susceptible to all beta-lactams); TL (resistant to aminopenicillins; amoxicillin-clavulanate susceptible and susceptible or intermediate to ampicillin-sulbactam); TI (resistant to aminopenicillins and ampicillin-sulbactam; susceptible to amoxicilin-clavulanate); TH-IRT (resistant to aminopenicillins; intermediate or resistant to amoxicillin-clavulanate; resistant to ampicillin-sulbactam); ESBL (resistant to aminopenicillins and oxyimino cephalosporins; positive results with the double-disc diffusion test); and CP (resistant to aminopenicillins, beta-lactam-beta-lactamase inhibitor combinations, oxyimino cephalosporins and cephamycins). Results showed that the TL phenotype was the commonest (40.2% of the isolates) followed by S (31%), TH-IRT (16.1%), ESBL and CP (3.4% each) and TI (2.3%). One isolate showed both ESBL and CP phenotypes while two isolates were classified as inconclusive. Representatives from each phenotype were further analysed for the presence of beta-lactamases which revealed a predominance of TEM and SHV enzyme producers. PCR-SSCP analysis of the SHV gene from all the ESBL and CP isolates revealed the predominance of SHV 5-type enzyme which was concurrent with our previous studies.
    Matched MeSH terms: Escherichia coli/classification; Escherichia coli/drug effects*; Escherichia coli/enzymology; Escherichia coli/genetics
  16. Fulltext Titanium Dioxide Nanoparticle-Based Interdigitated Electrodes: A Novel Current to Voltage DNA Biosensor Recognizes E. coli O157:H7
    Nadzirah Sh, Azizah N, Hashim U, Gopinath SC, Kashif M
    PLoS One, 2015;10(10):e0139766.
    PMID: 26445455 DOI: 10.1371/journal.pone.0139766
    Nanoparticle-mediated bio-sensing promoted the development of novel sensors in the front of medical diagnosis. In the present study, we have generated and examined the potential of titanium dioxide (TiO2) crystalline nanoparticles with aluminium interdigitated electrode biosensor to specifically detect single-stranded E.coli O157:H7 DNA. The performance of this novel DNA biosensor was measured the electrical current response using a picoammeter. The sensor surface was chemically functionalized with (3-aminopropyl) triethoxysilane (APTES) to provide contact between the organic and inorganic surfaces of a single-stranded DNA probe and TiO2 nanoparticles while maintaining the sensing system's physical characteristics. The complement of the target DNA of E. coli O157:H7 to the carboxylate-probe DNA could be translated into electrical signals and confirmed by the increased conductivity in the current-to-voltage curves. The specificity experiments indicate that the biosensor can discriminate between the complementary sequences from the base-mismatched and the non-complementary sequences. After duplex formation, the complementary target sequence can be quantified over a wide range with a detection limit of 1.0 x 10(-13)M. With target DNA from the lysed E. coli O157:H7, we could attain similar sensitivity. Stability of DNA immobilized surface was calculated with the relative standard deviation (4.6%), displayed the retaining with 99% of its original response current until 6 months. This high-performance interdigitated DNA biosensor with high sensitivity, stability and non-fouling on a novel sensing platform is suitable for a wide range of biomolecular interactive analyses.
    Matched MeSH terms: Escherichia coli Infections/microbiology; Escherichia coli O157/genetics*; Escherichia coli O157/isolation & purification*
  17. Fulltext Synthesis of new isoquinoline-base-oxadiazole derivatives as potent inhibitors of thymidine phosphorylase and molecular docking study
    Zaman K, Rahim F, Taha M, Wadood A, Shah SAA, Ahmed QU, et al.
    Sci Rep, 2019 11 05;9(1):16015.
    PMID: 31690793 DOI: 10.1038/s41598-019-52100-0
    Here in this study regarding the over expression of TP, which causes some physical, mental and socio problems like psoriasis, chronic inflammatory disease, tumor angiogenesis and rheumatoid arthritis etc. By this consideration, the inhibition of this enzyme is vital to secure life from serious threats. In connection with this, we have synthesized twenty derivatives of isoquinoline bearing oxadiazole (1-20), characterized through different spectroscopic techniques such as HREI-MS, 1H- NMR and 13C-NMR and evaluated for thymidine phosphorylase inhibition. All analogues showed outstanding inhibitory potential ranging in between 1.10 ± 0.05 to 54.60 ± 1.50 µM. 7-Deazaxanthine (IC50 = 38.68 ± 1.12 µM) was used as a positive control. Through limited structure activity relationships study, it has been observed that the difference in inhibitory activities of screened analogs are mainly affected by different substitutions on phenyl ring. The effective binding interactions of the most active analogs were confirmed through docking study.
    Matched MeSH terms: Escherichia coli/enzymology; Escherichia coli Proteins/metabolism; Escherichia coli Proteins/chemistry
  18. Genetic characterization of Escherichia coli O157: H7/- strains carrying the stx2 gene but not producing Shiga toxin 2
    Koitabashi T, Vuddhakul V, Radu S, Morigaki T, Asai N, Nakaguchi Y, et al.
    Microbiol. Immunol., 2006;50(2):135-48.
    PMID: 16490932
    Nine Escherichia coli O157: H7/- strains isolated primarily from non-clinical sources in Thailand and Japan carried the stx(2) gene but did not produce Stx2 toxin in a reversed passive latex agglutination (RPLA) assay. A strain (EDL933) bearing a stx(2) phage (933W) was compared to a strain (Thai-12) that was Stx2-negative but contained the stx(2) gene. To study the lack of Stx2 production, the Thai-12 stx(2) gene and its upstream nucleotide sequence were analyzed. The Thai-12 stx(2) coding region was intact and Stx2 was expressed from a cloned stx(2) gene using a plasmid vector and detected using RPLA. A lacZ fusion analysis found the Thai-12 stx(2) promoter non-functional. Because the stx(2) gene is downstream of the late promoter in the stx(2) phage genome, the antitermination activity of Q protein is essential for strong stx(2) transcription. Thai-12 had the q gene highly homologous to that of Phi21 phage but not to the 933W phage. High-level expression of exogenous q genes demonstrated Q antitermination activity was weak in Thai-12. Replication of stx(2) phage was not observed in Stx2-negative strains. The q-stx(2) gene sequence of Thai-12 was well conserved in all Stx2-negative strains. A PCR assay to detect the Thai-12 q-stx(2) sequence demonstrated that 30% of O157 strains from marketed Malaysian beef carried this sequence and they produced little or no Stx2. These results suggest that stx(2)-positive O157 strains that produce little or no Stx2 may be widely distributed in the Asian environment.
    Matched MeSH terms: Escherichia coli Infections/microbiology*; Escherichia coli O157/genetics*; Escherichia coli O157/metabolism*
  19. Fulltext Molecular analysis of asymptomatic bacteriuria Escherichia coli strain VR50 reveals adaptation to the urinary tract by gene acquisition
    Beatson SA, Ben Zakour NL, Totsika M, Forde BM, Watts RE, Mabbett AN, et al.
    Infect Immun, 2015 May;83(5):1749-64.
    PMID: 25667270 DOI: 10.1128/IAI.02810-14
    Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50afa and VR50afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50-pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder.
    Matched MeSH terms: Escherichia coli/genetics*; Escherichia coli/isolation & purification; Escherichia coli Infections/microbiology*
  20. Fingerprints of resistant Escherichia coli O157:H7 from vegetables and environmental samples
    Abakpa GO, Umoh VJ, Kamaruzaman S, Ibekwe M
    J Sci Food Agric, 2018 Jan;98(1):80-86.
    PMID: 28543177 DOI: 10.1002/jsfa.8441
    BACKGROUND: Some routes of transmission of Escherichia coli O157:H7 to fresh produce include contaminated irrigation water and manure polluted soils. The aim of the present study was to determine the genetic relationships of E. coli O157:H7 isolated from some produce growing region in Nigeria using enterobacterial repetitive intergenic consensus (ERIC) DNA fingerprinting analysis. A total of 440 samples comprising leafy greens, irrigation water, manure and soil were obtained from vegetable producing regions in Kano and Plateau States, Nigeria. Genes coding for the quinolone resistance-determinant (gyrA) and plasmid (pCT) coding for multidrug resistance (MDR) were determined using polymerase chain reaction (PCR) in 16 isolates that showed MDR.

    RESULTS: Cluster analysis of the ERIC-PCR profiles based on band sizes revealed six main clusters from the sixteen isolates analysed. The largest cluster (cluster 3) grouped isolates from vegetables and manure at a similarity coefficient of 0.72.

    CONCLUSION: The present study provides data that support the potential transmission of resistant strains of E. coli O157:H7 from vegetables and environmental sources to humans with potential public health implications, especially in developing countries. © 2017 Society of Chemical Industry.

    Matched MeSH terms: Escherichia coli O157/classification; Escherichia coli O157/drug effects; Escherichia coli O157/genetics; Escherichia coli O157/isolation & purification*
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