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1.
PLoS One ; 15(8): e0232305, 2020.
Article in English | MEDLINE | ID: mdl-32785271

ABSTRACT

Shiga toxin-producing Escherichia coli (STEC) that cause severe disease predominantly carry the toxin gene variant stx2a. However, the role of Shiga toxin in the ruminant reservoirs of this zoonotic pathogen is poorly understood and strains that cause severe disease in humans (HUSEC) likely constitute a small and atypical subset of the overall STEC flora. The aim of this study was to investigate the presence of stx2a in samples from cattle and to isolate and characterize stx2a-positive E. coli. In nationwide surveys in Sweden and Norway samples were collected from individual cattle or from cattle herds, respectively. Samples were tested for Shiga toxin genes by real-time PCR and amplicon sequencing and stx2a-positive isolates were whole genome sequenced. Among faecal samples from Sweden, stx1 was detected in 37%, stx2 in 53% and stx2a in 5% and in skin (ear) samples in 64%, 79% and 2% respectively. In Norway, 79% of the herds were positive for stx1, 93% for stx2 and 17% for stx2a. Based on amplicon sequencing the most common stx2 types in samples from Swedish cattle were stx2a and stx2d. Multilocus sequence typing (MLST) of 39 stx2a-positive isolates collected from both countries revealed substantial diversity with 19 different sequence types. Only a few classical LEE-positive strains similar to HUSEC were found among the stx2a-positive isolates, notably a single O121:H19 and an O26:H11. Lineages known to include LEE-negative HUSEC were also recovered including, such as O113:H21 (sequence type ST-223), O130:H11 (ST-297), and O101:H33 (ST-330). We conclude that E. coli encoding stx2a in cattle are ranging from strains similar to HUSEC to unknown STEC variants. Comparison of isolates from human HUS cases to related STEC from the ruminant reservoirs can help identify combinations of virulence attributes necessary to cause HUS, as well as provide a better understanding of the routes of infection for rare and emerging pathogenic STEC.


Subject(s)
Cattle/microbiology , Shiga Toxin 2/genetics , Shiga-Toxigenic Escherichia coli/genetics , Animals , Disease Reservoirs/microbiology , Escherichia coli Infections/epidemiology , Escherichia coli Infections/microbiology , Genetic Variation , Genome, Bacterial , Hemolytic-Uremic Syndrome/epidemiology , Hemolytic-Uremic Syndrome/microbiology , Humans , Multilocus Sequence Typing , Norway/epidemiology , Prevalence , Real-Time Polymerase Chain Reaction , Shiga-Toxigenic Escherichia coli/cytology , Shiga-Toxigenic Escherichia coli/isolation & purification , Sweden/epidemiology , Virulence/genetics , Zoonoses/epidemiology , Zoonoses/microbiology
2.
Anal Chem ; 90(15): 8989-8997, 2018 08 07.
Article in English | MEDLINE | ID: mdl-29939014

ABSTRACT

Shiga toxin (Stx)-producing Escherichia coli (STEC) and enterohemorrhagic E. coli (EHEC) as a human pathogenic subgroup of STEC are characterized by releasing Stx AB5-toxin as the major virulence factor. Worldwide disseminated EHEC strains cause sporadic infections and outbreaks in the human population and swine pathogenic STEC strains represent greatly feared pathogens in pig breeding and fattening plants. Among the various Stx subtypes, Stx1a and Stx2a are of eminent clinical importance in human infections being associated with life-threatening hemorrhagic colitis and hemolytic uremic syndrome, whereas Stx2e subtype is associated with porcine edema disease with a generalized fatal outcome for the animals. Binding toward the glycosphingolipid globotriaosylceramide (Gb3Cer) is a common feature of all Stx subtypes analyzed so far. Here, we report on the development of a matched strategy combining (i) miniaturized one-step affinity purification of native Stx subtypes from culture supernatant of bacterial wild-type strains using Gb3-functionalized magnetic beads, (ii) structural analysis and identification of Stx holotoxins by electrospray ionization ion mobility mass spectrometry (ESI MS), (iii) functional Stx-receptor real-time interaction analysis employing the surface acoustic wave (SAW) technology, and (iv) Vero cell culture assays for determining Stx-caused cytotoxic effects. Structural investigations revealed diagnostic tryptic peptide ions for purified Stx1a, Stx2a, and Stx2e, respectively, and functional analysis resulted in characteristic binding kinetics of each Stx subtype. Cytotoxicity studies revealed differing toxin-mediated cell damage ranked with Stx1a > Stx2a > Stx2e. Collectively, this matched procedure represents a promising clinical application for the characterization of life-endangering Stx subtypes at the protein level.


Subject(s)
Edema Disease of Swine/microbiology , Escherichia coli Infections/microbiology , Hemolytic-Uremic Syndrome/microbiology , Shiga-Toxigenic Escherichia coli/classification , Shiga-Toxigenic Escherichia coli/cytology , Spectrometry, Mass, Electrospray Ionization/methods , Animals , Chlorocebus aethiops , Humans , Immunomagnetic Separation/methods , Microbial Viability , Shiga-Toxigenic Escherichia coli/chemistry , Sound , Swine , Vero Cells
3.
J Enzyme Inhib Med Chem ; 33(1): 639-650, 2018 Dec.
Article in English | MEDLINE | ID: mdl-29536772

ABSTRACT

Oxidative stress may be the major cause of induction of Shiga toxin-converting (Stx) prophages from chromosomes of Shiga toxin-producing Escherichia coli (STEC) in human intestine. Thus, we aimed to test a series of novel antioxidant compounds for their activities against prophage induction, thus, preventing pathogenicity of STEC. Forty-six compounds (derivatives of carbazole, indazole, triazole, quinolone, ninhydrine, and indenoindole) were tested. Fifteen of them gave promising results and were further characterized. Eleven compounds had acceptable profiles in cytotoxicity tests with human HEK-293 and HDFa cell lines. Three of them (selected for molecular studies) prevent the prophage induction at the level of expression of specific phage genes. In bacterial cells treated with hydrogen peroxide, expression of genes involved in the oxidative stress response was significantly less efficient in the presence of the tested compounds. Therefore, they apparently reduce the oxidative stress, which prevents induction of Stx prophage in E. coli.


Subject(s)
Anti-Bacterial Agents/pharmacology , Antioxidants/pharmacology , Shiga Toxin/antagonists & inhibitors , Shiga-Toxigenic Escherichia coli/drug effects , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Antioxidants/chemical synthesis , Antioxidants/chemistry , Cell Line , Cell Survival/drug effects , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Molecular Structure , Oxidative Stress/drug effects , Shiga Toxin/genetics , Shiga Toxin/metabolism , Shiga-Toxigenic Escherichia coli/cytology , Shiga-Toxigenic Escherichia coli/metabolism , Structure-Activity Relationship
4.
Wei Sheng Wu Xue Bao ; 54(7): 737-45, 2014 Jul 04.
Article in Chinese | MEDLINE | ID: mdl-25252454

ABSTRACT

OBJECTIVE: The effect of flhDC, fliA, fliD and fliE genes involved in moving of Escherichia coli (E. coli) on the motility of lysogened strain by Stx2-encoding phage phiMin27 was explored by gene knockout and phage lysogenic conversion. METHODS: Using the lambda Red recombinase system, the mutant strains of E. coli MG1655 named MG1655 deltaflhDC, MG1655 deltafliA, MG1655 deltafliD and MG1655 deltafliE were constructed. Then the corresponding complemented strains by ligating amplified targeted genes into the low copy vector pUC18 at the BamHI and Hind III sites and transforming these plasmids into mutant strains were acquired. By lysogenic infection of Stx2-encoding phage phiMin27, the lysogens for mutants named MG1655 deltaflhDCphiMin27, MG1655 deltafliAdeltaMin27, MG1655 deltafliDphiMin27 and MG1655 deltafliEphiMin27 were achieved. Subsequently, the motility of wild strain, the mutants, the complemented strains and the lysogens were detected. The changes of expression of the other genes involved in motility between wild strain and the lysogens before and after flhDC deletion by qRT-PCR were analyzed. RESULTS: Lysogenic infection of Stx2-encoding phage phiMin27 could promote the expression of fliA and fliD gene and enhance the motility of MG1655. For flhDC deletion, higher expression of fliA and fliD gene of MG1655 appeared, but the motility had no change. However, lysogen for MG1655 deltaflhDC lost the swimming motility. By gene transcriptional level detection, the expression of fliA and fliD gene of MG1655 deltaflhDCphiMin27 was down-regulated significantly compared with MG1655 deltaflhDC, and no marked variation was observed for fliE gene. The single deletion of fliA, fliD and fliE gene had no effect on the motility of E. coli MG1655 and lysogened strain by Stx2-encoding phage phiMin27. CONCLUSION: The results show that fliA and fliD gene together participated the regulation for flagella motility and flhDC gene could affect the motility of the lysogened strain by phage. It provides the theoretical basis for further research on the mutual regulation between phage lysogenization and host genes.


Subject(s)
Bacteriophages/physiology , Shiga Toxin 2/biosynthesis , Shiga-Toxigenic Escherichia coli/cytology , Shiga-Toxigenic Escherichia coli/virology , Bacteriophages/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Flagella/genetics , Flagella/physiology , Gene Expression Regulation, Bacterial , Lysogeny , Shiga Toxin 2/genetics , Shiga-Toxigenic Escherichia coli/genetics , Shiga-Toxigenic Escherichia coli/metabolism
5.
Biomed Microdevices ; 16(3): 375-85, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24562605

ABSTRACT

This paper introduces a disposable battery-driven heating system for loop-mediated isothermal DNA amplification (LAMP) inside a centrifugally-driven DNA purification platform (LabTube). We demonstrate LabTube-based fully automated DNA purification of as low as 100 cell-equivalents of verotoxin-producing Escherichia coli (VTEC) in water, milk and apple juice in a laboratory centrifuge, followed by integrated and automated LAMP amplification with a reduction of hands-on time from 45 to 1 min. The heating system consists of two parallel SMD thick film resistors and a NTC as heating and temperature sensing elements. They are driven by a 3 V battery and controlled by a microcontroller. The LAMP reagents are stored in the elution chamber and the amplification starts immediately after the eluate is purged into the chamber. The LabTube, including a microcontroller-based heating system, demonstrates contamination-free and automated sample-to-answer nucleic acid testing within a laboratory centrifuge. The heating system can be easily parallelized within one LabTube and it is deployable for a variety of heating and electrical applications.


Subject(s)
Centrifugation/instrumentation , DNA/genetics , DNA/isolation & purification , Heating/economics , Heating/instrumentation , Polymerase Chain Reaction/instrumentation , Systems Integration , Automation , Disposable Equipment , Electric Power Supplies , Food Analysis , Shiga-Toxigenic Escherichia coli/cytology
6.
Anim Reprod Sci ; 127(3-4): 176-82, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21907505

ABSTRACT

The presence of bacteria in boar semen causes economic losses in artificial insemination (AI) centers, as a consequence of alterations on boar sperm quality. For this reason, the effects of different concentrations of enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC) on boar sperm quality were determined in this study, by conducting two experiments. The first one consisted of assessing these effects on boar sperm quality after incubating the inoculated doses at 37°C for a 96-h period, whereas the second inoculated doses were stored at 15°C during 11 days. In both experiments, the infective concentrations ranged from 10(8)cfu mL(-1) to 10(2)cfu mL(-1); the negative control being a non-inoculated dose. Twenty-four hours after inoculation, we checked by PCR for the presence of bacteria in all tubes. Sperm quality (sperm motility, sperm viability and sperm morphology) was assessed at 24h, 48h, 72h and 96h after inoculations in the first experiment (37°C), and after 3, 5, 7, 9 and 11 days in the second (15°C). Whereas no changes were observed in sperm morphology in both experiments, the percentages of progressive motile spermatozoa dramatically diminished after 24h of incubation at 37°C, the effect being more detrimental at the highest infective concentration of microbes. Moreover, a significant decrease in the percentage of viable spermatozoa in the tube inoculated with the highest concentration (10(8)cfu mL(-1)) was detected after 24h of incubating contaminated doses at 37°C. After 48h of incubation, the presence of infective concentrations of ETEC and VTEC from 10(8)cfu mL(-1) to 10(3)cfu mL(-1) resulted in a significant diminution in the percentage of viable spermatozoa. These results suggest that ETEC and VTEC PCR analyses should be done in doses destined for AI to minimize the use of doses with diminished sperm quality due to the presence of bacteria and to avoid the potential spread of infective diseases.


Subject(s)
Bacterial Load/physiology , Enterotoxigenic Escherichia coli/physiology , Shiga-Toxigenic Escherichia coli/physiology , Spermatozoa/cytology , Spermatozoa/microbiology , Swine , Acrosome Reaction , Animals , Cell Survival , Enterotoxigenic Escherichia coli/cytology , Enterotoxigenic Escherichia coli/isolation & purification , Escherichia coli Infections/microbiology , Escherichia coli Infections/physiopathology , Male , Mitochondria/physiology , Quality Control , Semen Analysis , Shiga-Toxigenic Escherichia coli/cytology , Shiga-Toxigenic Escherichia coli/isolation & purification , Spermatozoa/physiology , Swine/microbiology , Swine/physiology , Swine Diseases/microbiology , Swine Diseases/physiopathology
7.
Mol Cell Probes ; 24(4): 190-5, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20230890

ABSTRACT

We developed a completely homogeneous duplex loop-mediated isothermal amplification (LAMP) method. The present LAMP method employed a combination of a 6-carboxyfluorescein (FAM)-labeled primer (donor) for one target gene, a non-labeled primer for the other, and an intercalator ethidium bromide (EtBr) dye (acceptor) on the basis of fluorescence resonance energy transfer (FRET) between the FAM donor and EtBr acceptor. Measuring changes in fluorescence of FAM enabled the LAMP method to detect two different genes simultaneously. This method was used to detect Shiga toxin genes in Shiga toxigenic Escherichia coli isolates, demonstrating simultaneous detection of two different genes with rapidity and accuracy.


Subject(s)
DNA Primers/metabolism , Fluoresceins/metabolism , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Conformation , Shiga Toxin 1/genetics , Shiga Toxin 2/genetics , Shiga-Toxigenic Escherichia coli/isolation & purification , Ethidium/metabolism , Fluorescent Dyes/metabolism , Intercalating Agents/metabolism , Sensitivity and Specificity , Shiga-Toxigenic Escherichia coli/cytology , Shiga-Toxigenic Escherichia coli/genetics , Staining and Labeling , Temperature , Time Factors
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