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Sample records for heat-labile enterotoxin promotes

  1. Heat-labile enterotoxin of Escherichia coli promotes intestinal colonization of Salmonella enterica.

    PubMed

    Verbrugghe, Elin; Van Parys, Alexander; Leyman, Bregje; Boyen, Filip; Arnouts, Sven; Lundberg, Urban; Ducatelle, Richard; Van den Broeck, Wim; Yekta, Maryam Atef; Cox, Eric; Haesebrouck, Freddy; Pasmans, Frank

    2015-12-01

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of infantile and travellers' diarrhoea, which poses a serious health burden, especially in developing countries. In addition, ETEC bacteria are a major cause of illness and death in neonatal and recently weaned pigs. The production of a heat-labile enterotoxin (LT) promotes the colonization and pathogenicity of ETEC and may exacerbate co-infections with other enteric pathogens such as Salmonella enterica. We showed that the intraintestinal presence of LT dramatically increased the intestinal Salmonella Typhimurium load in experimentally inoculated pigs. This could not be explained by direct alteration of the invasion or survival capacity of Salmonella in enterocytes, in vitro. However, we demonstrated that LT affects the enteric mucus layer composition in a mucus-secreting goblet cell line by significantly decreasing the expression of mucin 4. The current results show that LT alters the intestinal mucus composition and aggravates a Salmonella Typhimurium infection, which may result in the exacerbation of the diarrhoeal illness.

  2. Adaptation of the staphylococcal coagglutination technique for detection of heat-labile enterotoxin of Escherichia coli.

    PubMed Central

    Brill, B M; Wasilauskas, B L; Richardson, S H

    1979-01-01

    Protein A-containing staphylococci coated with specific antiserum were tested for heat-labile enterotoxin of Escherichia coli. The immunological cross-reactivity of E. coli heat-labile enterotoxin with Vibrio cholerae toxin (choleragen) was the basis for sensitizing stabilized suspensions of the Cowan I strain of Staphylococcus aureus with anticholeragen. Unconcentrated culture supernatant fluid containing E. coli heat-labile enterotoxin produced macroscopic agglutination when mixed with sensitized staphylococci in capillary tubes. A total of 15 toxigenic and 61 nontoxigenic isolates were tested by the staphylococcal coagglutination technique in a coded fashion and found to be in agreement with previous results of the Chinese hamster ovary cell assay and the passive immune hemolysis test. The staphylococcal coagglutination technique is simple, relatively inexpensive to perform, and requires the immunoglobulin fraction of anticholeragen as the only specific reagent. The staphylococcal coagglutination technique appears to have potential for routine use in diagnostic microbiology laboratories. Images PMID:372214

  3. Immunological Interrelationships of Coliform Heat-Labile and Heat-Stable Enterotoxins

    DTIC Science & Technology

    1981-09-01

    FA, Engert RF: Immunological interrelationships between cholera toxin and the heat -labile and hoat-stable enterotoxins of coliform bacteria . Infec...When Date Enterd) -3- SUMMARY These investigations (a) established the fact that species of coliform bacteria other than ETEC strains of E. coZi...elaborate enterotoxins which alter gastrointestinal physiology, and (b) showed that immunization with either E. coli (ETEC) LT or ST toxin arouses an

  4. Expression of Escherichia coli heat-labile enterotoxin B subunit (LTB) in Saccharomyces cerevisiae.

    PubMed

    Rezaee, Mohammad Ahangarz; Rezaee, Abbas; Moazzeni, Seyed Mohammad; Salmanian, Ali Hatef; Yasuda, Yoko; Tochikubo, Kunio; Pirayeh, Shahin Najar; Arzanlou, Mohsen

    2005-08-01

    Heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli (ETEC) is both a strong mucosal adjuvant and immunogen. It is a subunit vaccine candidate to be used against ETEC-induced diarrhea. It has already been expressed in several bacterial and plant systems. In order to construct yeast expressing vector for the LTB protein, the eltB gene encoding LTB was amplified from a human origin enterotoxigenic E. coli DNA by PCR. The expression plasmid pLTB83 was constructed by inserting the eltB gene into the pYES2 shuttle vector immediately downstream of the GAL1 promoter. The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The LTB protein was detected in the total soluble protein of the yeast by SDS-PAGE analysis. Quantitative ELISA showed that the maximum amount of LTB protein expressed in the yeast was approximately 1.9% of the total soluble protein. Immunoblotting analysis showed the yeast-derived LTB protein was antigenically indistinguishable from bacterial LTB protein. Since the whole-recombinant yeast has been introduced as a new vaccine formulation the expression of LTB in S. cerevisiae can offer an inexpensive yet effective strategy to protect against ETEC, especially in developing countries where it is needed most.

  5. Detection of heat-labile enterotoxin-like activity in stools of patients with cholera and Escherichia coli diarrhea.

    PubMed Central

    Echeverria, P; Verheart, L; Ulyanco, C V; Santiago, L T

    1978-01-01

    The Y1 adrenal cell tissue culture assay was used to detect heat-labile enterotoxin-like activity in the stools of 14 of 74 patients with diarrhea. A positive effect of the stool on the adrenal cells was heat-labile and neutralized by cholera antitoxin. Enterotoxin-like activity was detected in the stools of 10 of 30 patients with cholera and in those of 2 of 4 from whom heat-labile Escherichia coli were isolated. None of the stools from nine individuals with Vibrio parahaemolyticus, Salmonella, or Shigella infections were positive. Two of 31 individuals from whom no pathogens were isolated had detectable toxin-like activity in their stools. The Y1 adrenal cell assay provides a rapid method of diagnosing heat-labile enterotoxigenic diarrhea and could be an adjunct in epidemiological studies of gastroenteritis. PMID:342414

  6. Comparison of Y1 mouse adrenal cell and coagglutination assays for detection of Escherichia coli heat labile enterotoxin.

    PubMed Central

    Chapman, P A; Daly, C M

    1989-01-01

    A commercial coagglutination assay (COA; Phadebact LT-ETEC) was compared with a Y1 mouse adrenal cell assay for detecting the heat labile enterotoxin of Escherichia coli. Of four different media evaluated for use with the COA, only one (modified blood agar) gave a positive result with all strains known to produce heat labile enterotoxin. With modified blood agar, the COA detected 74 (85%) of 87 such strains. Eighty six strains negative by cell culture assay were also negative by COA, and one strain positive by COA could not be confirmed by cell culture. The Phadebact LT-ETEC kit provides a simple, sensitive, and economical method for detecting E coli heat labile enterotoxin. PMID:2668342

  7. Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin.

    PubMed Central

    Spangler, B D

    1992-01-01

    Cholera and the related Escherichia coli-associated diarrheal disease are important problems confronting Third World nations and any area where water supplies can become contaminated. The disease is extremely debilitating and may be fatal in the absence of treatment. Symptoms are caused by the action of cholera toxin, secreted by the bacterium Vibrio cholerae, or by a closely related heat-labile enterotoxin, produced by Escherichia coli, that causes a milder, more common traveler's diarrhea. Both toxins bind receptors in intestinal epithelial cells and insert an enzymatic subunit that modifies a G protein associated with the adenylate cyclase complex. The consequent stimulated production of cyclic AMP, or other factors such as increased synthesis of prostaglandins by intoxicated cells, initiates a metabolic cascade that results in the excessive secretion of fluid and electrolytes characteristic of the disease. The toxins have a very high degree of structural and functional homology and may be evolutionarily related. Several effective new vaccine formulations have been developed and tested, and a growing family of endogenous cofactors is being discovered in eukaryotic cells. The recent elucidation of the three-dimensional structure of the heat-labile enterotoxin has provided an opportunity to examine and compare the correlations between structure and function of the two toxins. This information may improve our understanding of the disease process itself, as well as illuminate the role of the toxin in studies of signal transduction and G-protein function. Images PMID:1480112

  8. Heat-Labile Enterotoxin IIa, a Platform To Deliver Heterologous Proteins into Neurons

    PubMed Central

    Chen, Chen; Przedpelski, Amanda; Tepp, William H.; Pellett, Sabine; Johnson, Eric A.

    2015-01-01

    ABSTRACT Cholera toxin (CT) and the related heat-labile enterotoxins (LT) of Escherichia coli have been implicated as adjuvants in human therapies, but reactivity upon intranasal delivery dampened efforts to develop other clinical applications. However, each CT family member variant has unique biological properties that may warrant development as therapeutic platforms. In the current study, a nontoxic variant of the heat-labile enterotoxin IIa (LTIIa) was engineered to deliver heterologous, functional proteins into the cytosol of neurons. As proof of principle, the LTIIa variant delivered two cargos into neurons. LTIIa delivered β-lactamase efficiently into cells containing complex gangliosides, such as GD1b, as host receptors. LTIIa delivery of β-lactamase was sensitive to brefeldin A, an inhibitor that collapses the Golgi compartment into the endoplasmic reticulum, but not sensitive to treatment with botulinum neurotoxin D (BoNT/D), an inhibitor of synaptic vesicle cycling. LTIIa delivered a single-chain, anti-BoNT/A camelid antibody that inhibited SNAP25 cleavage during post-BoNT/A exposure of neurons. Delivery of functional, heterologous protein cargos into neurons demonstrates the potential of LTII variants as platforms to deliver therapies to inactivate toxins and microbial infections and to reverse the pathology of human neurodegenerative diseases. PMID:26265718

  9. Oral immunization of mice with attenuated Salmonella enteritidis containing a recombinant plasmid which codes for production of the B subunit of heat-labile Escherichia coli enterotoxin.

    PubMed Central

    Clements, J D; Lyon, F L; Lowe, K L; Farrand, A L; el-Morshidy, S

    1986-01-01

    We used Salmonella enteritidis serotype dublin strain SL1438, a nonreverting, aromatic-dependent, histidine-requiring mutant, as a recipient for a recombinant plasmid coding for production of the nontoxic B subunit of the heat-labile Escherichia coli enterotoxin. The S. enteritidis derivative EL23 produced heat-labile enterotoxin subunit B that was indistinguishable from heat-labile enterotoxin subunit B produced by strains of E. coli or Salmonella typhi harboring the same plasmid. Mice immunized orally with strain EL23 developed progressively increasing mucosal and serum antibody responses to both heat-labile enterotoxin subunit B and to the lipopolysaccharide of the vaccine strain. The mucosal antibody response was shown to be immunoglobulin A specific and to be capable of neutralizing the biological activities of both E. coli heat-labile enterotoxin and cholera enterotoxin in vitro. Images PMID:3527989

  10. Size-optimized galactose-capped gold nanoparticles for the colorimetric detection of heat-labile enterotoxin at nanomolar concentrations.

    PubMed

    Poonthiyil, Vivek; Golovko, Vladimir B; Fairbanks, Antony J

    2015-05-14

    The development of a galactose-capped gold nanoparticle-based colorimetric sensor for the detection of the lectin heat-labile enterotoxin is reported. Heat-labile enterotoxin is one of the pathogenic agents responsible for the intestinal disease called 'traveller's diarrhoea'. By means of specific interaction between galactose moieties attached to the surface of gold nanoparticles and receptors on the B-subunit of heat-labile enterotoxin (LTB), the gold nanoparticles reported here act as an efficient colorimetric sensor, which can detect the toxin at nanomolar concentrations. The effect of gold nanoparticle size on the detection sensitivity was investigated in detail. Amongst the various sizes of gold nanoparticles studied (2, 7, 12, and 20 nm), the 12 nm sized gold nanoparticles were found to be the most efficient, with a minimum heat-labile enterotoxin detection concentration of 100 nM. The red to purple colour change of the gold nanoparticle solution occurred within two minutes, indicating rapid toxin sensing.

  11. Rapid latex particle agglutination test for Escherichia coli strains of porcine origin producing heat-labile enterotoxin.

    PubMed Central

    Finkelstein, R A; Yang, Z S; Moseley, S L; Moon, H W

    1983-01-01

    A latex particle agglutination test previously shown to be suitable for the rapid identification of Escherichia coli strains of human origin producing heat-labile enterotoxin (R. A. Finkelstein and Z. Yang, J. Clin. Microbiol. 18:23-28) is equally applicable to strains of porcine origin. PMID:6361056

  12. Factors Affecting Release of Heat-Labile Enterotoxin by Enterotoxigenic Escherichia coli

    PubMed Central

    Kunkel, Steven L.; Robertson, Donald C.

    1979-01-01

    Various conditions affecting the release of heat-labile enterotoxin (LT) by enterotoxigenic Escherichia coli have been examined. The pH of a defined medium containing three amino acids, M-9 salts, and 0.5% glucose decreased to less than 7.0 in early log phase of growth, and no extracellular LT was detected. Adjustment of the pH at 8 h from 6.0 to 8.0 resulted in a concomitant increase in LT activity in culture supernatants. The release of cell-associated LT was significantly reduced by preincubation with protease inhibitors and increased by preincubation with trypsin. Cell-associated LT was not released by pH adjustment of cells grown at 21°C; however, polymyxin B treatment released a toxin species active in only the pigeon erythrocyte lysate (PEL) assay system. As the growth temperature was increased, polymyxin B released toxin species which exhibited both PEL and Y-1 adrenal tumor cell activity. Polymyxin B extracts of enterotoxigenic E. coli in early log phase grown at 37°C possessed only PEL activity, whereas extracts from cells in late-log and stationary phases had biological activity in both assay systems. Also, LT released by pH adjustment from mid-log to stationary phase was active in both PEL and Y-1 adrenal tumor cell assays. Gel electrophoresis of polymyxin B extracts revealed at least three molecular weight species active in either the PEL (22,000 daltons and 30,000 daltons) or both the PEL and the Y-1 adrenal tumor cell assay (72,000 daltons), depending on the growth temperature. These observations may help to explain the chemical and biological heterogeneity of most LT preparations and facilitate purification of LT by increasing the yield of enterotoxin. PMID:37162

  13. Inhibition of Escherichia coli heat-labile enterotoxin-induced diarrhea by Chaenomeles speciosa.

    PubMed

    Chen, Jaw-Chyun; Chang, Yuan-Shiun; Wu, Shih-Lu; Chao, De-Cheng; Chang, Chih-Shiang; Li, Chia-Cheng; Ho, Tin-Yun; Hsiang, Chien-Yun

    2007-09-05

    Enterotoxigenic Escherichia coli (ETEC) is responsible for millions of deaths in developing countries. Heat-labile enterotoxin (LT), the virulence factor of ETEC, induces diarrhea by initially binding to the G(M1) on the surface of intestinal epithelial cells and consequently leading to the massive loss of fluid and ions from cells. Fruit of Chaenomeles (FC), the dried fruit of Chaenomeles speciosa, has been used for diarrhea in China. However, the anti-diarrheal mechanism of FC is still unclear. In this study, we demonstrated that FC extract (FCE) inhibited the LT-induced diarrhea in mice by blocking the binding of the B subunit of LT (LTB) to G(M1). The ethyl acetate (EA) soluble fraction was the most active fraction of FC that significantly abolished the LTB and G(M1) interaction. Furthermore, the oleanolic acid, ursolic acid, and betulinic acid from EA fraction, blocked the toxin binding effects, resulting in the suppression of LT-induced diarrhea. Moreover, by docking techniques, these compounds fitted LTB well via hydrogen bonds and hydrophobic contacts with amino acid residues of LTB. In conclusion, our findings suggested that oleanolic acid, ursolic acid, and betulinic acid were the active constituents from FC and might be considered as lead therapeutic agents in the treatment of LT-induced diarrhea.

  14. Effect of fractions of Ethiopian And Norwegian colostrum on rotavirus and Escherichia coli heat-labile enterotoxin.

    PubMed Central

    Otnaess, A B; Orstavik, I

    1981-01-01

    Samples of colostrum from both Ethiopian and Norwegian women contained antirotavirus activities of immunoglobulin and non-immunoglobulin nature. No significant differences in rotavirus immunoglobulin A or in rotavirus-inhibiting activity were found between samples from the two countries. The non-immunoglobulin inhibitory activity was trypsin sensitive and heat stable (100 degrees C for 10 min). Escherichia coli heat-labile enterotoxin antibodies were measured in the colostrum samples by enzyme-linked immunosorbent assay. No E. coli enterotoxin-specific immunoglobulin A was detected, possibly due to the high background caused by the nonspecific adsorption of immunoglobulin A to the enzyme-linked immunosorbent assay plates in the absence of toxin. A total of 5 of 15 Ethiopian colostrum samples and 0 of 11 Norwegian colostrum samples neutralized the effect of E. coli heat-labile enterotoxin on YI adrenal cells. Both the Ethiopian and the Norwegian colostrum samples contained a non-immunoglobulin enterotoxin-inhibitory activity when the toxin was measured by enzyme-linked immunosorbent assay. This inhibitory activity was not trypsin sensitive, and extraction by chloroform-methanol indicated that the inhibitor was of a lipid nature. PMID:6268544

  15. Refined structure of Escherichia coli heat-labile enterotoxin, a close relative of cholera toxin.

    PubMed

    Sixma, T K; Kalk, K H; van Zanten, B A; Dauter, Z; Kingma, J; Witholt, B; Hol, W G

    1993-04-05

    Heat-labile enterotoxin (LT) from Escherichia coli is a bacterial protein toxin with an AB5 multimer structure, in which the B pentamer has a membrane binding function and the A subunit is needed for enzymatic activity. The LT crystal structure has been solved using a combination of multiple isomorphous replacement, fivefold averaging and molecular dynamics refinement. Phase combination using all these sources of phase information was of crucial importance for the chain tracing. The structure has now been refined to 1.95 A resolution, resulting in a model containing 6035 protein atoms and 293 solvent molecules with a crystallographic R-factor of 18.2% and good stereochemistry. The B subunits are arranged as a highly stable pentamer with a donut shape. Each subunit takes part in approximately 30 inter-subunit hydrogen bonds and six salt bridges with its two neighbors, whilst burying a large surface area. The A subunit has higher temperature factors and less well-defined secondary structure than the B subunits. It interacts with the B pentamer mainly via the C-terminal A2 fragment, which runs through the highly charged central pore of the B subunits. The pore contains at least 66 water molecules, which fill the space left by the A2 fragment. A detailed analysis of the contacts between A and B subunits showed that most specific contacts occur at the entrance of the central pore of the B pentamer, while the contacts within the pore are mainly hydrophobic and water mediated, with the exception of two salt bridges. Only a few contacts exist between the A1 fragment and the B pentamer, showing that the A2 fragment functions as a "linker" of the A and B parts of the protein. Interacting with the A subunit by the B subunits does not cause large deviations from a common B subunit structure, and the 5-fold symmetry is well maintained. A potential NAD(+)-binding site is located in an elongated crevice at the interface of two small sheets in the A1 fragment. At the back of this

  16. Analysis and modeling of heat-labile enterotoxins of Escherichia coli suggests a novel space with insights into receptor preference.

    PubMed

    Krishna Raja, M; Ghosh, Asit Ranjan; Vino, S; Sajitha Lulu, S

    2015-01-01

    Features of heat-labile enterotoxins of Escherichia coli which make them fit to use as novel receptors for antidiarrheals are not completely explored. Data-set of 14 different serovars of enterotoxigenic Escherichia coli producing heat-labile toxins were taken from NCBI Genbank database and used in the study. Sequence analysis showed mutations in different subunits and also at their interface residues. As these toxins lack crystallography structures, homology modeling using Modeller 9.11 led to the structural approximation for the E. coli producing heat-labile toxins. Interaction of modeled toxin subunits with proanthocyanidin, an antidiarrheal showed several strong hydrogen bonding interactions at the cost of minimized energy. The hits were subsequently characterized by molecular dynamics simulation studies to monitor their binding stabilities. This study looks into novel space where the ligand can choose the receptor preference not as a whole but as an individual subunit. Mutation at interface residues and interaction among subunits along with the binding of ligand to individual subunits would help to design a non-toxic labile toxin and also to improve the therapeutics.

  17. Immunologic Interrelationships of Coliform Heat-Labile and Heat-Stable Enterotoxins

    DTIC Science & Technology

    1980-01-15

    immunization program to prevent diarrheal disease due to Intestinal contamination by enterotoxigenic straing of coliform bacteria . We have found that...pnotobiotic rats are challenged by intestinal contamination with strains of E. coli which produce the heat-labile toxin , FOB W 13 -niouos wI, soov esis...extended protection against challenge with either this toxin or viable strains of LT-producing E. coZi. Current investigations are addressed at

  18. Evaluation of heat-labile enterotoxins type IIa and type IIb in the pathogenicity of enterotoxigenic Escherichia coli for neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Type II heat-labile enterotoxins (LT-II) have been reported in Escherichia coli isolates from humans, animals, food and water samples. The roles of the antigenically distinguishable LT-IIa and LT-IIb subtypes in pathogenesis and virulence of enterotoxigenic E. coli (ETEC) have not been previously re...

  19. Comparison of two GM1-erythrocyte assays to detect heat-labile Escherichia coli enterotoxin in stool specimens.

    PubMed Central

    Germani, Y; Guesdon, J L; Phalente, L; Begaud, E; Moreau, J P

    1988-01-01

    Two erythrocyte immunoassay techniques to detect the presence of Escherichia coli heat-labile enterotoxin (LTh) in stool supernatants and cell-free culture supernatants were compared. In the competitive assay, GM1 ganglioside was coated onto V-shaped-well microdilution plates and enterotoxin was coupled to sheep erythrocytes. As little as 0.8 ng of LTh per ml was detected by this method, which was based on the competition between the LTh of the test sample and the sensitized erythrocytes. The second assay made use of chimera antibody prepared by coupling polyclonal anti-LTh antibody to a monoclonal antibody specific for sheep erythrocytes. In this case, LTh, which was specifically bound to a GM1 ganglioside-coated plate, was detected by successively adding the chimera antibody and sheep erythrocytes. The limit of detection of the chimera antibody erythrocyte immunoassay was 0.2 ng/ml. Stool samples were collected from 167 infants hospitalized for diarrhea in the hospital of Noumea, New Caledonia. False-negative reactions due to proteases present in the stool samples were avoided by the addition of phenylmethylsulfonyl fluoride. Images PMID:3290242

  20. Preparation of biocompatible heat-labile enterotoxin subunit B-bovine serum albumin nanoparticles for improving tumor-targeted drug delivery via heat-labile enterotoxin subunit B mediation.

    PubMed

    Zhao, Liang; Su, Rongjian; Cui, Wenyu; Shi, Yijie; Liu, Liwei; Su, Chang

    2014-01-01

    Heat-labile enterotoxin subunit B (LTB) is a non-catalytic protein from a pentameric subunit of Escherichia coli. Based on its function of binding specifically to ganglioside GM1 on the surface of cells, a novel nanoparticle (NP) composed of a mixture of bovine serum albumin (BSA) and LTB was designed for targeted delivery of 5-fluorouracil to tumor cells. BSA-LTB NPs were characterized by determination of their particle size, polydispersity, morphology, drug encapsulation efficiency, and drug release behavior in vitro. The internalization of fluorescein isothiocyanate-labeled BSA-LTB NPs into cells was observed using fluorescent imaging. Results showed that BSA-LTB NPs presented a narrow size distribution with an average hydrodynamic diameter of approximately 254±19 nm and a mean zeta potential of approximately -19.95±0.94 mV. In addition, approximately 80.1% of drug was encapsulated in NPs and released in the biphasic pattern. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that BSA-LTB NPs exhibited higher cytotoxic activity than non-targeted NPs (BSA NPs) in SMMC-7721 cells. Fluorescent imaging results proved that, compared with BSA NPs, BSA-LTB NPs could greatly enhance cellular uptake. Hence, the results indicate that BSA-LTB NPs could be a potential nanocarrier to improve targeted delivery of 5-fluorouracil to tumor cells via mediation of LTB.

  1. Influence of Route of Administration on Immediate and Extended Protection in Rats Immunized with Escherichia coli Heat-Labile Enterotoxin

    PubMed Central

    Klipstein, Frederick A.; Engert, Richard F.

    1980-01-01

    The effect of route of administration, dosage, and number of boosts employed during immunization with the polymyxin-release form of Escherichia coli heat-labile (LT) enterotoxin on the degree and duration of protection afforded was evaluated in rats which were challenged by the ligated loop technique. Increasing the boosting dosage by fivefold from 50 to 250 μg resulted in a marked increase in protection against challenge with toxin in rats immunized either just by the parenteral route (i.p./i.p.) or by a parenteral prime, followed by peroral boosts (i.p./p.o.) in rats pretreated with cimetidine to ablate gastric secretions; such was not the case, however, even with a 50-fold increase in dosage in rats immunized just by the peroral route (p.o./p.o.). Four weekly peroral boosts were required to achieve the strongest degree of protection. Increasing the boosting dosage also increased the degree of protection against challenge with viable LT+/ST− and LT+/ST+ strains (ST indicates heat-stable enterotoxin) in rats immunized by the i.p./p.o., but not by the i.p./i.p., route; no protection was evident against an LT−/ST+ strain. Protection was lost within 3 weeks after immunization in rats immunized by the i.p./i.p. route. In contrast, protection was extended over the 3-month observation period in those immunized by the i.p./p.o. route; the degree of protection was enhanced in rats which received an additional boost at 2 months. These observations establish the fact that immunization with LT is similar to that with cholera toxin in that arousal of the local immune intestinal response by means of peroral immunization provides maximal extended protection. PMID:6987180

  2. Immunochromatographic detection of the heat-labile enterotoxin of enterotoxigenic Escherichia coli with cross-detection of cholera toxin.

    PubMed

    Arimitsu, Hideyuki; Sasaki, Keiko; Tsuji, Takao

    2017-01-01

    Here, we report the development of an immunochromatographic test strip that can detect heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli. Five types of monoclonal antibody (mAb)-producing hybridomas were isolated: three mAbs were A subunit specific and two were B subunit specific. Four mAbs also cross-reacted with both LT proteins derived from swine and human E. coli strains, but only one mAb 57B9 additionally cross-reacted with cholera toxin. Thus, mAb 57B9 was used to form a gold colloid-conjugated antibody for the immunochromatographic test by combination with polyclonal anti-LT rabbit IgG. This test strip detected not only LT in the culture supernatant of LT gene-positive strains, but also cholera toxin in the culture supernatant of Vibrio cholerae. These results indicate that this test strip is suitable for the diagnosis of both enterotoxigenic E. coli and V. cholerae infection.

  3. The suppressive activities of six sources of medicinal ferns known as gusuibu on heat-labile enterotoxin-induced diarrhea.

    PubMed

    Chang, Hung-Chi; Chen, Jaw-Chyun; Yang, Jiun-Long; Tsay, Hsin-Sheng; Hsiang, Chien-Yun; Ho, Tin-Yun

    2014-02-18

    Diarrheal disease is one of the most important worldwide health problems. Enterotoxigenic Escherichia coli (ETEC) is the most frequently isolated enteropathogen in diarrheal diseases. In developing countries, a very large number of people, especially children, suffer from diarrhea. To combat this problem, World Health Organization has constituted the Diarrhea Diseases Control Program which guides studies on traditional medicinal practices and preventive measures. Gusuibu, a traditional folk medicine, has been claimed to heal certain types of diarrhea. However, so far no scientific study has been carried out on the anti-diarrheal mechanism of Gusiubu. The present study was performed to examine the suppressive activities of ethanol extracts of six sources of folk medicinal ferns used as Gusuibu on heat-labile enterotoxin (LT)-induced diarrhea. Inhibitory effects of six sources were evaluated on the ETEC LT subunit B (LTB) and monosialotetrahexosylganglioside (GMI) interaction by GM1-enzyme linked immunosorbent assay and patent mouse gut assay. Our results indicated that Drynaria fortunei had no anti-diarrheal effect, while, among the remaining five folk medicinal ferns, four belonging to family Davalliaceae had significant abilities on both the blocking of LTB and GM1 interaction and the inhibition of LT-induced diarrhea. In conclusion, these findings suggested the potential application of Gusuibu as an anti-diarrheal remedy.

  4. Comparative Adjuvant Effects of Type II Heat-Labile Enterotoxins in Combination with Two Different Candidate Ricin Toxin Vaccine Antigens.

    PubMed

    Vance, David J; Greene, Christopher J; Rong, Yinghui; Mandell, Lorrie M; Connell, Terry D; Mantis, Nicholas J

    2015-12-01

    Type II heat-labile enterotoxins (HLTs) constitute a promising set of adjuvants that have been shown to enhance humoral and cellular immune responses when coadministered with an array of different proteins, including several pathogen-associated antigens. However, the adjuvant activities of the four best-studied HLTs, LT-IIa, LT-IIb, LT-IIb(T13I), and LT-IIc, have never been compared side by side. We therefore conducted immunization studies in which LT-IIa, LT-IIb, LT-IIb(T13I), and LT-IIc were coadministered by the intradermal route to mice with two clinically relevant protein subunit vaccine antigens derived from the enzymatic A subunit (RTA) of ricin toxin, RiVax and RVEc. The HLTs were tested with low and high doses of antigen and were assessed for their abilities to stimulate antigen-specific serum IgG titers, ricin toxin-neutralizing activity (TNA), and protective immunity. We found that all four HLTs tested were effective adjuvants when coadministered with RiVax or RVEc. LT-IIa was of particular interest because as little as 0.03 μg when coadministered with RiVax or RVEc proved effective at augmenting ricin toxin-specific serum antibody titers with nominal evidence of local inflammation. Collectively, these results justify the need for further studies into the mechanism(s) underlying LT-IIa adjuvant activity, with the long-term goal of evaluating LT-IIa's activity in humans.

  5. A Novel Method for Efficient Preparation of Mucosal Adjuvant Escherichia coli Heat-Labile Enterotoxin Mutant (LTm) by Artificially Assisted Self-Assembly In Vitro.

    PubMed

    Liu, Di; Zhang, Na; Zheng, Wenyun; Guo, Hua; Wang, Xiaoli; Wang, Tianwen; Wang, Ping; Ma, Xingyuan

    2016-04-01

    As well-known powerful mucosal adjuvant proteins, Escherichia coli heat-labile enterotoxin (LT) and its non-toxic or low-toxic mutants (LTm) are capable of promoting strong mucosal immune responses to co-administered antigens in various types of vaccines. However, due to the complex composition and special structure, the yield of LTm directly from the recombinant genetic engineering strains is quite low. Here, we put forward a novel method to prepare LTm protein which designed, expressed, and purified three kinds of component subunits respectively and assembled them into a hexamer structure in vitro by two combination modes. In addition, by simulated in vivo environment of polymer protein assembly, the factors of the protein solution system which include environment temperature, pH, ionic strength of the solution, and ratio between each subunit were taken into consideration. Finally, we confirmed the optimal conditions of two assembly strategies and prepared the hexamer holotoxin in vitro. These results are not only an important significance in promoting large-scale preparation of the mucosal adjuvant LTm but also an enlightening to produce other multi-subunit proteins.

  6. Arousal of mucosal secretory immunoglobulin A antitoxin in rats immunized with Escherichia coli heat-labile enterotoxin.

    PubMed Central

    Klipstein, F A; Engert, R F; Clements, J D

    1982-01-01

    Specific serum and mucosal antitoxin levels were determined by enzyme-linked immunosorbent assays in rats immunized with Escherichia coli heat-labile enterotoxin (LT). Immunization by means of a parenteral prime followed by peroral boosts was the only approach that aroused titers of both serum immunoglobulin G (IgG) antitoxin and mucosal secretory IgA antitoxin that were increased fourfold or more over control values. Primary parenteral immunization was effective when given either intraperitoneally or subcutaneously with either Freund complete adjuvant or alum as the adjuvant. The magnitude of the nucosal secretory IgA antitoxin response and the degree of protection against challenge with either LT or viable LT-producing organisms were related to the number and dosage of peroral boosts. LT antigenicity, as determined by enzyme-linked immunosorbent assay, was progressively reduced by toxoiding it with increasing amounts of glutaraldehyde or a carbodiimide; when LT antigenicity was reduced by greater than 50%, the effectiveness of the toxoid in stimulating mucosal antitoxin and providing protection was compromised. Strong protection extended for more than 6 weeks only in rats immunized with a sufficient peroral dosage of LT to arouse mucosal secretory IgA antitoxin titers at least fourfold greater than those of controls. These observations indicate that the ability of LT to stimulate a mucosal secretory IgA antitoxin response is dependent on the antigenicity, route, and dosage of this immunogen; they suggest that the duration of protection in animals immunized by the peroral route is related to the extent of arousal of mucosal secretory IgA antitoxin. PMID:7129629

  7. Development of a vaccine of cross-linked heat-stable and heat-labile enterotoxins that protects against Escherichia coli producing either enterotoxin.

    PubMed Central

    Klipstein, F A; Engert, R F; Clements, J D

    1982-01-01

    A vaccine of cross-linked heat-stable (ST) and heat-labile (LT) toxins that protects against heterologous serotypes of strains of Escherichia coli which produce either the LT or ST enterotoxin was developed by conjugating ST to LT by the carbodiimide reaction. Three interrelated factors were found to affect the composition and properties of the final conjugate: (i) the amount of carbodiimide added to the toxins, (ii) the initial ratio of ST to LT, and (iii) the duration of the conjugation reaction. Optimal conjugation conditions were identified as a carbodiimide-to-toxin ratio of 10:1 by weight, an initial molar ratio of ST to LT of 100:1, and a conjugation reaction time of 96 h. This approach yielded a conjugate that contained 96% by moles and 36% by weight pure ST, determined with radioiodinated pure ST, and 34% by weight semi-pure ST, determined by the Lowry protein method. The retained antigenicities of the conjugated toxins, as determined by enzyme-linked immunosorbent assays, was greater than or equal to 82%, and their toxicities, as determined by the Y1 adrenal cell assay for LT and by the suckling mouse assay for ST, were reduced to less than or equal to 0.15%. Immunization of rats with this cross-linked ST-LT vaccine provided strong protection against challenge with either the LT or the ST toxin or with viable heterologous strains which produce these toxins, either singly or together. These observations indicate that conjugation of ST to LT results in a unique new immunogen in that ST acquires immunogenicity as a function of the reaction, LT retains most of its antigenicity, and the toxic properties of each individual toxin are greatly reduced. PMID:6749682

  8. Identification of a Gene within a Pathogenicity Island of Enterotoxigenic Escherichia coli H10407 Required for Maximal Secretion of the Heat-Labile Enterotoxin

    PubMed Central

    Fleckenstein, James M.; Lindler, Luther E.; Elsinghorst, Eric A.; Dale, James B.

    2000-01-01

    Studies of the pathogenesis of enterotoxigenic Escherichia coli (ETEC) have largely centered on extrachromosomal determinants of virulence, in particular the plasmid-encoded heat-labile (LT) and heat-stable enterotoxins and the colonization factor antigens. ETEC causes illnesses that range from mild diarrhea to severe cholera-like disease. These differences in disease severity are not readily accounted for by our current understanding of ETEC pathogenesis. Here we demonstrate that Tia, a putative adhesin of ETEC H10407, is encoded on a large chromosomal element of approximately 46 kb that shares multiple features with previously described E. coli pathogenicity islands. Further analysis of the region downstream from tia revealed the presence of several candidate open reading frames (ORFs) in the same transcriptional orientation as tia. The putative proteins encoded by these ORFs bear multiple motifs associated with bacterial secretion apparatuses. An in-frame deletion in one candidate gene identified here as leoA (labile enterotoxin output) resulted in marked diminution of secretion of the LT enterotoxin and lack of fluid accumulation in a rabbit ileal loop model of infection. Although previous studies have suggested that E. coli lacks the capacity to secrete LT, our studies show that maximal release of LT from the periplasm of H10407 is dependent on one or more elements encoded on a pathogenicity island. PMID:10768971

  9. Humoral immune response to the heat-labile enterotoxin of Escherichia coli in naturally acquired diarrhea and antitoxin determination by passive immune hemolysis.

    PubMed Central

    Evans, D J; Ruiz-Palacios, G; Evans, D E; DuPont, H L; Pickering, L K; Olarte, J

    1977-01-01

    Acute- and convalescent-phase sera from 132 students attending a university in rural Mexico were assayed for antibody against the heat-labile enterotoxin (LT) of Escherichia coli by neutralization of LT activity in the Y-1 adrenal cell assay and by passive immune hemolysis of LT-sensitized sheep erythrocytes. The two titration methods produced comparable results with respect to antitoxin responses detected. An inverse relationship was found between acute geometric mean antitoxin titer and the occurrence of diarrhea associated with LT-producing E. coli, especially in newly arrived students from the U.S.A. A significant correlation (P less than 0.00 5) was found between a rise in antitoxin titer detectable by the passive immune hemolysis technique and diarrhea with LT-producing E. coli isolated. Thus, humoral antitoxin titers appear to be a useful indicator of immune status with respect to enterotoxigenic (LT) E. coli diarrhea. PMID:330395

  10. A two-year survey of the incidence of heat-labile enterotoxin-producing Escherichia coli and other enteric pathogens in travellers returning to the Sheffield area.

    PubMed Central

    Chapman, P. A.; Mitchelmore, D. L.

    1988-01-01

    A case-controlled study of the incidence of heat-labile enterotoxin-producing Escherichia coli (LT+ETEC) and other enteric pathogens in travellers returning to the Sheffield area was conducted from May 1984 to April 1986. LT+ETEC were found in 35 (5.8%) of 600 travellers to developed countries (mainly popular Mediterranean holiday resorts), 36 (11.3%) of 320 travellers to less-developed countries, and 11 (0.9%) of 1282 control patients whose illness was not associated with recent travel abroad. A seasonal peak of LT+ETEC infection was observed only in travellers to developed countries, with infections being significantly commoner in August to October. There was no significant deviation from expected age/sex distribution of LT+ETEC infection. Strains of LT+ETEC from travellers produced more toxin than strains from control patients, strains from travellers to less-developed countries producing most of all. PMID:3053217

  11. Local and systemic immune responses induced by a recombinant chimeric protein containing Mycoplasma hyopneumoniae antigens fused to the B subunit of Escherichia coli heat-labile enterotoxin LTB.

    PubMed

    Marchioro, Silvana Beutinger; Fisch, Andressa; Gomes, Charles K; Jorge, Sérgio; Galli, Vanessa; Haesebrouck, Freddy; Maes, Dominiek; Dellagostin, Odir; Conceição, Fabricio R

    2014-09-17

    A multi-antigen chimera composed of three antigens of Mycoplasma hyopneumoniae (R1, P42, and NrdF) and the mucosal adjuvant Escherichia coli heat-labile enterotoxin B subunit (LTB) was constructed, and its antigenic and immunogenic properties were evaluated in mice and pigs. In addition, we compared the effect of the fusion and co-administration of these proteins in mice. Antibodies against each subunit recognized the chimeric protein. Intranasal and intramuscular immunization of mice with the chimeric protein significantly increased IgG and IgA levels in the serum and tracheobronchial lavages, respectively, against some of the antigens present in the chimeric. Swine immunized with the chimeric protein developed an immune response against all M. hyopneumoniae antigens present in the fusion with a statistically significant difference (P<0.05). The adjuvant rLTB enhanced the immune response in both fused and co-administered antigens; however, better results were obtained with the chimeric protein. This multi-antigen is a promising vaccine candidate that may help control M. hyopneumoniae infection.

  12. Secretory IgA-mediated protection against V. cholerae and heat-labile enterotoxin-producing enterotoxigenic Escherichia coli by rice-based vaccine

    PubMed Central

    Tokuhara, Daisuke; Yuki, Yoshikazu; Nochi, Tomonori; Kodama, Toshio; Mejima, Mio; Kurokawa, Shiho; Takahashi, Yuko; Nanno, Masanobu; Nakanishi, Ushio; Takaiwa, Fumio; Honda, Takeshi; Kiyono, Hiroshi

    2010-01-01

    Cholera and enterotoxigenic Escherichia coli (ETEC) are among the most common causes of acute infantile gastroenteritis globally. We previously developed a rice-based vaccine that expressed cholera toxin B subunit (MucoRice-CTB) and had the advantages of being cold chain–free and providing protection against cholera toxin (CT)–induced diarrhea. To advance the development of MucoRice-CTB for human clinical application, we investigated whether the CTB-specific secretory IgA (SIgA) induced by MucoRice-CTB gives longstanding protection against diarrhea induced by Vibrio cholerae and heat-labile enterotoxin (LT)–producing ETEC (LT-ETEC) in mice. Oral immunization with MucoRice-CTB stored at room temperature for more than 3 y provided effective SIgA-mediated protection against CT- or LT-induced diarrhea, but the protection was impaired in polymeric Ig receptor–deficient mice lacking SIgA. The vaccine gave longstanding protection against CT- or LT-induced diarrhea (for ≥6 months after primary immunization), and a single booster immunization extended the duration of protective immunity by at least 4 months. Furthermore, MucoRice-CTB vaccination prevented diarrhea in the event of V. cholerae and LT-ETEC challenges. Thus, MucoRice-CTB is an effective long-term cold chain–free oral vaccine that induces CTB-specific SIgA-mediated longstanding protection against V. cholerae– or LT-ETEC–induced diarrhea. PMID:20421480

  13. Oral immunisation of mice with a recombinant rabies virus vaccine incorporating the heat-labile enterotoxin B subunit of Escherichia coli in an attenuated Salmonella strain.

    PubMed

    Wang, Xuelin; Liu, Juan; Wu, Xiuping; Yu, Lu; Chen, Haiying; Guo, Heng; Zhang, Maolin; Li, Huiping; Liu, Xue; Sun, Shumin; Zhao, Lijing; Zhang, Xinyue; Gao, Lifang; Liu, Mingyuan

    2012-10-01

    To investigate effective new rabies vaccines, a fusion protein consisting of the rabies virus (RV) glycoprotein and the heat-labile enterotoxin B subunit of Escherichia coli (LTB) was successfully constructed and delivered in a live attenuated Salmonella strain LH430. Mice were immunised with LH430 carrying pVAX1-G, pVAX1-G-LTB or pVAX1-ori-G-LTB. The antibody titres of mice immunised with oral LH430 carrying pVAX1-G-LTB or pVAX1-ori-G-LTB were significantly higher than those of pVAX1-G-immunised mice. The results of the challenge with the rabies virus standard strain (CVS-11) showed that the LH430 strain carrying the G-LTB gene induced immunity and elevated IL-2 levels in immunised mice ((∗∗)P<0.01), whereas LH430 carrying pVAX1-G did not contribute to protection. These results show that LH430 carrying recombinant G-LTB could provide overall immunity against challenge with CVS-11 and should be considered to be a potential rabies vaccine.

  14. Discovery of the cell-penetrating function of A2 domain derived from LTA subunit of Escherichia coli heat-labile enterotoxin.

    PubMed

    Liu, Di; Guo, Hua; Zheng, Wenyun; Zhang, Na; Wang, Tianwen; Wang, Ping; Ma, Xingyuan

    2016-06-01

    Heat-labile enterotoxin (LT) is a protein toxin produced by enterotoxigenic Escherichia coli (ETEC). As a bacterial toxin, LT holotoxin can enter intestinal epithelial cells and cause diarrhea. In addition, LT is also a powerful mucosal adjuvant capable of enhancing the strong immune responses to co-administered antigens. However, the LT immunological mechanism is still not clear in some aspects, especially with the respect to how the LTA subunit functions alone. Here, we discovered that the A2 domain of LTA could carry a fluorescent protein into cells, whose function is similar to a cell-penetrating peptide. The transmembrane-transporting ability of the A2 domain is non-specific in its cell-penetrating function, which was shown through testing with different cell types. Moreover, the LTA2 fusion protein penetrated a fluorescently labeled cell membrane that identified LTA2 internalization through membrane transport pathways, and showed it finally localized in the endoplasmic reticulum. Furthermore, low-temperature stress and pharmacological agent treatments showed that the LTA2 internalization route is a temperature-dependent process involving the clathrin-mediated endocytosis and the macropinocytosis pathways. These results could explain the internalization of the LTA subunit alone without the LTB pentamer, contributing to a better understanding of LTA working as a mucosal adjuvant; they also suggest that the A2 domain could be used as a novel transport vehicle for research and treatment of disease.

  15. The catalytic A1 domains of cholera toxin and heat-labile enterotoxin are potent DNA adjuvants that evoke mixed Th1/Th17 cellular immune responses.

    PubMed

    Bagley, Kenneth; Xu, Rong; Ota-Setlik, Ayuko; Egan, Michael; Schwartz, Jennifer; Fouts, Timothy

    2015-01-01

    DNA encoded adjuvants are well known for increasing the magnitude of cellular and/or humoral immune responses directed against vaccine antigens. DNA adjuvants can also tune immune responses directed against vaccine antigens to better protect against infection of the target organism. Two potent DNA adjuvants that have unique abilities to tune immune responses are the catalytic A1 domains of Cholera Toxin (CTA1) and Heat-Labile Enterotoxin (LTA1). Here, we have characterized the adjuvant activities of CTA1 and LTA1 using HIV and SIV genes as model antigens. Both of these adjuvants enhanced the magnitude of antigen-specific cellular immune responses on par with those induced by the well-characterized cytokine adjuvants IL-12 and GM-CSF. CTA1 and LTA1 preferentially enhanced cellular responses to the intracellular antigen SIVmac239-gag over those for the secreted HIVBaL-gp120 antigen. IL-12, GM-CSF and electroporation did the opposite suggesting differences in the mechanisms of actions of these diverse adjuvants. Combinations of CTA1 or LTA1 with IL-12 or GM-CSF generated additive and better balanced cellular responses to both of these antigens. Consistent with observations made with the holotoxin and the CTA1-DD adjuvant, CTA1 and LTA1 evoked mixed Th1/Th17 cellular immune responses. Together, these results show that CTA1 and LTA1 are potent DNA vaccine adjuvants that favor the intracellular antigen gag over the secreted antigen gp120 and evoke mixed Th1/Th17 responses against both of these antigens. The results also indicate that achieving a balanced immune response to multiple intracellular and extracellular antigens delivered via DNA vaccination may require combining adjuvants that have different and complementary mechanisms of action.

  16. Relative importance of heat-labile enterotoxin in the causation of severe diarrheal disease in the gnotobiotic piglet model by a strain of enterotoxigenic Escherichia coli that produces multiple enterotoxins.

    PubMed

    Berberov, Emil M; Zhou, You; Francis, David H; Scott, Michael A; Kachman, Stephen D; Moxley, Rodney A

    2004-07-01

    Enterotoxigenic Escherichia coli (ETEC) strains that produce multiple enterotoxins are important causes of severe dehydrating diarrhea in human beings and animals, but the relative importance of these enterotoxins in the pathogenesis is poorly understood. Gnotobiotic piglets were used to study the importance of heat-labile enterotoxin (LT) in infection with an ETEC strain that produces multiple enterotoxins. LT(-) (DeltaeltAB) and complemented mutants of an F4(+) LT(+) STb(+) EAST1(+) ETEC strain were constructed, and the virulence of these strains was compared in gnotobiotic piglets expressing receptors for F4(+) fimbria. Sixty percent of the piglets inoculated with the LT(-) mutant developed severe dehydrating diarrhea and septicemia compared to 100% of those inoculated with the nalidixic acid-resistant (Nal(r)) parent and 100% of those inoculated with the complemented mutant strain. Compared to piglets inoculated with the Nal(r) parent, the mean rate of weight loss (percent per hour) of those inoculated with the LT(-) mutant was 67% lower (P < 0.05) and that of those inoculated with the complemented strain was 36% higher (P < 0.001). Similarly, piglets inoculated with the LT(-) mutant had significant reductions in the mean bacterial colony count (CFU per gram) in the ileum; bacterial colonization scores (square millimeters) in the jejunum and ileum; and clinical pathology parameters of dehydration, electrolyte imbalance, and metabolic acidosis (P < 0.05). These results indicate the significance of LT to the development of severe dehydrating diarrhea and postdiarrheal septicemia in ETEC infections of swine and demonstrate that EAST1, LT, and STb may be concurrently expressed by porcine ETEC strains.

  17. A recombinant chimera composed of R1 repeat region of Mycoplasma hyopneumoniae P97 adhesin with Escherichia coli heat-labile enterotoxin B subunit elicits immune response in mice.

    PubMed

    Conceição, Fabricio Rochedo; Moreira, Angela Nunes; Dellagostin, Odir Antônio

    2006-07-17

    Swine mycoplasmal pneumonia (SMP), caused by fastidious bacterium Mycoplasma hyopneumoniae, is the most important respiratory disease in swine breeding. The commonly used vaccines to control this disease consist of inactivated whole cells (bacterins), whose production cost is high and the efficiency is limited. The objective of this study was to develop and to evaluate in BALB/c mice a recombinant subunit vaccine (rLTBR1) containing the R1 region of P97 adhesin of M. hyopneumoniae (R1) fused to the B subunit of the heat-labile enterotoxin of Escherichia coli (LTB). rLTBR1 formed functional oligomers that presented high affinity to GM1 ganglioside. Mice inoculated with rLTBR1 by intranasal (IN) or intramuscular (IM) route produced high levels of anti-R1 systemic and mucosal antibodies (IgA), which recognized the native P97. On the other hand, mice inoculated with the inactivated whole cell vaccine did not produce anti-R1 antibodies. The administration route influenced the modulation of the immune response by LTB, showing that IM rLTBR1 induced Th2-biased immune responses and IN rLTBR1 induced Th1-biased immune responses. rLTBR1 administrated by IN route also induced IFN-gamma secretion by lymphocytes. rLTBR1 may constitute a new strategy for preventing infection by M. hyopneumoniae and may have potential for developing vaccines against other infectious diseases as well.

  18. Functional Pentameric Formation via Coexpression of the Escherichia coli Heat-Labile Enterotoxin B Subunit and Its Fusion Protein Subunit with a Neutralizing Epitope of ApxIIA Exotoxin Improves the Mucosal Immunogenicity and Protection against Challenge by Actinobacillus pleuropneumoniae▿

    PubMed Central

    Kim, Jung-Mi; Park, Seung-Moon; Kim, Jung-Ae; Park, Jin-Ah; Yi, Min-Hee; Kim, Nan-Sun; Bae, Jong-Lye; Park, Sung Goo; Jang, Yong-Suk; Yang, Moon-Sik; Kim, Dae-Hyuk

    2011-01-01

    A coexpression strategy in Saccharomyces cerevisiae using episomal and integrative vectors for the Escherichia coli heat-labile enterotoxin B subunit (LTB) and a fusion protein of an ApxIIA toxin epitope produced by Actinobacillus pleuropneumoniae coupled to LTB, respectively, was adapted for the hetero-oligomerization of LTB and the LTB fusion construct. Enzyme-linked immunosorbent assay (ELISA) with GM1 ganglioside indicated that the LTB fusion construct, along with LTB, was oligomerized to make the functional heteropentameric form, which can bind to receptors on the mucosal epithelium. The antigen-specific antibody titer of mice orally administered antigen was increased when using recombinant yeast coexpressing the pentameric form instead of recombinant yeast expressing either the LTB fusion form or antigen alone. Better protection against challenge infection with A. pleuropneumoniae was also observed for coexpression in recombinant yeast compared with others. The present study clearly indicated that the coexpression strategy enabled the LTB fusion construct to participate in the pentameric formation, resulting in an improved induction of systemic and mucosal immune responses. PMID:22030372

  19. Comparison of a live attenuated Salmonella Enteritidis vaccine candidate secreting Escherichia coli heat-labile enterotoxin B subunit with a commercial vaccine for efficacy of protection against internal egg contamination by Salmonella in hens.

    PubMed

    Nandre, Rahul M; Eo, Seong Kug; Park, Sang Youel; Lee, John Hwa

    2015-07-01

    This study compared a new live attenuated Salmonella Enteritidis vaccine candidate secreting Escherichia coli heat-labile enterotoxin B subunit (SE-LTB) with a commercial Salmonella Enteritidis (SE) vaccine for efficacy of protection against SE infection in laying hens. Chickens were divided into 3 groups of 20 each. Group A chickens were inoculated orally with phosphate-buffered saline and served as controls, group B chickens were inoculated orally with the vaccine candidate, and group C chickens were inoculated intramuscularly with a commercial vaccine, the primary inoculation in groups B and C being at 10 wk of age and the booster at 16 wk. Groups B and C showed significantly higher titers of plasma immunoglobulin G, intestinal secretory immunoglobulin A, and egg yolk immunoglobulin Y antibodies compared with the control group, and both vaccinated groups showed a significantly elevated cellular immune response. After virulent challenge, group B had significantly lower production of thin-shelled and/or malformed eggs and a significantly lower rate of SE contamination of eggs compared with the control group. Furthermore, the challenge strain was detected significantly less in all of the examined organs of group B compared with the control group. Group C had lower gross lesion scores only in the spleen and had lower bacterial counts only in the spleen, ceca, and ovary. These findings indicate that vaccination with the SE-LTB vaccine candidate can efficiently reduce internal egg and internal organ contamination by Salmonella and has advantages over the commercial vaccine.

  20. Structure–activity correlations of variant forms of the B pentamer of Escherichia coli type II heat-labile enterotoxin LT-IIb with Toll-like receptor 2 binding

    SciTech Connect

    Cody, Vivian; Pace, Jim; Nawar, Hesham F.; King-Lyons, Natalie; Liang, Shuang; Connell, Terry D.; Hajishengallis, George

    2012-12-01

    Structural data for the S74D variant of the pentameric B subunit of type II heat-labile enterotoxin of Escherichia coli reveal a smaller pore opening that may explain its reduced Toll-like receptor binding affinity compared to that of the wild type enterotoxin. The explanation for the enhanced Toll-like receptor binding affinity of the S74A variant is more complex than simply being attributed to the pore opening. The pentameric B subunit of the type II heat-labile enterotoxin of Escherichia coli (LT-IIb-B{sub 5}) is a potent signaling molecule capable of modulating innate immune responses. It has previously been shown that LT-IIb-B{sub 5}, but not the LT-IIb-B{sub 5} Ser74Asp variant [LT-IIb-B{sub 5}(S74D)], activates Toll-like receptor (TLR2) signaling in macrophages. Consistent with this, the LT-IIb-B{sub 5}(S74D) variant failed to bind TLR2, in contrast to LT-IIb-B{sub 5} and the LT-IIb-B{sub 5} Thr13Ile [LT-IIb-B{sub 5}(T13I)] and LT-IIb-B{sub 5} Ser74Ala [LT-IIb-B{sub 5}(S74A)] variants, which displayed the highest binding activity to TLR2. Crystal structures of the Ser74Asp, Ser74Ala and Thr13Ile variants of LT-IIb-B{sub 5} have been determined to 1.90, 1.40 and 1.90 Å resolution, respectively. The structural data for the Ser74Asp variant reveal that the carboxylate side chain points into the pore, thereby reducing the pore size compared with that of the wild-type or the Ser74Ala variant B pentamer. On the basis of these crystallographic data, the reduced TLR2-binding affinity of the LT-IIb-B{sub 5}(S74D) variant may be the result of the pore of the pentamer being closed. On the other hand, the explanation for the enhanced TLR2-binding activity of the LT-IIb-B{sub 5}(S74A) variant is more complex as its activity is greater than that of the wild-type B pentamer, which also has an open pore as the Ser74 side chain points away from the pore opening. Data for the LT-IIb-B{sub 5}(T13I) variant show that four of the five variant side chains point to the outside

  1. Construction of a recombinant-attenuated Salmonella Enteritidis strain secreting Escherichia coli heat-labile enterotoxin B subunit protein and its immunogenicity and protection efficacy against salmonellosis in chickens.

    PubMed

    Nandre, Rahul M; Lee, John Hwa

    2014-01-09

    A live attenuated Salmonella Enteritidis (SE) strain secreting Escherichia coli heat-labile enterotoxin B subunit (LTB) protein was constructed as a new vaccine candidate. The comparative effect of this vaccine candidate was evaluated with a previously reported SE vaccine, JOL919. An asd+, p15A ori plasmid containing eltB-encoding LTB was introduced into a ΔlonΔcpxRΔasd SE strain, and designated as JOL1364. In a single immunization experiment, group A chickens were orally inoculated with phosphate-buffered saline as a control, group B chickens were orally immunized with JOL919, and group C chickens were orally immunized with JOL1364. The immunized groups B and C showed significantly higher systemic, mucosal and cellular immune responses as compared to those of the control group. In addition, the immunized group C showed significantly higher mucosal and cellular immune responses as compared to those of the immunized group B at the 1st week post-immunization. In the examination of protection efficacy, the immunized groups B and C showed lower gross lesion scores in the liver and spleen, and lower bacterial counts of SE challenge strain in the liver, spleen, and caeca as compared to those of the control group. The number of SE-positive birds was significantly lower in the immunized group C as compared to that of the control group at the 14th day post-challenge. In addition, the number of birds carrying the challenge strain in the caeca was significantly lower in the immunized group C than those in the immunized group B and control group at the 7th and 14th day post-challenge. These results indicate that immunization with the JOL1364 vaccine candidate can induce higher mucosal and cellular immune responses than those of the JOL919 for efficient protection against salmonellosis.

  2. Real-Time TaqMan PCR Assay for the Detection of Heat-Labile and Heat-Stable Enterotoxin Genes in a Geographically Diverse Collection of Enterotoxigenic Escherichia coli Strains and Stool Specimens.

    PubMed

    Pattabiraman, Vaishnavi; Parsons, Michele B; Bopp, Cheryl A

    2016-04-01

    Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrhea in children under the age of 5 years in developing countries and are the leading bacterial agent of traveler's diarrhea in persons traveling to these countries. ETEC strains secrete heat-labile (LT) and/or heat-stable (ST) enterotoxins that induce diarrhea by causing water and electrolyte imbalance. We describe the validation of a real-time TaqMan PCR (RT-PCR) assay to detect LT, ST1a, and ST1b enterotoxin genes in E. coli strains and in stool specimens. We validated LT/ST1b duplex and ST1a single-plex RT-PCR assay using a conventional PCR assay as a gold standard with 188 ETEC strains and 42 non-ETEC strains. We validated LT/ST1b duplex and ST1a single-plex RT-PCR assay in stool specimens (n = 106) using traditional culture as the gold standard. RT- PCR assay sensitivities for LT, ST1a, and ST1b detection in strains were 100%, 100%, and 98%; specificities were 95%, 98%, and 99%, and Pearson correlation coefficient r was 0.9954 between RT-PCR assay and the gold standard. In stool specimens, RT-PCR assay sensitivities for LT, ST1a, and ST1b detection were 97%, 100%, and 97%; and specificities were 99%, 94%, and 97%. Pearson correlation coefficient r was 0.9975 between RT-PCR results in stool specimens and the gold standard. Limits of detection of LT, ST1a, and ST1b by RT-PCR assay were 0.1 to1.0 pg/μL and by conventional PCR assay were 100 to1000 pg/μL. The accuracy, rapidity and sensitivity of this RT-PCR assay is promising for ETEC detection in public health/clinical laboratories and for laboratories in need of an independent method to confirm results of other culture independent diagnostic tests.

  3. Attenuated Escherichia coli strains expressing the colonization factor antigen I (CFA/I) and a detoxified heat-labile enterotoxin (LThK63) enhance clearance of ETEC from the lungs of mice and protect mice from intestinal ETEC colonization and LT-induced fluid accumulation.

    PubMed

    Byrd, Wyatt; Boedeker, Edgar C

    2013-03-15

    Although enterotoxigenic Escherichia coli (ETEC) infections are important causes of infantile and traveler's diarrhea there is no licensed vaccine available for those at-risk. Our goal is to develop a safe, live attenuated ETEC vaccine. We used an attenuated E. coli strain (O157:H7, Δ-intimin, Stx1-neg, Stx2-neg) as a vector (ZCR533) to prepare two vaccine strains, one strain expressing colonization factor antigen I (ZCR533-CFA/I) and one strain expressing CFA/I and a detoxified heat-labile enterotoxin (ZCR533-CFA/I+LThK63) to deliver ETEC antigens to mucosal sites in BALB/c mice. Following intranasal and intragastric immunization with the vaccine strains, serum IgG and IgA antibodies were measured to the CFA/I antigen, however, only serum IgG antibodies were detected to the heat-labile enterotoxin. Intranasal administration of the vaccine strains induced respiratory and intestinal antibody responses to the CFA/I and LT antigens, while intragastric administration induced only intestinal antibody responses with no respiratory antibodies detected to the CFA/I and LT antigens. Mice immunized intranasally with the vaccine strains showed enhanced clearance of wild-type (wt) ETEC bacteria from the lungs. Mice immunized intranasally and intragastrically with the vaccine strains were protected from intestinal colonization following oral challenge with ETEC wt bacteria. Mice immunized intragastrically with the ZCR533-CFA/I+LThK63 vaccine strain had less fluid accumulate in their intestine following challenge with ETEC wt bacteria or with purified LT as compared to the sham mice indicating that the immunized mice were protected from LT-induced intestinal fluid accumulation. Thus, mice intragastrically immunized with the ZCR533-CFA/I+LThK63 vaccine strain were able to effectively neutralize the activity of the LT enterotoxin. However, no difference in intestinal fluid accumulation was detected in the mice immunized intranasally with the vaccine strain as compared to the sham

  4. Enterotoxin

    MedlinePlus

    ... is harmful to your digestive system. It is produced by certain bacteria. The enterotoxin enters your stomach and intestines if you eat contaminated food or water. This causes symptoms such as cramps, nausea, vomiting, ...

  5. Investigation of ’Escherichia coli’ Enterotoxins

    DTIC Science & Technology

    1978-05-01

    E . coli diarrheal disease in man and domestic animals. Fundamentally, the design of the vaccine is based on the well- documented ability of cholera antitoxin to neutralize both cholera and heat- labile E . coli enterotoxins and on the ability of certain E . coli antigens to enhance the immune response to cholera toxoid and possibly whole-cell Cholera Vaccine, as

  6. Immunological Interrelationships of Coliform Heat-Labile and Heat-Stable Enterotoxins

    DTIC Science & Technology

    1981-01-01

    against direct challenge in ligated ileal loops of the immunized rats with either $he toxin itself or viable strains cf bacteria which produce L+T either... toxin or viable bacteria in ligated ileal loops end the serum antitoxin (AT) response was assayed by an enzyme-linked *Z immunosorbent assay (ELISA...protection against the toxin correlated with th&t against viable bacteria (LT+/ST- and LT+!ST+ strains) and with elevated serum AT titers. All seven

  7. Four foodborne disease outbreaks caused by a new type of enterotoxin-producing Clostridium perfringens.

    PubMed

    Monma, Chie; Hatakeyama, Kaoru; Obata, Hiromi; Yokoyama, Keiko; Konishi, Noriko; Itoh, Takeshi; Kai, Akemi

    2015-03-01

    The epidemiological and bacteriological investigations on four foodborne outbreaks caused by a new type of enterotoxin-producing Clostridium perfringens are described. C. perfringens isolated from patients of these outbreaks did not produce any known enterotoxin and did not carry the C. perfringens enterotoxin gene. However, the culture filtrates of these isolates induced the accumulation of fluid in rabbit ileal loop tests. The molecular weight of the new enterotoxin may be between 50,000 and 100,000, although the known C. perfringens enterotoxin is ca. 35,000. This new enterotoxin was heat labile, and its biological activities were inactivated by heating for 5 min at 60°C. The new enterotoxin was sensitive to pH values higher than 11.0 and protease treatment but was resistant to trypsin treatment. These results suggest that the new enterotoxin may be a protein. Although C. perfringens enterotoxin induced morphological changes in Vero cells, the changes induced by the new enterotoxin differed from those by the known C. perfringens enterotoxin. The new enterotoxin also induced morphological changes in L929 cells, whereas the known C. perfringens enterotoxin did not, because L929 cells lacked an appropriate enterotoxin receptor. Although C. perfringens enterotoxin is recognized as the only diarrheagenic toxin responsible for C. perfringens foodborne outbreaks, the results of the present study indicate that C. perfringens isolated from these four outbreaks produced a new type of enterotoxin.

  8. Seroepidemiology of heat-labile enterotoxigenic Escherichia coli and Norwalk virus infections in Panamanians, Canal Zone residents, Apache Indians, and United States Peace Corps volunteers.

    PubMed

    Ryder, R W; Greenberg, H; Singh, N; Oro, G; de Guardia, A; Sack, R B; Kapikian, A Z

    1982-09-01

    Serum antibody titrations against the heat-labile enterotoxin (LT) of Escherichia coli were carried out on Panamanians, U.S. citizens resident in the Panama Canal Zone, Apache Indians living on the reservation in Whiteriver, Arizona, and Peace Corps volunteers before they traveled overseas. Antibody titers to Norwalk virus were also carried out on serum from Panamanian and Canal Zone residents. A high prevalence of low-titer LT antibodies was found in infants and adults from Panama, the Canal Zone, and Whiteriver. Panamanian children aged 1 to 5 years had the highest LT antibody titers. Peace Corps volunteers had a low prevalence and titer of LT antibodies. Prevalence and titer of antibodies to Norwalk virus were generally higher in Panamanians compared with Canal Zone residents of the same age. In the populations we studied, various modes of transmission and mechanisms of immunity likely explain the differences which we observed in antibody prevalence and titer to these two enteric pathogens.

  9. Radiation-induced heat-labile sites that convert into DNA double-strand breaks

    NASA Technical Reports Server (NTRS)

    Rydberg, B.; Chatterjee, A. (Principal Investigator)

    2000-01-01

    The yield of DNA double-strand breaks (DSBs) in SV40 DNA irradiated in aqueous solution was found to increase by more than a factor of two as a result of postirradiation incubation of the DNA at 50 degrees C and pH 8.0 for 24 h. This is in agreement with data from studies performed at 37 degrees C that were published previously. Importantly, similar results were also obtained from irradiation of mammalian DNA in agarose plugs. These results suggest that heat-labile sites within locally multiply damaged sites are produced by radiation and are subsequently transformed into DSBs. Since incubation at 50 degrees C is typically employed for lysis of cells in commonly used pulsed-field gel assays for detection of DSBs in mammalian cells, the possibility that heat-labile sites are present in irradiated cells was also studied. An increase in the apparent number of DSBs as a function of lysis time at 50 degrees C was found with kinetics that was similar to that for irradiated DNA, although the magnitude of the increase was smaller. This suggests that heat-labile sites are also formed in the cell. If this is the case, a proportion of DSBs measured by the pulsed-field gel assays may occur during the lysis step and may not be present in the cell as breaks but as heat-labile sites. It is suggested that such sites consist mainly of heat-labile sugar lesions within locally multiply damaged sites. Comparing rejoining of DSBs measured with short and long lysis procedure indicates that the heat-labile sites are repaired with fast kinetics in comparison with repair of the bulk of DSBs.

  10. Role of various enterotoxins in Aeromonas hydrophila-induced gastroenteritis: generation of enterotoxin gene-deficient mutants and evaluation of their enterotoxic activity.

    PubMed

    Sha, Jian; Kozlova, E V; Chopra, A K

    2002-04-01

    Three enterotoxins from the Aeromonas hydrophila diarrheal isolate SSU have been molecularly characterized in our laboratory. One of these enterotoxins is cytotoxic in nature, whereas the other two are cytotonic enterotoxins, one of them heat labile and the other heat stable. Earlier, by developing an isogenic mutant, we demonstrated the role of a cytotoxic enterotoxin in causing systemic infection in mice. In the present study, we evaluated the role of these three enterotoxins in evoking diarrhea in a murine model by developing various combinations of enterotoxin gene-deficient mutants by marker-exchange mutagenesis. A total of six isogenic mutants were prepared in a cytotoxic enterotoxin gene (act)-positive or -negative background strain of A. hydrophila. We developed two single knockouts with truncation in either the heat-labile (alt) or the heat-stable (ast) cytotonic enterotoxin gene; three double knockouts with truncations of genes encoding (i) alt and ast, (ii) act and alt, and (iii) act and ast genes; and a triple-knockout mutant with truncation in all three genes, act, alt, and ast. The identity of these isogenic mutants developed by double-crossover homologous recombination was confirmed by Southern blot analysis. Northern and Western blot analyses revealed that the expression of different enterotoxin genes in the mutants was correspondingly abrogated. We tested the biological activity of these mutants in a diet-restricted and antibiotic-treated mouse model with a ligated ileal loop assay. Our data indicated that all of these mutants had significantly reduced capacity to evoke fluid secretion compared to that of wild-type A. hydrophila; the triple-knockout mutant failed to induce any detectable level of fluid secretion. The biological activity of selected A. hydrophila mutants was restored after complementation. Taken together, we have established a role for three enterotoxins in A. hydrophila-induced gastroenteritis in a mouse model with the greatest

  11. Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli

    PubMed Central

    Andrade, Fernanda B.; Nepomuceno, Roberto; Silva, Anderson; Munhoz, Danielle D.; Yamamoto, Bruno B.; Luz, Daniela; Abreu, Patrícia A. E.; Horton, Denise S. P. Q.; Elias, Waldir P.; Ramos, Oscar H. P.; Piazza, Roxane M. F.

    2015-01-01

    Background Diarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC) strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv) that were produced in E. coli against enterotoxins of ETEC strains. Methods and Findings Recombinant scFv were developed against ETEC heat-labile toxin (LT) and heat-stable toxin (ST), from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains. Conclusion The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis. PMID:26154103

  12. ApoFnr Binds as a Monomer to Promoters Regulating the Expression of Enterotoxin Genes of Bacillus cereus▿ †

    PubMed Central

    Esbelin, Julia; Jouanneau, Yves; Armengaud, Jean; Duport, Catherine

    2008-01-01

    Bacillus cereus Fnr is a member of the Crp/Fnr (cyclic AMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. It is essential for the expression of hbl and nhe enterotoxin genes independently of the oxygen tension in the environment. We studied aerobic Fnr binding to target sites in promoters regulating the expression of enterotoxin genes. B. cereus Fnr was overexpressed and purified as either a C-terminal His-tagged (FnrHis) fusion protein or an N-terminal fusion protein tagged with the Strep-tag (IBA BioTAGnology) (StrepFnr). Both recombinant Fnr proteins were produced as apoforms (clusterless) and occurred as mixtures of monomers and oligomers in solution. However, apoFnrHis was mainly monomeric, while apoStrepFnr was mainly oligomeric, suggesting that the His-tagged C-terminal extremity may interfere with oligomerization. The oligomeric state of apoStrepFnr was dithiothreitol sensitive, underlining the importance of a disulfide bridge for apoFnr oligomerization. Electrophoretic mobility shift assays showed that monomeric apoFnr, but not oligomeric apoFnr, bound to specific sequences located in the promoter regions of the enterotoxin regulators fnr, resDE, and plcR and the structural genes hbl and nhe. The question of whether apoFnr binding is regulated in vivo by redox-dependent oligomerization is discussed. PMID:18424517

  13. Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

    SciTech Connect

    Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2010-08-27

    Research highlights: {yields} Incubating PCR products at a high temperature causes smears in gel electrophoresis. {yields} Smears interfere with the interpretation of methylation analysis using COBRA. {yields} Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. {yields} The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 {sup o}C or 65 {sup o}C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.

  14. Different types of cell death induced by enterotoxins.

    PubMed

    Lin, Chiou-Feng; Chen, Chia-Ling; Huang, Wei-Ching; Cheng, Yi-Lin; Hsieh, Chia-Yuan; Wang, Chi-Yun; Hong, Ming-Yuan

    2010-08-01

    The infection of bacterial organisms generally causes cell death to facilitate microbial invasion and immune escape, both of which are involved in the pathogenesis of infectious diseases. In addition to the intercellular infectious processes, pathogen-produced/secreted enterotoxins (mostly exotoxins) are the major weapons that kill host cells and cause diseases by inducing different types of cell death, particularly apoptosis and necrosis. Blocking these enterotoxins with synthetic drugs and vaccines is important for treating patients with infectious diseases. Studies of enterotoxin-induced apoptotic and necrotic mechanisms have helped us to create efficient strategies to use against these well-characterized cytopathic toxins. In this article, we review the induction of the different types of cell death from various bacterial enterotoxins, such as staphylococcal enterotoxin B, staphylococcal alpha-toxin, Panton-Valentine leukocidin, alpha-hemolysin of Escherichia coli, Shiga toxins, cytotoxic necrotizing factor 1, heat-labile enterotoxins, and the cholera toxin, Vibrio cholerae. In addition, necrosis caused by pore-forming toxins, apoptotic signaling through cross-talk pathways involving mitochondrial damage, endoplasmic reticulum stress, and lysosomal injury is discussed.

  15. Plesiomonas shigelloides exports a lethal cytotoxic-enterotoxin (LCE) by membrane vesicles.

    PubMed

    Ludovico, Marilucia Santos; Martins, Luciano Moura; Bianco, Juares Ednaldo Romero; Andrade, Célia Guadalupe Tardelli de Jesus; Falcon, Rosabel; Joazeiro, Paulo Pinto; Gatti, Maria Silvia Viccari; Yano, Tomomasa

    Plesiomonas shigelloides isolated from water in Brazil was previously described as a hemorrhagic heat-labile cytotoxic-enterotoxin producer. We purified this toxin from culture supernatants using ion metallic affinity chromatography (IMAC) followed by molecular exclusion chromatography. The pure toxin presented molecular mass of 50kDa and isoelectric point (pI) around 6.9 by 2D electrophoresis. When injected intravenously, the purified cytotoxic-enterotoxin induced also severe spasms followed by sudden death of mice. Hence, we entitled it as lethal cytotoxic-enterotoxin (LCE). The presence of membrane vesicles (MVs) on cell surfaces of P. shigelloides was observed by scan electron microscopy (SEM). From these MVs the LCE toxin was extracted and confirmed by biological and serological assays. These data suggest that P. shigelloides also exports this cytotoxic-enterotoxin by membrane vesicles, a different mechanism of delivering extra cellular virulence factors, so far not described in this bacterium.

  16. Detection of Escherichia coli enterotoxins in stools.

    PubMed Central

    Merson, M H; Yolken, R H; Sack, R B; Froehlich, J L; Greenberg, H B; Huq, I; Black, R W

    1980-01-01

    We determined whether enterotoxigenic Escherichia coli diarrhea could be diagnosed by direct examination of stools for heat-labile (LT) and heat-stable (ST) enterotoxins. The Y-1 adrenal cell and an enzyme-linked immunosorbent assay (ELISA) detected LT in 85 and 93%, respectively, of stool specimens obtained from adults with acute diarrhea from whom an LT- and ST-producing organism had been isolated. Furthermore, the ELISA assay detected LT in 8 of 35 stool specimens from which no LT-producing E. coli had been isolated. The infant mouse assay was utilized to detect ST in these stool specimens and was found to be an insensitive method, showing positive results in only 36% of the specimens from which an ST-producing organism was isolated. Further studies are warranted to determine the diagnostic value of direct detection of LT in stools, especially by the ELISA method. PMID:6995331

  17. Positive Feedback Cycle of TNFα Promotes Staphylococcal Enterotoxin B-Induced THP-1 Cell Apoptosis

    PubMed Central

    Zhang, Xiaopeng; Shang, Weilong; Yuan, Jizhen; Hu, Zhen; Peng, Huagang; Zhu, Junmin; Hu, Qiwen; Yang, Yi; Liu, Hui; Jiang, Bei; Wang, Yinan; Li, Shu; Hu, Xiaomei; Rao, Xiancai

    2016-01-01

    Staphylococcal enterotoxin B (SEB) has been demonstrated to be of importance in Staphylococcus aureus related diseases, such as atopic dermatitis (AD). Dysregulated apoptosis in AD is remarkable, and SEB can induce apoptosis of various cell types. However, the mechanisms by which SEB induces apoptosis and influences disease processes remain unclear. In this study, the recombinant SEB-induced THP-1 monocyte apoptosis was demonstrated in the absence of preliminary cell activation in a time- and dose-dependent manner. SEB could up-regulate the expression of tumor necrosis factor alpha (TNFα) in THP-1 cells and induce apoptosis via an extrinsic pathway. TNFα could in turn increase the expression of HLA-DRa, the SEB receptor on the cell surface. As a result, a positive feedback cycle of TNFα was established. TNFα expression and SEB-induced apoptosis were decreased by knocking down the expression of either HLA-DRa or TNFR1. Therefore, the feedback cycle of TNFα is crucial for SEB functions. This work provides insights into the mechanisms of SEB-induced monocyte apoptosis and emphasizes the major role of TNFα in future related studies. PMID:27709104

  18. Alkaline pH Is a signal for optimal production and secretion of the heat labile toxin, LT in enterotoxigenic Escherichia coli (ETEC).

    PubMed

    Gonzales, Lucia; Ali, Zahra Bagher; Nygren, Erik; Wang, Zhiyun; Karlsson, Stefan; Zhu, Baoli; Quiding-Järbrink, Marianne; Sjöling, Åsa

    2013-01-01

    Enterotoxigenic Escherichia coli (ETEC) cause secretory diarrhea in children and travelers to endemic areas. ETEC spreads through the fecal-oral route. After ingestion, ETEC passes through the stomach and duodenum before it colonizes the lower part of the small intestine, exposing bacteria to a wide range of pH and environmental conditions. This study aimed to determine the impact of external pH and activity of the Cyclic AMP receptor protein (CRP) on the regulation of production and secretion of heat labile (LT) enterotoxin. ETEC strain E2863wt and its isogenic mutant E2863ΔCRP were grown in LBK media buffered to pH 5, 7 and 9. GM1 ELISA, cDNA and cAMP analyses were carried out on bacterial pellet and supernatant samples derived from 3 and 5 hours growth and from overnight cultures. We confirm that CRP is a repressor of LT transcription and production as has been shown before but we show for the first time that CRP is a positive regulator of LT secretion both in vitro and in vivo. LT secretion increased at neutral to alkaline pH compared to acidic pH 5 where secretion was completely inhibited. At pH 9 secretion of LT was optimal resulting in 600 percent increase of secreted LT compared to unbuffered LBK media. This effect was not due to membrane leakage since the bacteria were viable at pH 9. The results indicate that the transition to the alkaline duodenum and/or exposure to high pH close to the epithelium as well as activation of the global transcription factor CRP are signals that induce secretion of the LT toxin in ETEC.

  19. Immunization of swine with heat-stable Escherichia coli enterotoxin coupled to a carrier protein does not protect suckling pigs against an Escherichia coli strain that produces heat-stable enterotoxin.

    PubMed Central

    Moon, H W; Baetz, A L; Giannella, R A

    1983-01-01

    Pregnant swine were immunized parenterally with purified heat-stable Escherichia coli enterotoxin that was made antigenic by coupling it to bovine immunoglobulin G. Immunized swine had high titers of antitoxin in serum and colostrum as measured by radioimmunoassay. However, the heat-stable enterotoxin neutralizing titers of the serum and colostrum from immunized swine were comparatively low. Newborn pigs suckling their immunized dams were not protected against challenge with porcine enterotoxigenic E. coli that produce heat-stable toxin but do not produce heat-labile toxin. PMID:6339398

  20. Induction of heat-labile sites in DNA of mammalian cells by the antitumor alkylating drug CC-1065

    SciTech Connect

    Zsido, T.J.; Woynarowski, J.M.; Baker, R.M.; Gawron, L.S.; Beerman, T.A. )

    1991-04-16

    CC-1065 is a very potent antitumor antibiotic capable of covalent and noncovalent binding to the minor groove of naked DNA. Upon thermal treatment, covalent adducts formed between CC-1065 and DNA generate strand break. The authors have shown that this molecular damage can be detected following CC-1065 treatment of mammalian whole cells. Using alkaline sucrose gradient analysis, They observe thermally induced breakage of ({sup 14}C)thymidine-prelabeled DNA from drug-treated African green monkey kidney BSC-1 cells. Very little damage to cellular DNA by CC-1065 can be detected without first heating the drug-treated samples. CC-1065 can also generate heat-labile sites within DNA during cell lysis and heating, subsequent to the exposure of cells to drug, suggesting that a pool of free and noncovalently bound drug is available for posttreatment adduct formation. This effect was controlled for by mixing ({sup 3}H)thymidine-labeled untreated cells with the ({sup 14}C)thymidine-labeled drug-treated samples. The lowest drug dose at which heat-labile sites were detected was 3 nM CC-1065 (3 single-stranded breaks/10{sup 6} base pairs). This concentration reduced survival of BSC-1 cells to 0.1% in cytotoxicity assays. The generation of CC-1065-induced lesions in cellular DNA is time dependent (the frequency of lesions caused by a 60 nM treatment reaching a plateau at 2 h) and is not readily reversible. The results of this study demonstrate that CC-1065 does generate heat-labile sites with the cellular DNA of intact cells and suggest that a mechanism of cytotoxic action of CC-1065 involves formation of covalent adducts to DNA.

  1. The discovery of cholera - like enterotoxins produced by Escherichia coli causing secretory diarrhoea in humans

    PubMed Central

    Sack, R. Bradley

    2011-01-01

    Non-vibrio cholera has been recognized as a clinical entity for as long as cholera was known to be caused by Vibrio cholerae. Until 1968, the aetiologic agent of this syndrome was not known. Following a series of studies in patients with non-vibrio cholera it was found that these patients had large concentrations of Escherichia coli in the small bowel and stools which produced cholera toxin-like enterotoxins, and had fluid and electrolyte transport abnormalities in the small bowel similar to patients with documented cholera. Furthermore, these patients developed antibodies to the cholera-like enterotoxin. Later studies showed that these strains, when fed to volunteers produced a cholera-like disease and that two enterotoxins were found to be produced by these organisms: a heat-labile enterotoxin (LT) which is nearly identical to cholera toxin, and a heat-stable enterotoxin (ST), a small molecular weight polypeptide. E. coli that produced one or both of these enterotoxins were designated enterotoxigenic E. coli (ETEC). ETEC are now known not only to cause a severe cholera-like illness, but to be the most common bacterial cause of acute diarrhoea in children in the developing world, and to be the most common cause of travellers’ diarrhoea in persons who visit the developing world. PMID:21415491

  2. Treatment with a hybrid between the synapsin ABC domains and the B subunit of E. coli heat-labile toxin reduces frequency of proinflammatory cells and cytokines in the central nervous system of rats with EAE.

    PubMed

    Bibolini, M J; Scerbo, M J; Roth, G A; Monferran, C G

    2014-09-26

    Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are crucially dependent on the invasion of activated autoreactive lymphocytes and blood macrophages into the central nervous system (CNS). Proinflammatory mononuclear cells and activated local microglia mediate inflammation, demyelination and axonal damage at the target organ. Previously, we observed that the administration of a hybrid between the synapsin ABC domains and the B subunit of Escherichia coli heat labile-enterotoxin (LTBABC) to rats with EAE ameliorated disease by modulating the peripheral Th1 response to myelin basic protein (MBP). In the present study, we investigated the effect of LTBABC administration on proinflammatory cell frequency in the CNS of rats with EAE. Treatment with the hybrid in the inductive phase of EAE attenuated disease severity and diminished histological inflammatory infiltrates and demyelination in the spinal cord of rats with acute EAE. Lower frequencies of infiltrating and local macrophages as well as CD4+ T cells that produce the proinflammatory cytokines interferon-gamma (IFN-γ) and interleukin (IL)-17 were found at the target organ. Concomitantly, low levels of INF-γ and IL-17 and increased levels of IL-10 were measured in cultures of CNS infiltrating cells and spinal cord tissue. An increased frequency of CD4+CD25+Foxp3 cells was observed at the disease peak and at the beginning of the recovery stage. These results provide further evidence for the immunomodulatory properties of the fusion protein LTBABC in autoimmune demyelinating disease affecting the central nervous system.

  3. Analysis and application of a neutralizing linear epitope on liable toxin B of enterotoxin Escherichia coli.

    PubMed

    Guan, Weikun; Liu, Wenxin; Bao, Jun; Li, Jinping; Yuan, Chaowen; Tang, Jie; Shi, Dongfang

    2015-07-01

    Heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) is one of the major virulence factors for causing diarrhea in piglets, and LT is a strong immunogen. Thus, LT represents an important target for development of vaccines and diagnostic tests. In this study, bioinformatic tools were used to predict six antigenic B cell epitopes in the B subunit of LT protein (LTB) of ETEC strains. Then, seven antigenic B cell epitopes of LTB were identified by polyclonal antisera (polyclonal antibody (PAb)) using a set of LTB-derived peptides expressed as maltose-binding protein (MBP) fusion protein. In addition, one LTB-specific monoclonal antibody (MAb) was generated and defined its corresponding epitope as mentioned above. This MAb was able to specifically bind with native LT toxin and has no cross-reaction with LT-II (type II heat-labile enterotoxin), Stx1 (Shiga toxin I), Stx2 (Shiga toxin II), STa (heat-stable enterotoxin I), and STb (heat-stable enterotoxin II) toxins. Further, this MAb was able to interrupt LT toxin specific binding to GM1 receptor, indicating that the corresponding epitope is the specific binding region to GM1 receptor. Moreover, in vitro and in vivo assay showed that the MAb was able to neutralize the native LT toxin. Diarrheal suckling pigs challenged with LT-positive ETEC strain recovered when an enema with this purified MAb was administered. This study will provide the foundation for further studies about the interaction between LT toxin and GM1 receptor and about the developing of epitope-based vaccines and specific therapeutic agent.

  4. Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 or PARP

    SciTech Connect

    Stenerlöw, Bo; Karlsson, Karin H.; Radulescu, Irina; Rydberg, Bjorn; Stenerlow, Bo

    2008-04-29

    Ionizing radiation induces a variety of different DNA lesions: in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have previously shown that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites (HLS) within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of HLS on DSB induction and repair, four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for bi-phasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements the fraction of fast rejoining decreased to less than 50% of the total. However, neither the half-times of the fast (t{sub 1/2} = 7-8 min) or slow (t{sub 1/2} = 2.5 h) DSB rejoining were changed significantly. At t=0 the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSB/cell/Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all tested cells, including M059K cells treated with wortmannin or DNA-PKcs defect M059J cells. Furthermore, cells lacking XRCC1 or Poly(ADP-ribose) polymerase-1 (PARP-1) rejoined both total DSBs and heat-released DSBs similar to normal cells. In summary, the presence of heat-labile sites have a substantial impact on DSB induction yields and DSB rejoining rates measured by pulsed-field gel electrophoresis, and HLS repair is independent of DNA-PKcs, XRCC1 and PARP.

  5. Expression of a bioactive fusion protein of Escherichia coli heat-labile toxin B subunit to a synapsin peptide.

    PubMed

    Julia Scerbo, M; Bibolini, Mario J; Barra, José L; Roth, German A; Monferran, Clara G

    2008-06-01

    The B subunit of Escherichia coli heat-labile toxin (LTB) may function as an efficient carrier molecule for the delivery of genetically coupled antigens across the mucosal barrier. We constructed vectors for the expression of LTB and LTBSC proteins. LTBSC is a fusion protein that comprises the amino acid sequence from the C-domain of rat synapsin fused to the C-terminal end of LTB. Both constructions have a coding sequence for a 6His-tag fused in-frame. LTBSC was expressed in E. coli as inclusion bodies. The inclusion bodies were isolated and purified by Ni2+-chelating affinity chromatography under denaturing condition. Purified LTBSC was diluted in several refolding buffers to gain a soluble and biologically active protein. Refolded LTBSC assembled as an active oligomer which binds to the GM1 receptor in an enzyme-linked immunosorbent assay (ELISA). Soluble LTB in the E. coli lysate was also purified by Ni2+-chelating affinity chromatography and the assembled pentamer was able to bind with high affinity to GM1 in vitro. LTBSC and LTB were fed to rats and the ability to induce antigen-specific tolerance was tested. LTBSC inhibited the specific delayed-type hypersensitivity (DTH) response and induced decreased antigen-specific in vivo and in vitro cell proliferation more efficiently than LTB. Thus, the novel hybrid molecule LTBSC when orally delivered was able to elicit a systemic immune response. These results suggest that LTBSC could be suitable for exploring further therapeutic treatment of autoimmune inflammatory diseases involving antigens from central nervous system.

  6. Suppression of enterotoxin-induced intestinal fluid secretion by wood creosote.

    PubMed

    Ataka, K; Ogata, N; Kuge, T; Shibata, T

    1996-08-01

    Wood creosote suppresses intestinal fluid secretion induced by heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC). When rabbit jejunum is ligated into a 5-cm segment and LT is administered locally, it actively induces intestinal fluid secretion in a dose-dependent manner. Local administration of wood creosote together with a fixed dose of LT suppressed the LT-induced fluid secretion in a dose-dependent manner. At a 50-ng/segment dose of LT, 7.4 +/- 1.1 ml (n = 5) of fluid is secreted into an intestinal segment; coadministration of wood creosote (150 micrograms/segment) significantly (p < 0.01) suppressed the fluid secretion to 2.4 +/- 2.3 ml. Based on these results, we conclude that the antidiarrheal activity of wood creosote is attributable to its antisecretory or proabsorptive effect (or both) on the intestine.

  7. Investigation of E. coli Enterotoxins.

    DTIC Science & Technology

    In the course of investigating E . coli enterotoxins, it was discovered that trypsin treatment of partially purified enterotoxin from strain H197 (078...loops) did exhibit elevated PF titers compared with uninoculated controls. These findings are consistent with the hypothesis that E . coli enterotoxins

  8. Isolation and characterization of the subunits of a heat-labile alpha-amylase inhibitor from Phaseolus vulgaris white kidney bean.

    PubMed

    Yamaguchi, H

    1993-02-01

    The heat-labile one of the two alpha-amylase inhibitors of the white kidney bean (Phaseolus vulgaris) was found to be composed of three kinds of subunits, and they were isolated and characterized. The alpha-subunit was free from tryptophan and cysteine and the beta-subunit contained no methionine or cysteine. There was no marked resemblance in tryptic peptide maps between the alpha- and beta-subunit polypeptides. The alpha-subunit contained 30% by weight of carbohydrate, mainly made up of high mannose-oligosaccharides, and the sugar moiety of the beta-subunit amounted 7% and appeared to be predominantly composed of xylomannose-type oligosaccharides. The largest subunit, gamma, was very similar in molecular features to a postulated alpha beta-dimer and its N-terminal sequence coincided with that of the alpha-subunit. The molecular weights of the polypeptides of alpha, beta-, and gamma-subunits were shown to be 7,800, 14,000, and 22,000, respectively, by SDS-PAGE. It seemed likely that the alpha- and beta-subunits are common to both of the inhibitors and that the heat-lability of this inhibitor arises from the gamma-subunit.

  9. Effect of Escherichia coli on Fluid Transport across Canine Small Bowel MECHANISM AND TIME-COURSE WITH ENTEROTOXIN AND WHOLE BACTERIAL CELLS

    PubMed Central

    Guerrant, R. L.; Ganguly, U.; Casper, A. G. T.; Moore, E. J.; Pierce, N. F.; Carpenter, C. C. J.

    1973-01-01

    An Escherichia coli strain isolated from a patient with severe cholera-like diarrhea elaborates a partly heat-labile enterotoxin shown to cause prompt adenyl cyclase stimulation and isotonic fluid secretion by canine jejunum. Both responses disappear upon removal of the enterotoxin. The duration of action of a submaximal dose of this E. coli enterotoxin was brief, despite sustained exposure to the jejunum, suggesting inactivation of the enterotoxin by its interaction with the mucosa. Inoculation of whole bacterial cultures of this E. coli strain into canine duodenum was followed by bacterial survival and induction of net secretion after 4-7 h. The onset of fluid production was associated with increasing gut mucosal adenyl cyclase activity. Washed bacterial cells could also produce fluid secretion. In vivo multiplication of this enterotoxin-producing E. coli was demonstrated 6-12 h after intraduodenal inoculation of approximately 106 organisms. This was associated with fluid secretion. Intestinal fluid production occurred without microscopic pathology in the mucosa. Images PMID:4578157

  10. Protection of mice against enterotoxigenic E. coli by immunization with a polyvalent enterotoxin comprising a combination of LTB, STa, and STb.

    PubMed

    You, Jiansong; Xu, Yongping; He, Maolong; McAllister, Tim A; Thacker, Philip A; Li, Xiaoyu; Wang, Tingting; Jin, Liji

    2011-03-01

    Currently available enterotoxigenic Escherichia coli (ETEC) vaccines are based on colonization factors and/or the heat-labile enterotoxin B subunit (LTB). However, the induction of antitoxic responses against heat-stable enterotoxin a (STa) and b (STb) has merit as these two poorly immunogenic toxins are frequently associated with ETEC strains. In this study, we genetically constructed a trivalent enterotoxin fusion protein (STa-LTB-STb, abbreviated to SLS) in an effort to develop a single toxoid containing these three enterotoxins for vaccination against ETEC. Mutagenesis at one disulfide-bridge-forming cysteine in STa led to a dramatic reduction in the STa toxicity of SLS; however, the fusion peptide retained the STb-associated toxicity. Immunization of mice with SLS protein elicited significant antibody responses to LTB, STa, and STb. Significantly, the mice antisera were able to neutralize the biological activity of both STa and STb. In the experiment to assess the protective effect of SLS immunization, the mortality of mice receiving SLS was significantly lower than their control cohorts (P < 0.01) after intraperitoneal challenge with ETEC. These results show that the trivalent fusion enterotoxin SLS has the potential to serve as a useful toxin-based vaccine against ETEC-induced diarrheal disease via a single immunogen.

  11. Molecular Characterization of Enterotoxin-Producing Escherichia coli Collected in 2011–2012, Russia

    PubMed Central

    Kartsev, Nikolay N.; Fursova, Nadezhda K.; Pachkunov, Dmitry M.; Bannov, Vasiliy A.; Eruslanov, Boris V.; Svetoch, Edward A.; Dyatlov, Ivan A.

    2015-01-01

    Enterotoxin-producing Escherichia coli (ETEC) are one of the main causative agents of diarrhea in children especially in developing countries and travel diarrhoea in adults. Pathogenic properties of ETEC associated with their ability to produce a heat-stable (ST) and/or heat-labile (LT) enterotoxins, as well as adhesins providing bacterial adhesion to intestinal epithelial cells. This study presents the molecular characterization of the ETEC isolates collected from the Central and Far-Eastern regions of Russia in 2011–2012. It was shown that all ETEC under study (n=18) had the heat-labile enterotoxin-coding operon elt, and had no the genes of the heat-stable enterotoxin operon est. DNA sequencing revealed two types of nucleotide exchanges in the eltB gene coding subunit B of LT in isolates collected from Cherepovets city (Central region, Russia) and Vladivostok city (Far-East region, Russia). Only one ETEC strain carried genes cfaA, cfaB, cfaC and cfaD coding adhesion factor CFA/I. Expression of LT in four ETEC isolates in the agglutination reaction was detected using a latex test-system. The isolates were assigned to serogroups O142 (n = 6), О6 (n = 4), О25 (n = 5), О26 (n = 2), and O115 (n = 1). Genotyping showed that they belonged to an earlier described sequence-type ST4 (n = 3) as well as to 11 novel sequence-types ST1043, ST1312, ST3697, ST3707, ST3708, ST3709, ST3710, ST3755, ST3756, ST3757 and ST4509. The ETEC isolates displayed different levels of antimicrobial resistance. Eight isolates were resistant to only one drug, three isolates—to two drugs, one isolate—to three drugs, two isolates—to four antibacterials, and only one isolate to each of the five, six and ten antibacterials simultaneously. Genetic determinants of the resistance to beta-lactams and other classes of antibacterials on the ETEC genomes were identified. There are blaTEM (n = 10), blaCTX-M-15 (n = 1), class 1 integron (n = 3) carrying resistance cassettes to aminoglycosides and

  12. Molecular Characterization of Enterotoxin-Producing Escherichia coli Collected in 2011-2012, Russia.

    PubMed

    Kartsev, Nikolay N; Fursova, Nadezhda K; Pachkunov, Dmitry M; Bannov, Vasiliy A; Eruslanov, Boris V; Svetoch, Edward A; Dyatlov, Ivan A

    2015-01-01

    Enterotoxin-producing Escherichia coli (ETEC) are one of the main causative agents of diarrhea in children especially in developing countries and travel diarrhoea in adults. Pathogenic properties of ETEC associated with their ability to produce a heat-stable (ST) and/or heat-labile (LT) enterotoxins, as well as adhesins providing bacterial adhesion to intestinal epithelial cells. This study presents the molecular characterization of the ETEC isolates collected from the Central and Far-Eastern regions of Russia in 2011-2012. It was shown that all ETEC under study (n=18) had the heat-labile enterotoxin-coding operon elt, and had no the genes of the heat-stable enterotoxin operon est. DNA sequencing revealed two types of nucleotide exchanges in the eltB gene coding subunit B of LT in isolates collected from Cherepovets city (Central region, Russia) and Vladivostok city (Far-East region, Russia). Only one ETEC strain carried genes cfaA, cfaB, cfaC and cfaD coding adhesion factor CFA/I. Expression of LT in four ETEC isolates in the agglutination reaction was detected using a latex test-system. The isolates were assigned to serogroups O142 (n = 6), О6 (n = 4), О25 (n = 5), О26 (n = 2), and O115 (n = 1). Genotyping showed that they belonged to an earlier described sequence-type ST4 (n = 3) as well as to 11 novel sequence-types ST1043, ST1312, ST3697, ST3707, ST3708, ST3709, ST3710, ST3755, ST3756, ST3757 and ST4509. The ETEC isolates displayed different levels of antimicrobial resistance. Eight isolates were resistant to only one drug, three isolates-to two drugs, one isolate-to three drugs, two isolates-to four antibacterials, and only one isolate to each of the five, six and ten antibacterials simultaneously. Genetic determinants of the resistance to beta-lactams and other classes of antibacterials on the ETEC genomes were identified. There are blaTEM (n = 10), blaCTX-M-15 (n = 1), class 1 integron (n = 3) carrying resistance cassettes to aminoglycosides and

  13. Binding of Escherichia coli heat-stable enterotoxin to rat intestinal cells and brush border membranes.

    PubMed Central

    Frantz, J C; Jaso-Friedman, L; Robertson, D C

    1984-01-01

    The association of heat-stable enterotoxin (STa) produced by enterotoxigenic Escherichia coli 431 with isolated rat intestinal epithelial cells and brush border membranes was characterized. Specific binding of strain 431 125I-STa to a single class of specific high-affinity receptors was saturable and temperature dependent and reached a maximum between 5 and 10 min. A 1,000-fold excess of unlabeled 431 STa competitively displaced 90 to 95% of radiolabeled enterotoxin bound to brush border membranes. In contrast, specific binding of 431 125I-STa to intestinal cells ranged from 40 to 65%. The number of STa-specific receptors on rat intestinal cells determined by Scatchard analysis was 47,520 +/- 14,352 (mean +/- standard error of the mean) per cell, with affinity constants (KaS) of 2.55 X 10(11)and 4.32 x 10(11) liters/mol determined for intestinal cells and brush border membranes, respectively. Villus intestinal cells appeared to possess about twice as many STa receptors as did crypt cells. Dissociation of specifically bound 431 125I-STa from intestinal cells and brush border membranes was minimal (2 to 5%). In addition, neither the rate nor the extent of dissociation was increased by a 1,000-fold excess of unlabeled homologous 431 Sta. Binding experiments with 431 125I-STa and brush border membranes showed that purified unlabeled STas from enterotoxigenic E. coli strains 667 (class 1 porcine enteropathogen), B-41 (bovine enteropathogen), and human strains 213C2 (Mexico) and 153961-2 (Dacca, Bangledesh) exhibited patterns of competitive inhibition similar to those of homologous unlabeled 431 STa (class 2 enteropathogen). A lipid extract which contained gangliosides and glycolipids exhibited dose-dependent competitive inhibition of heat-labile enterotoxin binding to brush border membranes but did not inhibit binding of 431 125I-STa. Purified heat-labile enterotoxin from strain 286C2 did not inhibit binding of 431 STa to brush border membranes. Pronase treatment of

  14. Role of heat-stable enterotoxins in the induction of early immune responses in piglets after infection with enterotoxigenic Escherichia coli.

    PubMed

    Loos, Michaela; Geens, Marisa; Schauvliege, Stijn; Gasthuys, Frank; van der Meulen, Jan; Dubreuil, J Daniel; Goddeeris, Bruno M; Niewold, Theo; Cox, Eric

    2012-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains that produce heat-stable (ST) and/or heat-labile (LT) enterotoxins are cause of post-weaning diarrhea in piglets. However, the relative importance of the different enterotoxins in host immune responses against ETEC infection has been poorly defined. In the present study, several isogenic mutant strains of an O149:F4ac(+), LT(+) STa(+) STb(+) ETEC strain were constructed that lack the expression of LT in combination with one or both types of ST enterotoxins (STa and/or STb). The small intestinal segment perfusion (SISP) technique and microarray analysis were used to study host early immune responses induced by these mutant strains 4 h after infection in comparison to the wild type strain and a PBS control. Simultaneously, net fluid absorption of pig small intestinal mucosa was measured 4 h after infection, allowing us to correlate enterotoxin secretion with gene regulation. Microarray analysis showed on the one hand a non-toxin related general antibacterial response comprising genes such as PAP, MMP1 and IL8. On the other hand, results suggest a dominant role for STb in small intestinal secretion early after post-weaning infection, as well as in the induced innate immune response through differential regulation of immune mediators like interleukin 1 and interleukin 17.

  15. Etiologic diagnosis of diarrheal disease of calves: frequency and methods for detecting enterotoxin and K99 antigen production by Escherichia cola.

    PubMed

    Moon, H W; Whipp, S C; Skartvedt, S M

    1976-09-01

    Escherichia coli isolated from calves in Minnesota and Montana were tested for enterotoxigenicity via bio-assay of cell-free broth culture fluid and for K99 antigen via a serum agglutination test. Infant mice were used to assay for heat-stable enterotoxin (ST), and adrenal cells in culture were used to assay for heat-labile enterotoxin (LT). Forty-six of the 345 E coli isolates produced ST enterotoxin, but none produced LT enterotoxin. Thirty-five of the 46 enterotoxigenic isolates had K99 antigen, and only 9 of 66 nonenterotoxigenic isolates so tested had this antigen. The enterotoxigenicity of 28 additional E coli isolates known or suspected to be calf enteropathogens and provided by investigators from 3 different laboratories was also tested. All isolates from 2 laboratories produced ST but not LT. All isolates from the 3rd laboratory produced LT but not ST. Escherichia coli organisms that were positive in the infant mouse assay also caused positive ligated, jejunal-loop responses in calves and in 9-day-old (but not in 5-week-old) pigs. It was concluded that the infant mouse and adrenal cell tests for ST and LT, combined with the agglutination test for K99, would be useful in the diagnosis of enteric enterotoxic colibacillosis of calves.

  16. Serological studies of HL-A on continuous lymphoblastoid cell lines (CLC) and the definition of an antigen on CLC determined by a heat-labile antibody*

    PubMed Central

    Dumble, Lynette; Jack, I.; Morris, P. J.

    1974-01-01

    Continuous lymphoblastoid cell lines (CLC) are more reactive with HL-A antisera in a complement-dependent cytotoxic test than are peripheral blood lymphocytes (PBL). This additional reactivity leads to assignment to a given CLC of more than four HL-A antigens, the maximum allowable under the two locus concept of the genetic control of HL-A. However, absorption of antisera by CLC shows that no more than four HL-A antigens exist on any of the CLC used in this laboratory. The additional reactivity of these cells lines can be explained in three ways. Firstly, it may be due to the presence of sublytic amounts of HL-A antibody in operationally monospecific antisera. Secondly, it may be due to cross-reactivity between HL-A antigens. Both these findings can be explained on the basis of the increased quantity of HL-A antigens on CLC compared to PBL. Thirdly, it may be due to the presence of a heat-labile (56° for 30 min) complement-dependent cytotoxic antibody which is present in 90% of normal human sera, and detects an antigen group tentatively labelled `D'. PMID:4466611

  17. Intranasal immunization with live recombinant Lactococcus lactis combined with heat-labile toxin B subunit protects chickens from highly pathogenic avian influenza H5N1 virus.

    PubMed

    Lei, Han; Peng, Xiaojue; Shu, Handing; Zhao, Daxian

    2015-01-01

    Development of safe and effective vaccines to prevent highly pathogenic avian influenza H5N1 virus infection is a challenging goal. Lactococcus lactis (L. lactis) is an ideal delivery vector for vaccine development, and it has been shown previously that oral immunization of encapsulated secretory L. lactis-hemagglutinin (HA) could provide complete protection against homologous H5N1 virus challenge in the mice model. While intranasal immunization is an appealing approach, it is now reported that secretory L. lactis-HA combined with mucosal adjuvant heat-labile toxin B subunit (LTB) could provide protective immunity in the chicken model. As compared to intranasal immunization with L. lactis-HA alone, L. lactis-HA combined with LTB (L. lactis-HA + LTB) could elicit robust neutralizing antibody responses and mucosal IgA responses, as well as strong cellular immune responses in the vaccinated chickens. Importantly, intranasal immunization with L. lactis-HA + LTB could provide 100% protection against H5N1 virus challenge. Taken together, these results suggest that intranasal immunization with L. lactis-HA + LTB can be considered as an effective approach for preventing and controlling infection of H5N1 virus in poultry during an avian influenza A/H5N1 pandemic.

  18. In vitro effects of wood creosote on enterotoxin-induced secretion measured electrophysiologically in the rat jejunum and colon.

    PubMed

    Kuge, T; Venkova, K; Greenwood-Van Meerveld, B

    2001-06-01

    Secretory diarrhea occurs when the balance between intestinal absorption and secretion is disturbed by excessive secretion caused by enterotoxins produced by the pathogen. Wood creosote has long been used as a traditional antidiarrheal remedy. The goal of our study was to extend our knowledge about the antisecretory action of wood creosote against Escherichia coli enterotoxin-induced secretion in the small intestine and colon. Experiments were performed in mucosal sheets of rat jejunum and colon which were stripped of the external muscle layers to eliminate interactions with smooth muscle activity and local blood flow. Mucosal sheets were placed in modified Ussing chambers and hypersecretory conditions were induced by heat-labile (LT) or heat-stable (STa) E. coli enterotoxins added cumulatively (0.01-10 microg/ml) to the mucosal bathing solution. Intestinal secretion was monitored electrophysiologically as transmucosal short circuit current (Isc). LT induced a concentration-dependent increase in Isc in the rat jejunum, with no effect in the colon. In contrast, STa induced a significant increase in colonic Isc, without causing any change in Isc across the jejunum. In separate experiments the effects of increasing concentrations of wood creosote (0.1-50 microg/ml), added to the mucosal or serosal bathing solution, were examined against the secretory responses induced by LT or STa. In the small intestine the antisecretory activity of wood creosote against LT-induced secretion was more potent following serosal application, whereas in the colon wood creosote inhibited STa-induced secretion with equal potency following either serosal or mucosal addition. In summary, our findings demonstrate that wood creosote possesses antidiarrheal activity suppressing E. coli enterotoxin-induced secretion in both the small intestine and colon.

  19. Prevalence of Pilus Antigens, Enterotoxin Types, and Enteropathogenicity Among K88-Negative Enterotoxigenic Escherichia coli from Neonatal Pigs

    PubMed Central

    Moon, H. W.; Kohler, E. M.; Schneider, R. A.; Whipp, S. C.

    1980-01-01

    Enterotoxigenic Escherichia coli (ETEC) that were isolated from neonatal pigs and that did not react in preliminary tests for pilus antigen K88 were subjected to additional tests for K88 and for pilus antigens K99 and 987P. Four such isolates produced K88, 9 isolates produced K99, 55 isolates produced 987P, and the remaining 43 isolates produced none of the three pilus antigens (3P−). Immunofluorescence tests of ileal sections from pigs were more sensitive for 987P detection than was serum agglutination of bacteria grown from the ileum. Most ETEC that produced K88, K99, or 987P were enteropathogenic (adhered to ileal villi, colonized intensively, and caused profuse diarrhea) when given to neonatal pigs. In contrast, only 3 of the 43 ETEC that produced none of the pilus antigens were enteropathogenic. The isolates were also tested for the type of enterotoxin produced. The K88+ isolates all produced heat-labile enterotoxin (LT) detectable in cultured adrenal cells (i.e., were LT+). None of the 987P+, K99+, or enterpathogenic 3P− isolates produced LT. However (except for a single K99+ isolate), they all produced heat-stable enterotoxin detectable in infant mice (STa+). Sixteen isolates produced neither LT nor STa but did produce enterotoxin detectable in ligated intestinal loops of pigs (STb). Most of these LT− STa− STb+ isolates were also K88−, K99−, and 987P− and non-enteropathogenic. One of them was K99+ and enteropathogenic. Our conclusions are as follows. (i) Most enteropathogenic ETEC from neonatal pigs produce either K88, 987P, or K99; however, there are some that produce none of the three antigens. (ii) Immunofluorescence tests for pilus antigens produced in vivo are recommended for the diagnosis of ETEC infections. (iii) Reports of LT− STa− STb+ swine ETEC are confirmed; furthermore, such isolates can be enteropathogenic. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6102079

  20. Quantitative Proteomic Analysis of Escherichia coli Heat-Labile Toxin B Subunit (LTB) with Enterovirus 71 (EV71) Subunit VP1

    PubMed Central

    Liu, Lin; Ma, Yongping; Zhou, Huicong; Wu, Mingjun

    2016-01-01

    The nontoxic heat-labile toxin (LT) B subunit (LTB) was used as mucosal adjuvant experimentally. However, the mechanism of LTB adjuvant was still unclear. The LTB and enterovirus 71 (EV71) VP1 subunit (EVP1) were constructed in pET32 and expressed in E. coli BL21, respectively. The immunogenicity of purified EVP1 and the adjuvanticity of LTB were evaluated via intranasal immunization EVP1 plus LTB in Balb/c mice. In order to elucidate the proteome change triggered by the adjuvant of LTB, the proteomic profiles of LTB, EVP1, and LTB plus EVP1 were quantitatively analyzed by iTRAQ-LC-MS/MS (isobaric tags for relative and absolute quantitation; liquid chromatography-tandem mass spectrometry) in murine macrophage RAW264.7. The proteomic data were analyzed by bioinformatics and validated by western blot analysis. The predicted protein interactions were confirmed using LTB pull-down and the LTB processing pathway was validated by confocal microscopy. The results showed that LTB significantly boosted EVP1 specific systematic and mucosal antibodies. A total of 3666 differential proteins were identified in the three groups. Pathway enrichment of proteomic data predicted that LTB upregulated the specific and dominant MAPK (mitogen-activated protein kinase) signaling pathway and the protein processing in endoplasmic reticulum (PPER) pathway, whereas LTB or EVP1 did not significantly upregulate these two signaling pathways. Confocal microscopy and LTB pull-down assays confirmed that the LTB adjuvant was endocytosed and processed through endocytosis (ENS)-lysosomal-endoplasmic reticulum (ER) system. PMID:27618897

  1. Quantitative Proteomic Analysis of Escherichia coli Heat-Labile Toxin B Subunit (LTB) with Enterovirus 71 (EV71) Subunit VP1.

    PubMed

    Liu, Lin; Ma, Yongping; Zhou, Huicong; Wu, Mingjun

    2016-08-27

    The nontoxic heat-labile toxin (LT) B subunit (LTB) was used as mucosal adjuvant experimentally. However, the mechanism of LTB adjuvant was still unclear. The LTB and enterovirus 71 (EV71) VP1 subunit (EVP1) were constructed in pET32 and expressed in E. coli BL21, respectively. The immunogenicity of purified EVP1 and the adjuvanticity of LTB were evaluated via intranasal immunization EVP1 plus LTB in Balb/c mice. In order to elucidate the proteome change triggered by the adjuvant of LTB, the proteomic profiles of LTB, EVP1, and LTB plus EVP1 were quantitatively analyzed by iTRAQ-LC-MS/MS (isobaric tags for relative and absolute quantitation; liquid chromatography-tandem mass spectrometry) in murine macrophage RAW264.7. The proteomic data were analyzed by bioinformatics and validated by western blot analysis. The predicted protein interactions were confirmed using LTB pull-down and the LTB processing pathway was validated by confocal microscopy. The results showed that LTB significantly boosted EVP1 specific systematic and mucosal antibodies. A total of 3666 differential proteins were identified in the three groups. Pathway enrichment of proteomic data predicted that LTB upregulated the specific and dominant MAPK (mitogen-activated protein kinase) signaling pathway and the protein processing in endoplasmic reticulum (PPER) pathway, whereas LTB or EVP1 did not significantly upregulate these two signaling pathways. Confocal microscopy and LTB pull-down assays confirmed that the LTB adjuvant was endocytosed and processed through endocytosis (ENS)-lysosomal-endoplasmic reticulum (ER) system.

  2. Associations between Heat-Stable (O) and Heat-Labile (HL) Serogroup Antigens of Campylobacter jejuni: Evidence for Interstrain Relationships within Three O/HL Serovars

    PubMed Central

    Jackson, C. J.; Fox, A. J.; Jones, D. M.; Wareing, D. R. A.; Hutchinson, D. N.

    1998-01-01

    A comparative examination of the heat-stable (O) and heat-labile (HL) serogrouping results for 9,024 sporadic human isolates of Campylobacter jejuni revealed conserved associations between specific O and HL antigens (O/HL serovars). Forty-nine percent of the isolates which grouped for both O and HL antigens belonged to one of three serovars: O 4 complex/HL 1 (17.9%), O 1/HL 2 (16.8%), or O 50/HL 7 (14.5%). Other common serovars were O 2/HL 4 (8.3%), O 6/HL 6 (8.1%), O 53/HL 11 (4.5%), O 19/HL 17 (3.3%), O 5/HL 9 (3.3%), O 9/HL 9 (3.2%), and O 23/HL 5 (3.1%). These 10 serovars accounted for 83.1% of the serogroupable isolates. A large number of strains (41.3%) could be typed by only one of the two methods or could not be serogrouped (11%). Strains belonging to three serovars, O 2/HL 4, O 50/HL 7, and O 23/HL 5, were further characterized by combining data from expressed features (O/HL serogroups, phage groups, and biotypes) with restriction fragment length polymorphism genotypes. These polyphasic data demonstrated that within each serovar, individual isolates showed substantial conservation of both genomic and phenotypic characteristics. The essentially clonal nature of the three serovars confirmed the potential of combined O and HL serogrouping as a practical and phylogenetically valid method for investigating the epidemiology of sporadic C. jejuni infection. PMID:9665996

  3. Avirulent K88 (F4)+ Escherichia coli strains constructed to express modified enterotoxins protect young piglets from challenge with a virulent enterotoxigenic Escherichia coli strain that expresses the same adhesion and enterotoxins.

    PubMed

    Santiago-Mateo, Kristina; Zhao, Mojun; Lin, Jun; Zhang, Weiping; Francis, David H

    2012-10-12

    Virulence of enterotoxigenic Escherichia coli (ETEC) is associated with fimbrial adhesins and enterotoxins such as heat-labile (LT) and/or heat-stable (ST) enterotoxins. Previous studies using a cell culture model suggest that exclusion of ETEC from attachment to epithelial cells requires expression of both an adhesin such as K88 (F4) fimbriae, and LT. To test the ability of non-pathogenic E. coli constructs to exclude virulent ETEC sufficiently to prevent clinical disease, we utilized a piglet ETEC challenge model. Thirty-nine 5-day-old piglets were inoculated with a placebo (control), or with either of the three K88(+)E. coli strains isogenic with regard to modified LT expression: 8017 (pBR322 plasmid vector control), non-toxigenic mutant 8221 (LT(R192G)) in pBR322, or 8488, with the LT gene fused to the STb gene in pBR322 (LT(R192G)-STb). Piglets were challenged with virulent ETEC Strain 3030-2 (K88(+)/LT/STb) 24h post-inoculation. K88ac receptor-positive piglets in the control group developed diarrhea and became dehydrated 12-24h post-challenge. Piglets inoculated with 8221 or 8488 did not exhibit clinical signs of ETEC disease; most piglets inoculated with 8017 showed diarrhea. Control pigs exhibited significant weight loss, increased blood total protein, and higher numbers of colony-forming units of 3030-2 E. coli in washed ileum and jejunum than treated pigs. This study shows for the first time that pre-inoculation with an avirulent strain expressing adhesive fimbriae and a non-toxic form of LT provides significant short term protection from challenge with a virulent ETEC strain that expresses the same fimbrial adhesion and enterotoxin.

  4. Escherichia coli Heat-Stable Enterotoxin Mediates Na+/H+ Exchanger 4 Inhibition Involving cAMP in T84 Human Intestinal Epithelial Cells.

    PubMed

    Beltrán, Ana R; Carraro-Lacroix, Luciene R; Bezerra, Camila N A; Cornejo, Marcelo; Norambuena, Katrina; Toledo, Fernando; Araos, Joaquín; Pardo, Fabián; Leiva, Andrea; Sanhueza, Carlos; Malnic, Gerhard; Sobrevia, Luis; Ramírez, Marco A

    2015-01-01

    The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl-release in intestinal cells, including the human colonic carcinoma T84 cell line, involving increased cGMP and membrane alkalization due to reduced Na+/H+ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pHi), and NHE1, NHE2, and NHE4 are expressed in T84 cells, we characterized the STa role as modulator of these exchangers. pHi was assayed by the NH4Cl pulse technique and measured by fluorescence microscopy in BCECF-preloaded cells. pHi recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 μmol/L STa (30 minutes), 25 μmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 μmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 μmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 μmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H+ efflux (JH+) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and JH+ (~63%), without altering basal pHi (range 7.144-7.172). STa did not alter ßi value in a range of 1.6 pHi units. The dpHi/dt and JH+ was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa-decreased dpHi/dt and JH+ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T84 cells with STa results in reduced NHE4 activity leading to a lower capacity of pHi recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human

  5. Escherichia coli Heat-Stable Enterotoxin Mediates Na+/H+ Exchanger 4 Inhibition Involving cAMP in T84 Human Intestinal Epithelial Cells

    PubMed Central

    Beltrán, Ana R.; Carraro-Lacroix, Luciene R.; Bezerra, Camila N. A.; Cornejo, Marcelo; Norambuena, Katrina; Toledo, Fernando; Araos, Joaquín; Pardo, Fabián; Leiva, Andrea; Sanhueza, Carlos; Malnic, Gerhard; Sobrevia, Luis; Ramírez, Marco A.

    2015-01-01

    The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl-release in intestinal cells, including the human colonic carcinoma T84 cell line, involving increased cGMP and membrane alkalization due to reduced Na+/H+ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pHi), and NHE1, NHE2, and NHE4 are expressed in T84 cells, we characterized the STa role as modulator of these exchangers. pHi was assayed by the NH4Cl pulse technique and measured by fluorescence microscopy in BCECF–preloaded cells. pHi recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 μmol/L STa (30 minutes), 25 μmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 μmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 μmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 μmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H+ efflux (JH+) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and JH+ (~63%), without altering basal pHi (range 7.144–7.172). STa did not alter ßi value in a range of 1.6 pHi units. The dpHi/dt and JH+ was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa–decreased dpHi/dt and JH+ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T84 cells with STa results in reduced NHE4 activity leading to a lower capacity of pHi recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting

  6. Purification and characterization of Vibrio cholerae non-O1 heat-stable enterotoxin.

    PubMed Central

    Arita, M; Takeda, T; Honda, T; Miwatani, T

    1986-01-01

    A toxin which causes rapid fluid accumulation in a suckling mouse assay and which was produced by Vibrio cholerae non-O1 was investigated. The toxin was purified from the culture supernatant of V. cholerae non-O1 (strain A-5) by ammonium sulfate fractionation, hydroxyapatite treatment, ethanol extraction, column chromatographies on SP-Sephadex C-50 and DEAE-Sephadex A-25, and high-pressure liquid chromatography on a Lichrosorb RP-8 column. About 1.4 X 10(5)-fold purification was achieved, with a recovery of about 12%. Although the crude preparation was heat labile, the purified toxin was heat stable. The minimum effective dose of purified toxin was about 5 ng in the suckling mouse assay. The amino acid composition of the purified toxin was determined to be Asp(3), Glu(1), Ala(1), half-Cys(6), Ile(2), Leu(1), Phe(1), and Pro(1). These data show the production of a new type of heat-stable enterotoxin (NAG-ST) by V. cholerae non-O1. PMID:3957432

  7. E. coli heat-stable enterotoxin and guanylyl cyclase C: new functions and unsuspected actions.

    PubMed Central

    Giannella, Ralph A.; Mann, Elizabeth A.

    2003-01-01

    Some E. coli cause diarrhea by elaborating heat-labile and heat-stable (ST) enterotoxins which stimulate intestinal secretion. E. coli ST's are small peptides which bind to intestinal luminal epithelial cell receptors. The ST receptor, one of a family of receptor-cyclases called guanylyl cyclase C (GC-C), is a membrane spanning protein containing an extracellular binding domain and intracellular protein kinase and catalytic domains. The intestine synthesizes and secretes homologous peptides, guanylin and uroguanylin. The kidney also synthesizes uroguanylin. ST, guanylin or uroguanylin binding to GC-C results in increased cGMP, phosphorylation of the CFTR Cl- channel and secretion. Proguanylin and prouroguanylin circulate in blood and bind to receptors in intestine, kidney, liver, brain etc. In the kidney, they stimulate the excretion of Na+ and K+. Study of GC-C "knock-out" mice reveal that GC-C is important to intestinal salt and water secretion, duodenal bicarbonate secretion, recovery from CCl4-induced liver injury, and to intestinal polyp formation in Min mice lacking GC-C. PMID:12813912

  8. Genetic Fusions of a CFA/I/II/IV MEFA (Multiepitope Fusion Antigen) and a Toxoid Fusion of Heat-Stable Toxin (STa) and Heat-Labile Toxin (LT) of Enterotoxigenic Escherichia coli (ETEC) Retain Broad Anti-CFA and Antitoxin Antigenicity

    PubMed Central

    Ruan, Xiaosai; Sack, David A.; Zhang, Weiping

    2015-01-01

    Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTaA14Q-dmLT or 3xSTaN12S-dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STaA14Q-dmLT and CFA/I/II/IV-STaN12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTaN12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTaN12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent

  9. Detection of Clostridium perfringens enterotoxin in human fecal samples and anti-enterotoxin in sera.

    PubMed Central

    Naik, H S; Duncan, C L

    1978-01-01

    By using counterimmunoelectrophoresis (CIEP), Clostridium perfringens enterotoxin was successfully demonstrated in fecal samples collected within 1 day of attack from sick individuals involved in a bacteriologically and epidemiologically proven outbreak of C. perfringens food poisoning. In contrast, enterotoxin was not demonstrable in fecal samples of apparently healthy individuals both at high- and low-risk exposure to the organism and enterotoxin or in fecal samples collected 4 to 5 days after a food poisoning outbreak. A 100% prevalence of C. perfringens anti-enterotoxin in sera of human volunteers at high- as well as low-risk exposure to the organism and enterotoxin was recorded with CIEP. PMID:211142

  10. Investigation of E. coli Enterotoxins.

    DTIC Science & Technology

    1976-08-01

    It has been determined that representative culture filtrates from two different strains (H197 and 74-114) of enterotoxigenic E . coli contain at least...for E . coli entorotoxin (soluble) and that trypsin-activated insol ECT is more antigenic than unactivated insol ECT. In contrast, it was determined...that cholera (ga) toxoid, with or without adjuvant, stimulates antitoxin capable of neutralizing both cholera and E . coli enterotoxins. It has been

  11. Detection and sequences of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene in enterotoxigenic E. coli strains isolated from piglets and calves with diarrhea.

    PubMed

    Yamamoto, T; Nakazawa, M

    1997-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains isolated from piglets and calves with diarrhea were tested for the presence of the enteroaggregative E. coli enterotoxin 1 (EAST1) gene sequences by PCR and colony hybridization. The EAST1 gene was found in most porcine ETEC strains with adherence factor K88, especially in those elaborating heat-labile enterotoxin. One porcine ETEC strain with adherence factor K99 was also positive for the EAST1 gene. In contrast, 987P-positive (987P+) ETEC strains from piglets, K99+ ETEC strains from calves, and K99+ F41+ or F41+ ETEC strains from piglets and calves were negative for the EAST1 gene. The K88ab+ or K88ac+ ETEC strains tested possessed the EAST1 gene on a plasmid that was distinct from a K88-encoding plasmid. The EAST1 gene sequences of the K88+ ETEC strains were identical to each other and 99.1 and 98.3% homologous to the previously reported sequences of ETEC strains colonizing humans and enteroaggregative E. coli strains, respectively. The data indicate that the EAST1 gene is distributed among porcine ETEC strains in association with the adherence factor type.

  12. A nontoxic chimeric enterotoxin adjuvant induces protective immunity in both mucosal and systemic compartments with reduced IgE antibodies.

    PubMed

    Kweon, Mi-Na; Yamamoto, Masafumi; Watanabe, Fumiko; Tamura, Shinichi; Van Ginkel, Frederik W; Miyauchi, Akira; Takagi, Hiroaki; Takeda, Yoshifumi; Hamabata, Takashi; Fujihashi, Kohtaro; McGhee, Jerry R; Kiyono, Hiroshi

    2002-11-01

    A novel nontoxic form of chimeric mucosal adjuvant that combines the A subunit of mutant cholera toxin E112K with the pentameric B subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli was constructed by use of the Brevibacillus choshinensis expression system (mCTA/LTB). Nasal immunization of mice with tetanus toxoid (TT) plus mCTA/LTB elicited significant TT-specific immunoglobulin A responses in mucosal compartments and induced high serum immunoglobulin G and immunoglobulin A anti-TT antibody responses. Although TT plus native CT induced high total and TT-specific immunoglobulin E responses, use of the chimera molecule as mucosal adjuvant did not. Furthermore, all mice immunized with TT plus mCTA/LTB were protected from lethal systemic challenge with tetanus toxin. Importantly, the mice were completely protected from influenza virus infection after nasal immunization with inactivated influenza vaccine together with mCTA/LTB. These results show that B. choshinensis-derived mCTA/LTB is an effective and safe mucosal adjuvant for the induction of protective immunity against potent bacterial exotoxin and influenza virus infection.

  13. In vitro inhibition of ETEC K88 adhesion by pea hulls and of LT enterotoxin binding by faba bean hulls.

    PubMed

    Becker, P M; van der Meulen, J; Jansman, A J M; van Wikselaar, P G

    2012-12-01

    Enterotoxigenic Escherichia coli (ETEC) expressing K88 (F4) adhesins are associated with post-weaning diarrhoea in piglets. Different grain fractions from pea (Pisum sativum) and faba bean (Vicia faba) were tested in vitro for their capacity to counteract aetiological factors, which contribute to the development of diarrhoea. In detail, adhesion of E. coli O149:K91:K88ac (ETEC K88ac) to grain legume products, intended to impair the colonization of the host, was studied as well as interference with receptor binding of the pathogen's heat-labile enterotoxin LT, intended to reduce toxin-inflicted gut cell damage. When comparing different pea and faba bean products tested for their binding capacity of ETEC K88ac, especially pea hulls, but also whole pea meal, starch-enriched and protein-enriched pea meal, and digestion-resistant pea hull and meal fractions showed a higher binding of ETEC K88ac than faba bean products. In contrast to the ETEC K88ac adhesion results, bean hulls proved more effective than pea hulls in preventing GM1 receptor binding of LT. Previous small intestinal segment perfusion experiments we performed with ETEC K88ac-challenged piglets indicated that both pea and bean hulls have the potential for successful application in diarrhoea prophylaxis and treatment, which is in agreement with and refined by our detection of their different modes of functioning.

  14. Detection of Escherichia coli Shiga toxin (stx) and enterotoxin (estA and elt) genes in fecal samples from non-diarrheic and diarrheic greyhounds.

    PubMed

    Staats, Jacque J; Chengappa, M M; DeBey, Mary C; Fickbohm, Barry; Oberst, Richard D

    2003-07-30

    Virulence factors responsible for acute diarrhea in greyhounds have not been well established. The objective of this study was to determine if a correlation exists between disease and the presence of the Escherichia coli toxin genes in non-diarrheic and diarrheic greyhound feces. DNA extracted from broth cultures was evaluated for the presence of Shiga toxin and enterotoxin genes and broth samples were evaluated for Shiga toxin and heat-labile enterotoxin. Shiga toxin (stx1 and stx2) and enterotoxin (et and estA) genes were identified in both non-diarrheic and diarrheic samples after in vitro cultured of swabs at 37 degrees C for 16-24h. The stx1 gene was present in 3% of non-diarrheic and 15% diarrheic samples and the stx2 gene was identified in 36 and 23%, non-diarrheic and diarrheic samples, respectively. Shiga toxin was present in 48% diarrheic and 25% of the non-diarrheic in vitro cultured samples. The elt gene was detected in vitro cultured swabs in 12% of the non-diarrheic and 7% of the diarrheic samples. Labile toxin was present in the feces of small numbers of both groups of dogs. A significant correlation existed between the presence of both stx1 genes and Shiga toxin in feces, and lack of disease in non-diarrheic (P=0.01) and presence of disease in diarrheic (P=0.024) greyhounds. Correlation between production of Shiga toxin and detection of stx1 or stx2 was significant in both the diarrheic and non-diarrheic feces (P=0.03); however, only the presence of stx1 correlated with diarrhea in both groups of samples (P<0.008). The incidence of toxigenic E. coli in both non-diarrheic and diarrheic greyhounds indicates a zoonotic potential from dogs to humans and requires further study.

  15. Production and partial purification of Salmonella enterotoxin.

    PubMed Central

    Sedlock, D M; Koupal, L R; Deibel, R H

    1978-01-01

    By using a strain of Salmonella typhimurium, we detected the presence of an enterotoxin, as determined by the rabbit ileal loop assay, in various complex and defined media. The enterotoxin was concentrated by ultrafiltration of culture supernatant fluids and eluted in and adjacent to the void volume of a Sephadex G-100 column. This suggested that the enterotoxic factor was of a relatively high molecular weight, and additional evidence indicated it was heterogeneous in size. Further chromatography, using a diethylaminoethyl-cellulose anion exchanger, facilitated at least a 50-fold purification of the Salmonella enterotoxin. PMID:352941

  16. Escherichia coli STb enterotoxin dislodges claudin-1 from epithelial tight junctions.

    PubMed

    Nassour, Hassan; Dubreuil, J Daniel

    2014-01-01

    Enterotoxigenic Escherichia coli produce various heat-labile and heat-stable enterotoxins. STb is a low molecular weight heat-resistant toxin responsible for diarrhea in farm animals, mainly young pigs. A previous study demonstrated that cells having internalized STb toxin induce epithelial barrier dysfunction through changes in tight junction (TJ) proteins. These modifications contribute probably to the diarrhea observed. To gain insight into the mechanism of increased intestinal permeability following STb exposure we treated human colon cells (T84) with purified STb toxin after which cells were harvested and proteins extracted. Using a 1% Nonidet P-40-containing solution we investigated the distribution of claudin-1, a major structural and functional TJ protein responsible for the epithelium impermeability, between membrane (NP40-insoluble) and the cytoplasmic (NP-40 soluble) location. Using immunoblot and confocal microscopy, we observed that treatment of T84 cell monolayers with STb induced redistribution of claudin-1. After 24 h, cells grown in Ca++-free medium treated with STb showed about 40% more claudin-1 in the cytoplasm compare to the control. Switching from Ca++-free to Ca++-enriched medium (1.8 mM) increased the dislodgement rate of claudin-1 as comparable quantitative delocalization was observed after only 6 h. Medium supplemented with the same concentration of Mg++ or Zn++ did not affect the dislodgement rate compared to the Ca++-free medium. Using anti-phosphoserine and anti-phosphothreonine antibodies, we observed that the loss of membrane claudin-1 was accompanied by dephosphorylation of this TJ protein. Overall, our findings showed an important redistribution of claudin-1 in cells treated with STb toxin. The loss of phosphorylated TJ membrane claudin-1 is likely to be involved in the increased permeability observed. The mechanisms by which these changes are brought about remain to be elucidated.

  17. [Staphylococcus enterotoxins, their properties and role as pathogenicity factors].

    PubMed

    Fluer, F S

    2012-01-01

    Data on staphylococci species producing staphylococcus enterotoxins (SE) are presented in the review. Genetics of toxin formation, SE biosynthesis, factors influencing SE formation (pH, temperature, effect of inductors and repressors), physical-chemical properties of SE, influence of temperature on enterotoxin stability, enterotoxin structure, immunologic properties, super antigen properties, SE mechanism of action, role of SE in nosocomial infections, intestine dysbacteriosis, atopic dermatitis, enterotoxin toxicity, clinical manifestations are examined.

  18. A quantitative study of enterotoxin production by sheep milk staphylococci.

    PubMed Central

    Bautista, L; Gaya, P; Medina, M; Nuñez, M

    1988-01-01

    Of 124 staphylococcal strains isolated from sheep milk, 78 produced enterotoxin A, B, C, or D when evaluated by an enzyme-linked immunosorbent assay. Enterotoxins A and D, elaborated by 44 and 43 strains, respectively, showed the highest incidence. Enterotoxin production by coagulase-negative strains (one Staphylococcus cohnii, three S. epidermidis, five S. haemolyticus, and four S. xylosus) was detected. Linear and logarithmic-logarithmic regressions of optical density on enterotoxin concentration yielded the best-fitting equations for enterotoxin quantitation. A significantly higher incidence of enterotoxin producers and significantly higher levels of enterotoxins produced were recorded for coagulase-positive, thermostable nuclease-positive, hemolysis-positive, or mannitol-positive strains. Mannitol utilization was the best test for discriminating between enterotoxigenic and nonenterotoxigenic staphylococci. PMID:3355142

  19. Natural Indoles, Indole-3-Carbinol (I3C) and 3,3’-Diindolylmethane (DIM), Attenuate Staphylococcal Enterotoxin B-Mediated Liver Injury by Downregulating miR-31 Expression and Promoting Caspase-2-Mediated Apoptosis

    PubMed Central

    Busbee, Philip B.; Nagarkatti, Mitzi; Nagarkatti, Prakash S.

    2015-01-01

    Staphylococcal enterotoxin B (SEB) is a potent superantigen capable of inducing inflammation characterized by robust immune cell activation and proinflammatory cytokine release. Exposure to SEB can result in food poisoning as well as fatal conditions such as toxic shock syndrome. In the current study, we investigated the effect of natural indoles including indole-3-carbinol (I3C) and 3,3’-diindolylmethane (DIM) on SEB-mediated liver injury. Injection of SEB into D-galactosamine-sensitized female C57BL/6 mice resulted in liver injury as indicated by an increase in enzyme aspartate transaminase (AST) levels, induction of inflammatory cytokines, and massive infiltration of immune cells into the liver. Administration of I3C and DIM (40mg/kg), by intraperitonal injection, attenuated SEB-induced acute liver injury, as evidenced by decrease in AST levels, inflammatory cytokines and cellular infiltration in the liver. I3C and DIM triggered apoptosis in SEB-activated T cells primarily through activation of the intrinsic mitochondrial pathway. In addition, inhibitor studies involving caspases revealed that I3C and DIM-mediated apoptosis in these activated cells was dependent on caspase-2 but independent of caspase-8, 9 and 3. In addition, I3C and DIM caused a decrease in Bcl-2 expression. Both compounds also down-regulated miR-31, which directly targets caspase-2 and influences apoptosis in SEB-activated cells. Our data demonstrate for the first time that indoles can effectively suppress acute hepatic inflammation caused by SEB and that this may be mediated by decreased expression of miR-31 and consequent caspase-2-dependent apoptosis in T cells. PMID:25706292

  20. Purification and biochemical properties of Clostridium perfringens type A enterotoxin.

    PubMed

    Stark, R L; Duncan, C L

    1972-11-01

    The sporulation-specific enterotoxin of Clostridium perfringens type A, which is the toxin active in human food poisoning, has been purified from extracts of sporulating cells. Highly purified enterotoxin was obtained by treatment of crude cell extract with ribonuclease for 30 min, followed by sequential chromatography on Sephadex G-100, Cellex T cellulose, and hydroxylapatite. Recovery was 65 to 75% of the initial activity. Enterotoxin purity was > 99% as indicated by sedimentation velocity, sedimentation equilibrium, disc electrophoresis, and serological methods. Purified enterotoxin focused at pH 4.3 during isoelectric focusing. Molecular weights of 34,000 and 35,000 were obtained by Sephadex G-100 chromatography and sedimentation equilibrium, respectively. An S(20,w) of 3.08 was obtained for the purified enterotoxin. The enterotoxin precipitated heavily at its isoelectric point and at concentrations greater than 4 mg/ml.

  1. Expression of staphylococcal enterotoxin C1 in Escherichia coli.

    PubMed Central

    Bohach, G A; Schlievert, P M

    1987-01-01

    The structural gene encoding staphylococcal enterotoxin C1 was cloned into Escherichia coli and localized on a 1.5-kilobase HindIII-ClaI DNA fragment by subcloning. The toxin was partially purified from E. coli clones and shown to be immunologically identical to enterotoxin C1 from Staphylococcus aureus. The cloned toxin also had the same molecular weight (26,000) and charge heterogeneity as staphylococcus-derived enterotoxin. Toxins from both sources were equally biologically active. Images PMID:3542834

  2. Mechanisms of staphylococcal enterotoxin-induced emesis.

    PubMed

    Hu, Dong-Liang; Nakane, Akio

    2014-01-05

    Pathogenic bacteria use various strategies to interact with the host organisms. Among them, toxin production constitutes an efficient way to alter specific functions of target cells. Various enterotoxins interact with the enteric nervous system, by stimulating afferent neurons or inducing neurotransmitter release from enterochromaffin cells which result either in vomiting, diarrhea, or in the intestinal inflammation process. Staphylococcus aureus produces a wide variety of toxins including staphylococcal enterotoxins (SEs) with demonstrated emetic activity; and staphylococcal enterotoxin-like (SEl) proteins, which are not emetic in a primate model or have yet to be tested. SEs and SEls have been traditionally subdivided into classical (SEA to SEE) and new (SEG to SElX) types. These toxins possess superantigenic activity and are highly resistant to denaturation which allows them to remain intact in contaminated foods and trigger food poisoning outbreaks. Symptoms are of rapid onset, and include nausea and violent vomiting. SEA is the most recognizable toxin causing food poisoning in humans throughout the world. However, it remains unclear how SEs induce emesis and via which signal pathway. This review is divided into four parts, and will focus on the following: (1) how bacterial toxins interact with the nervous system, (2) biological characteristics of SEs and SEls, (3) mechanisms of SE-induced emesis, and (4) use of a vaccine for the prevention of SE-induced emesis.

  3. Staphylococcal aureus Enterotoxin C and Enterotoxin-Like L Associated with Post-partum Mastitis

    PubMed Central

    Franck, Kristina T.; Gumpert, Heidi; Olesen, Bente; Larsen, Anders R.; Petersen, Andreas; Bangsborg, Jette; Albertsen, Per; Westh, Henrik; Bartels, Mette D.

    2017-01-01

    Denmark is a low prevalence country with regard to methicillin resistant Staphylococcus aureus (MRSA). In 2008 and 2014, two neonatal wards in the Copenhagen area experienced outbreaks with a typical community acquired MRSA belonging to the same spa type and sequence type (t015:ST45) and both were PVL and ACME negative. In outbreak 1, the isolates harbored SCCmec IVa and in outbreak 2 SCCmec V. The clinical presentation differed between the two outbreaks, as none of five MRSA positive mothers in outbreak 1 had mastitis vs. five of six MRSA positive mothers in outbreak 2 (p < 0.02). To investigate if whole-genome sequencing could identify virulence genes associated with mastitis, t015:ST45 isolates from Denmark (N = 101) were whole-genome sequenced. Sequence analysis confirmed two separate outbreaks with no sign of sustained spread into the community. Analysis of the accessory genome between isolates from the two outbreaks revealed a S. aureus pathogenicity island containing enterotoxin C and enterotoxin-like L only in isolates from outbreak 2. Enterotoxin C and enterotoxin-like L carrying S. aureus are associated with bovine mastitis and our findings indicate that these may also be important virulence factors for human mastitis. PMID:28223977

  4. Staphylococcal aureus Enterotoxin C and Enterotoxin-Like L Associated with Post-partum Mastitis.

    PubMed

    Franck, Kristina T; Gumpert, Heidi; Olesen, Bente; Larsen, Anders R; Petersen, Andreas; Bangsborg, Jette; Albertsen, Per; Westh, Henrik; Bartels, Mette D

    2017-01-01

    Denmark is a low prevalence country with regard to methicillin resistant Staphylococcus aureus (MRSA). In 2008 and 2014, two neonatal wards in the Copenhagen area experienced outbreaks with a typical community acquired MRSA belonging to the same spa type and sequence type (t015:ST45) and both were PVL and ACME negative. In outbreak 1, the isolates harbored SCCmec IVa and in outbreak 2 SCCmec V. The clinical presentation differed between the two outbreaks, as none of five MRSA positive mothers in outbreak 1 had mastitis vs. five of six MRSA positive mothers in outbreak 2 (p < 0.02). To investigate if whole-genome sequencing could identify virulence genes associated with mastitis, t015:ST45 isolates from Denmark (N = 101) were whole-genome sequenced. Sequence analysis confirmed two separate outbreaks with no sign of sustained spread into the community. Analysis of the accessory genome between isolates from the two outbreaks revealed a S. aureus pathogenicity island containing enterotoxin C and enterotoxin-like L only in isolates from outbreak 2. Enterotoxin C and enterotoxin-like L carrying S. aureus are associated with bovine mastitis and our findings indicate that these may also be important virulence factors for human mastitis.

  5. The enterotoxin of Bacteroides fragilis is a metalloprotease.

    PubMed Central

    Moncrief, J S; Obiso, R; Barroso, L A; Kling, J J; Wright, R L; Van Tassell, R L; Lyerly, D M; Wilkins, T D

    1995-01-01

    During the past decade, strains of Bacteroides fragilis that produce an enterotoxin have been implicated in diarrheal disease in animals and humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (M(r), approximately 20,000). Single specific primer-PCR was used to clone a portion of the B. fragilis enterotoxin gene. The recombinant protein expressed by the cloned gene fragment reacted with monospecific antibodies to B. fragilis enterotoxin by enzyme-linked immunosorbent assay and immunoblot analysis. The deduced amino acid sequence revealed a signature zinc-binding consensus motif (HEXXHXXGXXH/Met-turn) characteristic of metalloproteases termed metzincins. Sequence comparisons showed close identity to matrix metalloproteases (e.g., human fibroblast collagenase) within the zinc-binding and Met-turn region. Purified enterotoxin contained 1 g-atom of Zn2+ per molecule and hydrolyzed gelatin, azocoll, actin, tropomyosin, and fibrinogen. The enterotoxin also underwent autodigestion. The N-terminal amino acid sequences of two autodigestion products were identical to the deduced amino acid sequence of the recombinant enterotoxin and revealed cleavage at Cys-Leu and Ser-Leu peptide bonds. Gelatinase (type IV collagenase) activity comigrated with the toxin when analyzed by gel fractionation and zymography, indicating that protease activity is due to the enterotoxin and not to a contaminating protease(s). Optimal proteolytic activity occurred at 37 degrees C and pH 6.5. Primary proteolytic cleavage sites in actin were identified, revealing cleavage at Gly-Met and Thr-Leu peptide bonds. Enzymatic activity was inhibited by metal chelators but not by inhibitors of other classes of proteases. Additionally, cytotoxic activity of the enterotoxin on human carcinoma HT-29 cells was inhibited by acetoxymethyl ester EDTA. The metalloprotease activity of the enterotoxin suggests a possible mechanism for enterotoxicity and may have additional

  6. The chromosomal nature of LT-II enterotoxins solved: a lambdoid prophage encodes both LT-II and one of two novel pertussis-toxin-like toxin family members in type II enterotoxigenic Escherichia coli

    PubMed Central

    Jobling, Michael G.

    2016-01-01

    Heat-labile enterotoxins (LT) of enterotoxigenic Escherichia coli (ETEC) are structurally and functionally related to cholera toxin (CT). LT-I toxins are plasmid-encoded and flanked by IS elements, while LT-II toxins of type II ETEC are chromosomally encoded with flanking genes that appear phage related. Here, I determined the complete genomic sequence of the locus for the LT-IIa type strain SA53, and show that the LT-IIa genes are encoded by a 51 239 bp lambdoid prophage integrated at the rac locus, the site of a defective prophage in E. coli K12 strains. Of 50 LT-IIa and LT-IIc, 46 prophages also encode one member of two novel two-gene ADP-ribosyltransferase toxin families that are both related to pertussis toxin, which I named eplBA or ealAB, respectively. The eplBA and ealAB genes are syntenic with the Shiga toxin loci in their lambdoid prophages of the enteric pathogen enterohemorrhagic E. coli. These novel AB5 toxins show pertussis-toxin-like activity on tissue culture cells, and like pertussis toxin bind to sialic acid containing glycoprotein ligands. Type II ETEC are the first mucosal pathogens known to simultaneously produce two ADP-ribosylating toxins predicted to act on and modulate activity of both stimulatory and inhibitory alpha subunits of host cell heterotrimeric G-proteins. PMID:26755534

  7. The chromosomal nature of LT-II enterotoxins solved: a lambdoid prophage encodes both LT-II and one of two novel pertussis-toxin-like toxin family members in type II enterotoxigenic Escherichia coli.

    PubMed

    Jobling, Michael G

    2016-04-01

    Heat-labile enterotoxins (LT) of enterotoxigenic Escherichia coli (ETEC) are structurally and functionally related to cholera toxin (CT). LT-I toxins are plasmid-encoded and flanked by IS elements, while LT-II toxins of type II ETEC are chromosomally encoded with flanking genes that appear phage related. Here, I determined the complete genomic sequence of the locus for the LT-IIa type strain SA53, and show that the LT-IIa genes are encoded by a 51 239 bp lambdoid prophage integrated at the rac locus, the site of a defective prophage in E. coli K12 strains. Of 50 LT-IIa and LT-IIc, 46 prophages also encode one member of two novel two-gene ADP-ribosyltransferase toxin families that are both related to pertussis toxin, which I named eplBA or ealAB, respectively. The eplBA and ealAB genes are syntenic with the Shiga toxin loci in their lambdoid prophages of the enteric pathogen enterohemorrhagic E. coli. These novel AB(5) toxins show pertussis-toxin-like activity on tissue culture cells, and like pertussis toxin bind to sialic acid containing glycoprotein ligands. Type II ETEC are the first mucosal pathogens known to simultaneously produce two ADP-ribosylating toxins predicted to act on and modulate activity of both stimulatory and inhibitory alpha subunits of host cell heterotrimeric G-proteins.

  8. Heat-stable Escherichia coli enterotoxin production in vivo.

    PubMed Central

    Whipp, S C; Moon, H W; Lyon, N C

    1975-01-01

    Hysterectomy-derived, colostrum-deprived piglets were infected with enterotoxigenic Escherichia coli on day 4 of life. Samples of feces and intestinal contents were collected and tested in infant mice for enterotoxic activity. Positive enterotoxic responses were observed in mice given filtrates of feces and intestinal contents from piglets infected withe enterotoxigenic E. coli known to produce heat-stable enterotoxin but not heat-liabile enterotoxin in vitro. It is concluded that heat-stable enterotoxigenic E. coli induce diarrhea by production of heat-stable enterotoxin in vivo. PMID:1097335

  9. Binding of flavonoids to staphylococcal enterotoxin B.

    PubMed

    Benedik, Evgen; Skrt, Mihaela; Podlipnik, Crtomir; Ulrih, Nataša Poklar

    2014-12-01

    Staphylococcal enterotoxins are metabolic products of Staphylococcus aureus that are responsible for the second-most-commonly reported type of food poisoning. Polyphenols are known to interact with proteins to form complexes, the properties of which depend on the structures of both the polyphenols and the protein. In the present study, we investigated the binding of four flavonoid polyphenols to Staphylococcal enterotoxin B (SEB) at pH 7.5 and 25 °C: (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), kaempferol-3-glucoside (KAM-G) and kaempferol (KAM). Fluorescence emission spectrometry and molecular docking were applied to compare experimentally determined binding parameters with molecular modeling. EGCG showed an order of magnitude higher binding constant (1.4 × 10(5) M(-1)) than the other studied polyphenols. Our blind-docking results showed that EGCG and similar polyphenolic ligands is likely to bind to the channel at the surface of SEB that is responsible for the recognition of the T-cell beta chain fragment and influence the adhesion of SEB to T cells.

  10. Crystal structure of the superantigen staphylococcal enterotoxin type A.

    PubMed Central

    Schad, E M; Zaitseva, I; Zaitsev, V N; Dohlsten, M; Kalland, T; Schlievert, P M; Ohlendorf, D H; Svensson, L A

    1995-01-01

    Staphylococcal enterotoxins are prototype superantigens characterized by their ability to bind to major histocompatibility complex (MHC) class II molecules and subsequently activate a large fraction of T-lymphocytes. The crystal structure of staphylococcal enterotoxin type A (SEA), a 27 kDa monomeric protein, was determined to 1.9 A resolution with an R-factor of 19.9% by multiple isomorphous replacement. SEA is a two domain protein composed of a beta-barrel and a beta-grasp motif demonstrating the same general structure as staphylococcal enterotoxins SEB and TSST-1. Unique for SEA, however, is a Zn2+ coordination site involved in MHC class II binding. Four amino acids including Ser1, His187, His225 and Asp227 were found to be involved in direct coordination of the metal ion. SEA is the first Zn2+ binding enterotoxin that has been structurally determined. Images PMID:7628431

  11. Enterotoxin production, phage typing and serotyping of Staphylococcus aureus strains isolated from clinical materials and food.

    PubMed Central

    Melconian, A. K.; Brun, Y.; Fleurette, J.

    1983-01-01

    The production of enterotoxins A, B, C and F by strains of Staphylococcus aureus isolated from various clinical sources and from isolates implicated in food poisoning was investigated. One hundred and ninety one of the 374 clinical strains (51.1%) were found to be enterotoxigenic; of these, 81 (27.7%) strains produced enterotoxin A, 57 (15.3%) strains produced enterotoxin B, 23 (6.2%) strains produced enterotoxin C, and 64 (17.1%) strains produced enterotoxin F. These enterotoxigenic strains were most frequently lysed by phages of group III (21.5%) or were not typable (22%). Eighteen of the 29 strains implicated in food poisoning were enterotoxigenic. The correlation of antigens and bacteriophage patterns with enterotoxigenicity was determined: enterotoxin A being related to a4 antigen, enterotoxin B to phages of 94/96 complex with c1, o antigens, and enterotoxin F to phages of group I with 2632, k1k2, m antigens. PMID:6227656

  12. Comparison of different purification procedure for extraction of staphylococcal enterotoxin A from foods.

    PubMed Central

    Niskanen, A; Lindroth, S

    1976-01-01

    Different procedures commonly used for extraction, purification, and concentration of staphylococcal enterotoxins from foods were investigated with 131I- and 125I-labeled staphylococcal enterotoxin A. Loss of labeled enterotoxin A was compared with loss of total nitrogen. The results showed that in most of the common procedures, such as gel filtration, ion exchange, and heat treatment, the percentage of loss of labeled enterotoxin A was greater than the loss of total nitrogen. Chloroform extraction and acid precipitation with hydrochloric acid had nearly the same effect on the purification of both labeled enterotoxin A and total nitrogen. Ammonium sulfate precipitation proved to be practical and was successfully used for purification of enterotoxin A from sausage extract. Simultaneous use of trypsin and Pseudomonas peptidase for treatment of food extracts considerably reduced food proteins capable of interfering with serological detection of enterotoxins but did not essentailly influence the loss of enterotoxin A. PMID:984824

  13. Thermal Inactivation of Staphylococcal Enterotoxin B in Veronal Buffer

    PubMed Central

    Read, R. B.; Bradshaw, J. G.

    1966-01-01

    The times and temperatures required to inactivate staphylococcal enterotoxin B were studied by use of the double-gel-diffusion technique to assay enterotoxin. Enterotoxin B (99 +% pure) was suspended in 0.04 M Veronal buffer, dispensed into borosilicate vials, and the vials were sealed and heated in an oil bath. An amount of 30 μg/ml of this toxin was reduced to less than 0.7 μg/ml in 103.0, 87.1, 70.5, 57.2, 39.1, 27.6, 16.4, and 12.0 min, respectively, at temperatures of 96, 99, 101.7, 104.4, 110, 115.6, 121, and 126.7 C. The end point for enterotoxin inactivation by gel diffusion was identical to that by intravenous injection of cats. Limited studies with crude enterotoxin B showed that the crude preparation was slightly more thermostable. The respective D values of crude and purified enterotoxin B were 64.5 and 52.3, 40.5 and 34.4, 29.7 and 23.5, 18.8 and 16.6, and 11.4 and 9.9 min at temperatures of 99, 104.4, 110, 115.6, and 121 C. The z value for purified enterotoxin B was 32.4 C. The experimental activation energy was 20,700 cal/g mole, standard enthalpy of activation at 120 C was 19,900 cal/g mole, standard entropy of activation at 120 C was -21.4 cal/g mole K, and the standard free energy of activation at 120 C was 28,200 cal/g mole. PMID:4958146

  14. Rapid cell-based assay for detection and quantification of active staphylococcal enterotoxin type D

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Food poisoning by Staphylococcus aureus is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by this bacterium and is a major source of foodborne illness. Staphylococcal enterotoxin D (SED) is one of the predominant enterotoxins recovered in Staphylococcal food poisoning incidences...

  15. Production and properties of crude enterotoxin of Pseudomonas aeruginosa.

    PubMed

    Grover, S; Batish, V K; Srinivasan, R A

    1990-05-01

    Pseudomonas aeruginosa CTM-3 was found to be the most potentially enterotoxigenic strain out of the 12 isolates recovered from milk, as a high fluid length ratio, i.e. F/L (1.1) in rabbit gut and a strong permeability response in rabbit skin (38.5 mm2 necrotic zone) was obtained with this culture. No clear-cut relationship between the two tests was observed. Six of the ethidium bromide (300 micrograms/ml) cured variants of this culture completely lost their ability to produce enterotoxin indicating the possible involvement of a plasmid in enterotoxin synthesis. The crude enterotoxin from P. aeruginosa CTM-3 was completely inactivated in 15 s at 72 degrees C. However, it was fairly stable at pH values in the range 4.5-7.5. Both pepsin and trypsin inactivated the enterotoxin activity at a concentration of 40 micrograms/ml. Organic acids, formalin and hydrogen peroxide had no significant effect on the enterotoxin activity. The need for further investigations with purified preparations is emphasized.

  16. Distribution of food-borne Staphylococcus aureus enterotoxin genes.

    PubMed

    Hu, W D

    2016-01-29

    We identified and analyzed 5 new-type enterotoxin genes, including SEj, SEl, SEq, SEm, and SEr, to explore the distribution of 5 enterotoxin genes in Staphylococcus aureus of different origins as well as their correlations and differences. We examined the distribution of the S. aureus enterotoxin genes and their pathogenic mechanisms. A total of 660 specimens were collected from January 2011 to December 2014, and 217 strains of S. aureus were isolated. The template DNA of S. aureus was extracted. The Primer6.0 and Oligo7 software were used to design and synthesize polymerase chain reaction primers. Amplification results were analyzed by electrophoresis, and the amplification products were recovered and sequenced. Thirty-six bacterial strains contained the SEj gene (16.6%), including 15, 8, 8, 4, and 1 strains in fresh meat, quick-frozen food, raw milk, human purulent tissue, and living environment, respectively. Thirty-one bacterial strains contained the SEr gene (14.3%), including 16, 9, and 6 strains in fresh meat, quick-frozen food, and raw milk, respectively. Twenty-one bacterial strains contained the enterotoxin SEq gene (9.7%), including 8, 6, 6, and 1 strains in fresh meat, quick-frozen food, raw milk, and human purulent tissue, respectively. No SEm and SEl genes were detected. Different types of foods carry different types of enterotoxins, providing a basis for quick tracing for food poisoning. Three enterotoxin genes, SEj, SEr, and SEq, showed the highest carrier rate in quick-frozen food. It is imperative to improve their detection in quick-frozen food.

  17. Non-hemolytic enterotoxin of Bacillus cereus induces apoptosis in Vero cells.

    PubMed

    Liu, Xiaoye; Ding, Shuangyang; Shi, Peijie; Dietrich, Richard; Märtlbauer, Erwin; Zhu, Kui

    2016-10-20

    Bacillus cereus is an opportunistic pathogen that often causes foodborne infectious diseases and food poisoning. Non-hemolytic enterotoxin (Nhe) is the major toxin found in almost all enteropathogenic B. cereus and B. thuringiensis isolates. However, little is known about the cellular response after Nhe triggered pore formation on cell membrane. Here, we demonstrate that Nhe induced cell cycle arrest at G0 /G1 phase and provoked apoptosis in Vero cells, most likely associated with mitogen-activated protein kinase (MAPK) and death receptor pathways. The influx of extracellular calcium ions and increased level of reactive oxygen species in cytoplasm were sensed by apoptosis signal-regulating kinase 1 (ASK1) and p38 MAPK. Extrinsic death receptor Fas could also promote the activation of p38 MAPK. Subsequently, ASK1 and p38 MAPK triggered downstream caspase-8 and 3 to initiate apoptosis. Our results clearly demonstrate that ASK1, and Fas-p38 MAPK-mediated caspase-8 dependent pathways are involved in apoptotic cell death provoked by the pore-forming enterotoxin Nhe.

  18. Food Poisoning and Staphylococcus aureus Enterotoxins

    PubMed Central

    Argudín, María Ángeles; Mendoza, María Carmen; Rodicio, María Rosario

    2010-01-01

    Staphylococcus aureus produces a wide variety of toxins including staphylococcal enterotoxins (SEs; SEA to SEE, SEG to SEI, SER to SET) with demonstrated emetic activity, and staphylococcal-like (SEl) proteins, which are not emetic in a primate model (SElL and SElQ) or have yet to be tested (SElJ, SElK, SElM to SElP, SElU, SElU2 and SElV). SEs and SEls have been traditionally subdivided into classical (SEA to SEE) and new (SEG to SElU2) types. All possess superantigenic activity and are encoded by accessory genetic elements, including plasmids, prophages, pathogenicity islands, vSa genomic islands, or by genes located next to the staphylococcal cassette chromosome (SCC) implicated in methicillin resistance. SEs are a major cause of food poisoning, which typically occurs after ingestion of different foods, particularly processed meat and dairy products, contaminated with S. aureus by improper handling and subsequent storage at elevated temperatures. Symptoms are of rapid onset and include nausea and violent vomiting, with or without diarrhea. The illness is usually self-limiting and only occasionally it is severe enough to warrant hospitalization. SEA is the most common cause of staphylococcal food poisoning worldwide, but the involvement of other classical SEs has been also demonstrated. Of the new SE/SEls, only SEH have clearly been associated with food poisoning. However, genes encoding novel SEs as well as SEls with untested emetic activity are widely represented in S. aureus, and their role in pathogenesis may be underestimated. PMID:22069659

  19. Promotion

    PubMed Central

    Alam, Hasan B.

    2013-01-01

    This article gives an overview of the promotion process in an academic medical center. A description of different promotional tracks, tenure and endowed chairs, and the process of submitting an application is provided. Finally, some practical advice about developing skills and attributes that can help with academic growth and promotion is dispensed. PMID:24436683

  20. Staphylococcus enterotoxin profile of China isolates and the superantigenicity of some novel enterotoxins.

    PubMed

    Shen, Menglu; Li, Yi; Zhang, Linlin; Dai, Songbao; Wang, Jiashun; Li, Yongqing; Zhang, Lei; Huang, Jinhai

    2017-02-24

    The genus of staphylococcus widely distributes in environments and contributes to a variety of animal and human diseases. The enterotoxins (SEs) secreted by this type of pathogen have been the leading cause of bacterial toxic shock syndrome and food poisoning, and thus present a substantial concern to public health. In this study, we analyzed the superantigen profile of 122 staphylococcus strains isolated from diverse sources. When screened for the presence and prevalence of 17 known se or se-like (sel) genes, except selj, all other genes were detected in these isolates. In particular, 95.9% of the isolates harbored at least one se/sel gene. Moreover, 47.5% of them bore at least 5. Remarkably, several non-pathogenic species of animal- and environment-origin were also found to carry multiple se/sels. The most frequent genes detected were tsst (62.3%), sei (54.1%), and seb (46.7%), followed by some sel genes (selo, selu, and selm), which also were present at relatively high frequency (20-30%). The generated data improved understanding of strain-specific differences in enterotoxin expression. The gene products of the latter (selo and selu) were subsequently analyzed for their antigenicity in a mouse model using purified E. coli-based recombinant proteins. The studies revealed a strong activity for SEO in induction of T-lymphocyte proliferation and production of various inflammatory cytokines either in vivo or in vitro. In contrast, SEU exhibited little superantigenic effects. The molecular basis for the difference in antigenicity was analyzed by 3D homology remodeling, which revealed a difference in binding and affinities for MHC-II molecules and TCR Vβ region.

  1. Rapid Quantitative Serological Assay of Staphylococcal Enterotoxin B

    PubMed Central

    Weirether, Francis J.; Lewis, Evelyn E.; Rosenwald, Albert J.; Lincoln, Ralph E.

    1966-01-01

    A simple, rapid method, based on the Oudin single diffusion technique, is described for the quantitative assay of staphylococcal enterotoxin B. The method yields reproducible results without close control of such assay variables as temperature, antiserum dilution, and assay time, provided that the ionic strength is maintained above 0.2 n sodium chloride equivalent. PMID:4959985

  2. Development and evaluation of a rapid strategy to determine enterotoxin gene content in Staphylococcus aureus.

    PubMed

    Fischer, Adrien; Francois, Patrice; Holtfreter, Silva; Broeker, Barbara; Schrenzel, Jacques

    2009-05-01

    Enterotoxins of S. aureus are important molecules displaying superantigenic properties. To date no less than 18 enterotoxins have been identified in S. aureus and their role has been documented in very diverse diseases. Using available nucleotide sequence information, we developed a rapid and automated PCR-based approach to evaluate enterotoxin content in S aureus. We studied a collection of S. aureus strains previously analyzed for enterotoxins gene content and report a perfect correlation between simplex and multiplex PCR assays for the presence of all enterotoxin genes described so far. The determination of enterotoxin content relies on 4 multiplex PCR tubes whose amplification products are resolved by a rapid microcapillary electrophoresis. Automated analysis of the PCR profiles evaluates for the presence of the 18 enterotoxin genes in less than 3 h and at moderate cost. Finally, the use of enterotoxin gene content for genotyping purpose was compared to multi-locus variable number of tandem repeat assay and spa genotyping. Analysis revealed an important homogeneity of the genetic backgrounds for strains harboring the egc cluster as well as a large diversity for strains harboring other enterotoxins but lacking the egc cluster. A combined genotyping method that includes rapid enterotoxin content determination appears informative for various epidemiological survey purposes.

  3. Enterotoxin production by Bacillus cereus under gastrointestinal conditions and their immunological detection by commercially available kits.

    PubMed

    Ceuppens, Siele; Rajkovic, Andreja; Hamelink, Stefanie; Van de Wiele, Tom; Boon, Nico; Uyttendaele, Mieke

    2012-12-01

    Currently, three commercial kits for Bacillus cereus enterotoxins Nhe and/or Hbl detection are available, namely, the Bacillus diarrheal enterotoxin visual immunoassay (BDE VIA™) kit (3M Tecra), B. cereus enterotoxin reversed passive latex agglutination (BCET-RPLA) kit (Oxoid), and the Duopath(®) Cereus Enterotoxins (Merck). The performance of the kits and their applicability to gastrointestinal simulation samples were evaluated. Then, the stability and production of enterotoxins Hbl and Nhe under gastrointestinal conditions were investigated. Enterotoxin production was absent or impaired at acidic pH, i.e., in gastric medium with pH 5.0 and lasagne verde with pH 5.5. B. cereus did produce enterotoxins Nhe and Hbl during anaerobic growth in intestinal medium at pH 7.0, but the toxins were instantly degraded by the enzymes in the host's digestive secretions. Preformed enterotoxins did not withstand gastrointestinal passage under the simulated conditions, which suggests that preformed enterotoxins in food do not contribute to the diarrheal food poisoning syndrome. In conclusion, diarrhea is probably caused by de novo enterotoxin production by B. cereus cells located closely to the host's intestinal epithelium.

  4. Nasal Vaccination with the 40-Kilodalton Outer Membrane Protein of Porphyromonas gingivalis and a Nontoxic Chimeric Enterotoxin Adjuvant Induces Long-Term Protective Immunity with Reduced Levels of Immunoglobulin E Antibodies▿

    PubMed Central

    Momoi, Fumiki; Hashizume, Tomomi; Kurita-Ochiai, Tomoko; Yuki, Yoshikazu; Kiyono, Hiroshi; Yamamoto, Masafumi

    2008-01-01

    In this study, we demonstrated that the 40-kDa outer membrane protein of Porphyromonas gingivalis (40-kDa OMP) nasally administered with a nontoxic chimeric adjuvant that combines the A subunit of mutant cholera toxin E112K with the pentameric B subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli (mCTA/LTB) elicited a long-term protective immune response. Immunization with the 40-kDa OMP and mCTA/LTB induced high levels of 40-kDa-OMP-specific immunoglobulin G (IgG) and IgA antibodies (Abs) in sera and elicited a significant IgA anti-40-kDa OMP Ab response in saliva. These Ab responses were maintained for at least 1 year after the immunization. Although using adjuvant mCTA/LTB gave Ab responses in the saliva comparable to those obtained using native cholera toxin (nCT) as the adjuvant, the levels of total IgE and 40-kDa-OMP-specific IgE Abs as well as interleukin-4 levels induced by the immunization with mCTA/LTB were lower than those induced by the immunization with nCT. Importantly, IgG Abs generated by nasal immunization with the 40-kDa OMP plus mCTA/LTB inhibited the coaggregation and hemagglutinin activities of P. gingivalis. Furthermore, the mice given nasal 40-kDa OMP plus mCTA/LTB showed a significant reduction of alveolar bone loss caused by oral infection with P. gingivalis even 1 year after the immunization compared to the loss in unimmunized mice. Because mCTA/LTB is nontoxic, nasally administered 40-kDa OMP together with mCTA/LTB should be an effective and safe mucosal vaccine against P. gingivalis infection in humans and may be an important tool for the prevention of chronic periodontitis. PMID:18411288

  5. Modulation of the humoral and cellular immune response in Abeta immunotherapy by the adjuvants monophosphoryl lipid A (MPL), cholera toxin B subunit (CTB) and E. coli enterotoxin LT(R192G).

    PubMed

    Maier, Marcel; Seabrook, Timothy J; Lemere, Cynthia A

    2005-10-25

    Abeta vaccination or passive transfer of human-specific anti-Abeta antibodies are approaches under investigation to prevent and/or treat Alzheimer's disease (AD). Successful active Abeta vaccination requires a strong and safe adjuvant to induce anti-Abeta antibody formation. We compared the adjuvants monophosphoryl lipid A (MPL)/trehalose dicorynomycolate (TDM), cholera toxin B subunit (CTB) and Escherichia coli heat-labile enterotoxin LT(R192G) for their ability to induce a humoral and cellular immune reaction, using fibrillar Abeta1-40/42 as a common immunogen in wildtype B6D2F1 mice. Subcutaneous (s.c.) administration with MPL/TDM resulted in anti-Abeta antibodies levels up to four times higher compared to s.c. LT(R192G). Using MPL/TDM, the anti-Abeta antibodies induced were mainly IgG2b, IgG1 and lower levels of IgG2a and IgM, with a moderate splenocyte proliferation and IFN-gamma production in vitro upon stimulation with Abeta1-40/42. LT(R192G), previously shown by us to induce robust titers of anti-Abeta antibodies, generated predominantly IgG2b and IgG1 anti-Abeta antibodies with very low splenocyte proliferation and IFN-gamma production. Weekly intranasal (i.n.) administration over 11 weeks of Abeta40/42 with CTB induced only moderate levels of antibodies. All immunogens generated antibodies that recognized mainly the Abeta1-7 epitope and specifically detected amyloid plaques on AD brain sections. In conclusion, MPL/TDM, in addition to LT(R192G), is an effective adjuvant when combined with Abeta40/42 and may aid in the design of Abeta immunotherapy.

  6. Specificity of Interaction between Clostridium perfringens Enterotoxin and Claudin-Family Tight Junction Proteins

    PubMed Central

    Mitchell, Leslie A.; Koval, Michael

    2010-01-01

    Clostridium perfringens enterotoxin (CPE), a major cause of food poisoning, forms physical pores in the plasma membrane of intestinal epithelial cells. The ability of CPE to recognize the epithelium is due to the C-terminal binding domain, which binds to a specific motif on the second extracellular loop of tight junction proteins known as claudins. The interaction between claudins and CPE plays a key role in mediating CPE toxicity by facilitating pore formation and by promoting tight junction disassembly. Recently, the ability of CPE to distinguish between specific claudins has been used to develop tools for studying roles for claudins in epithelial barrier function. Moreover, the high affinity of CPE to selected claudins makes CPE a useful platform for targeted drug delivery to tumors expressing these claudins. PMID:22069652

  7. [The detection of a choleriform thermolabile enterotoxin in clinical strains of Proteus isolated in different infections].

    PubMed

    Gabidullin, Z G; Zhukova, S L; Ezepchuk, Iu V; Bondarenko, V M

    1989-12-01

    The capacity of Proteus strains, isolated from patients with purulent inflammatory, urological and enteric infections, for the production of choleriform thermolabile enterotoxin was studied by means of the enzyme immunoassay (EIA) with the use of antitoxic serum to Escherichia coli enterotoxin. Out of 125 strains, 27 (21.6%) showed the capacity for producing choleriform thermolabile enterotoxin in EIA experiments. The results thus obtained indicate that EIA techniques can be used, in principle, for detecting the capacity of Proteus for the production of choleriform thermolabile enterotoxin.

  8. Sensitive, rapid, quantitative and in vitro method for the detection of biologically active staphylococcal enterotoxin type E

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcus aureus is a major bacterial pathogen which causes clinical infections and food poisoning. This bacterium produces a group of enterotoxins (SEs). These enterotoxins have two separate but related biological activities. They cause gastroenteritis and function as superantigens that activa...

  9. Monkey Feeding Assay for Testing Emetic Activity of Staphylococcal Enterotoxin.

    PubMed

    Seo, Keun Seok

    2016-01-01

    Staphylococcal enterotoxins (SEs) are unique bacterial toxins that cause gastrointestinal toxicity as well as superantigenic activity. Since systemic administration of SEs induces superantigenic activity leading to toxic shock syndrome that may mimic enterotoxic activity of SEs such as vomiting and diarrhea, oral administration of SEs in the monkey feeding assay is considered as a standard method to evaluate emetic activity of SEs. This chapter summarizes and discusses practical considerations of the monkey feeding assay used in studies characterizing classical and newly identified SEs.

  10. Coat and enterotoxin-related proteins in Clostridium perfringens spores.

    PubMed

    Ryu, S; Labbe, R G

    1989-11-01

    Coat proteins from mature spores of two enterotoxin-positive (Ent+) and two enterotoxin-negative (Ent-) strains of Clostridium perfringens were solubilized using 50 mM-dithiothreitol and 1% sodium dodecyl sulphate at pH 9.7, and alkylated using 110 mM-iodoacetamide to prevent aggregation. The coat proteins and C. perfringens type A enterotoxin (CPE) were separated by SDS-PAGE and analysed by Western blotting using anti-CPE antibody. As previously reported, CPE aggregated in the presence of SDS, but no aggregation occurred at concentrations below 15 micrograms CPE ml-1. Two CPE-related proteins (34 and 48 kDa) were found in the solubilized spore coat protein of Ent+ strains while only the 48 kDa CPE-related protein was found in the spore coat fraction of Ent- strains. CPE-related proteins comprised 2.7% and 0.8% of the total solubilized coat protein of Ent+ and Ent- strains respectively. CPE-related proteins could be extracted from the spores with 1% SDS alone. They could also be released by disruption of whole spores, indicating that the CPE-related proteins may be in the spore core or trapped between the core and coat layers. The results suggest that CPE is not a major structural component of the coat fraction of C. perfringens spores.

  11. Staphylococcal enterotoxins bind H-2Db molecules on macrophages

    NASA Technical Reports Server (NTRS)

    Beharka, A. A.; Iandolo, J. J.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    We screened a panel of monoclonal antibodies against selected macrophage cell surface molecules for their ability to inhibit enterotoxin binding to major histocompatibility complex class II-negative C2D (H-2b) macrophages. Two monoclonal antibodies, HB36 and TIB126, that are specific for the alpha 2 domain of major histocompatibility complex class I, blocked staphylococcal enterotoxins A and B (SEA and SEB, respectively) binding to C2D macrophages in a specific and concentration-dependent manner. Inhibitory activities were haplotype-specific in that SEA and SEB binding to H-2k or H-2d macrophages was not inhibited by either monoclonal antibody. HB36, but not TIB126, inhibited enterotoxin-induced secretion of cytokines by H-2b macrophages. Lastly, passive protection of D-galactosamine-sensitized C2D mice by injection with HB36 antibody prevented SEB-induced death. Therefore, SEA and SEB binding to the alpha 2 domain of the H-2Db molecule induces biological activity and has physiological consequences.

  12. Staphylococcal enterotoxin B-specific electrochemiluminescence and lateral flow device assays cross-react with staphylococcal enterotoxin D.

    PubMed

    Tallent, Sandra M; Hait, Jennifer; Bennett, Reginald W

    2014-01-01

    Guam school children and faculty members experienced symptoms of vomiting, nausea, abdominal cramps, and diarrhea shortly after eating breakfast prepared by contracted caterers. The first illness was reported within an hour after breakfast, affecting 295 students and two faculty members. Local hospitals treated 130 people, and 61 were admitted for further treatment. Reported symptoms were consistent with staphylococcal food poisoning. Initial food testing using a lateral flow device and electrochemiluminescence method incorrectly implicated staphylococcal enterotoxin B as the causative agent, prompting partial activation of Guam's Emergency Response Center. Traditional ELISAs proved that the food poisoning agent was staphylococcal enterotoxin D. More specific and sensitive assays would have alleviated the issues and confusion that surrounded the reporting and investigation of this outbreak.

  13. Surface plasmon resonance (SPR) detection of Staphylococcal Enterotoxin A in food samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An automated and rapid method for detection of staphylococcal enterotoxins (SE) is needed. A sandwich assay was developed using a surface plasmon resonance (SPR) biosensor for detection of staphylococcal enterotoxin A (SEA) at subpicomolar concentration. Assay conditions were optimized for capturing...

  14. TNF as biomarker for rapid quantification of active Staphylococcus enterotoxin A in food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcus aureus is a major bacterial pathogen which causes clinical infection and food poisoning. This bacterium produces a group of twenty-one enterotoxins (SEs). These enterotoxins have two separate but related biological activities. They cause gastroenteritis and function as superantigens t...

  15. Comparison of enterotoxin production and phenotypic characteristics between emetic and enterotoxic Bacillus cereus.

    PubMed

    Kim, Jung-Beom; Kim, Jai-Moung; Kim, So-Yeong; Kim, Jong-Hyun; Park, Yong-Bae; Choi, Na-Jung; Oh, Deog-Hwan

    2010-07-01

    Bacillus cereus was divided into emetic toxin (cereulide)- and enterotoxin-producing strains, but emetic toxin-producing B. cereus is difficult to detect immunochemically. Screening methods for emetic toxin-producing B. cereus are needed. The objectives of this study were to identify and detect emetic toxin-producing B. cereus among 160 B. cereus strains, and to compare enterotoxin production and phenotypic characteristics between the emetic toxin-producing and enterotoxin-producing strains. Forty emetic toxin-producing B. cereus strains were determined with high-pressure liquid chromatography-mass spectrometry analysis. Among the emetic toxin-producing strains (n = 40), 31 (77.5%) and 3 (7.5%) strains produced nonhemolytic enterotoxin (NHE) and hemolysin BL (HBL) enterotoxins, respectively. In addition, 107 (89.2%) and 100 (83.3%) strains produced NHE and HBL enterotoxins among the enterotoxin-producing strains (n = 120). The number of strains positive for starch hydrolysis, salicin fermentation, and hemolysis among the emetic toxin-producing strains were 3 (7.5%), 3 (7.5%), and 26 (65.0%), respectively, and among enterotoxin-producing strains, these numbers were 101 (84.2%), 100 (83.3%), and 111 (92.5%), respectively. In particular, the three emetic toxin-producing B. cereus strains (JNHE 6, JNHE 36, and KNIH 28) produced the HBL and NHE enterotoxins and were capable of starch hydrolysis and salicin fermentation. The absence of HBL enterotoxin and certain phenotypic properties, such as starch hydrolysis and salicin fermentation, indicates that these properties were not critical characteristics of the emetic toxin-producing B. cereus tested in this study.

  16. Inhibition of small-intestinal sugar and amino acid transport by the enterotoxin of Shigella dysenteriae I.

    PubMed

    Binder, H J; Whiting, D S

    1977-05-01

    The enterotoxin of Shigella dysenteriae I produces fluid and electrolyte secretion in the rabbit ileum. These present studies were designed to evaluate nonelectrolyte transport in rabbit ileal mucosa exposed to Shigella enterotoxin. Both 10 mM galactose and 5 mM L-alanine absorptions were significantly impaired in enterotoxin-exposed ileal mucosa compared with control mucosa. L-Alanine influx was not imparied in two other secretory processes: that induced by cholera enterotoxin and hyperosmolarity. These studies provide evidence that both surgar and amino acid absorptions are diminished in the small intestine by the enterotoxin of S. dysenteriae I.

  17. Inhibition of small-intestinal sugar and amino acid transport by the enterotoxin of Shigella dysenteriae I.

    PubMed Central

    Binder, H J; Whiting, D S

    1977-01-01

    The enterotoxin of Shigella dysenteriae I produces fluid and electrolyte secretion in the rabbit ileum. These present studies were designed to evaluate nonelectrolyte transport in rabbit ileal mucosa exposed to Shigella enterotoxin. Both 10 mM galactose and 5 mM L-alanine absorptions were significantly impaired in enterotoxin-exposed ileal mucosa compared with control mucosa. L-Alanine influx was not imparied in two other secretory processes: that induced by cholera enterotoxin and hyperosmolarity. These studies provide evidence that both surgar and amino acid absorptions are diminished in the small intestine by the enterotoxin of S. dysenteriae I. PMID:324910

  18. Purified Shigella enterotoxin does not alter intestinal motility.

    PubMed Central

    Fernandez, A; Sninsky, C A; O'Brien, A D; Clench, M H; Mathias, J R

    1984-01-01

    A purified Shigella enterotoxin (pST) and a cell-free lysate with pST removed (CFL-pST) from the whole-cell lysate of Shigella dysenteriae 60 R were used to study their effect on the myoelectric activity and mucosal integrity of rabbit ileal segments. We have previously defined two myoelectric patterns: the migrating action potential complex and repetitive bursts of action potentials that occur in response to certain bacteria and their enterotoxins. The in vivo model consisted of isolated ileal segments in male New Zealand White rabbits. The segments were infused with sterile saline (1 ml/h), pST (2.4-micrograms injection), or CFL-pST (1 ml/h). Myoelectric activity in the segments exposed to pST was similar to that with the saline infusion, but CFL-pST induced significant alterations in myoelectric activity in the form of repetitive bursts of action potentials. The mucosa of the segments exposed to pST showed only mild inflammatory changes. In contrast, CFL-pST caused moderate to severe inflammatory changes with enterocyte necrosis. These studies show that pST, a known enterotoxin, did not alter myoelectric activity and had no significant effect on the integrity of ileal mucosa, as determined by light microscopy. CFL-pST caused both inflammation and tissue necrosis with significant alterations in motor activity. These studies suggest that S. dysenteriae 60 R produces a substance or substances other than pST that cause florid in vivo cytotoxicity and alter myoelectric activity. Images PMID:6363286

  19. A Tripartite Fusion, FaeG-FedF-LT192A2:B, of Enterotoxigenic Escherichia coli (ETEC) Elicits Antibodies That Neutralize Cholera Toxin, Inhibit Adherence of K88 (F4) and F18 Fimbriae, and Protect Pigs against K88ac/Heat-Labile Toxin Infection ▿

    PubMed Central

    Ruan, Xiaosai; Liu, Mei; Casey, Thomas A.; Zhang, Weiping

    2011-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 (F4) or F18 fimbriae and heat-labile (LT) and/or heat-stable (ST) toxins are the major cause of diarrhea in young pigs. Effective vaccines inducing antiadhesin (anti-K88 and anti-F18) and antitoxin (anti-LT and anti-ST) immunity would provide broad protection to young pigs against ETEC. In this study, we genetically fused nucleotides coding for peptides from K88ac major subunit FaeG, F18 minor subunit FedF, and LT toxoid (LT192) A2 and B subunits for a tripartite adhesin-adhesin-toxoid fusion (FaeG-FedF-LT192A2:B). This fusion was used for immunizations in mice and pigs to assess the induction of antiadhesin and antitoxin antibodies. In addition, protection by the elicited antiadhesin and antitoxin antibodies against a porcine ETEC strain was evaluated in a gnotobiotic piglet challenge model. The data showed that this FaeG-FedF-LT192A2:B fusion elicited anti-K88, anti-F18, and anti-LT antibodies in immunized mice and pigs. In addition, the anti-porcine antibodies elicited neutralized cholera toxin and inhibited adherence against both K88 and F18 fimbriae. Moreover, immunized piglets were protected when challenged with ETEC strain 30302 (K88ac/LT/STb) and did not develop clinical disease. In contrast, all control nonvaccinated piglets developed severe diarrhea and dehydration after being challenged with the same ETEC strain. This study clearly demonstrated that this FaeG-FedF-LT192A2:B fusion antigen elicited antibodies that neutralized LT toxin and inhibited the adherence of K88 and F18 fimbrial E. coli strains and that this fusion could serve as an antigen for vaccines against porcine ETEC diarrhea. In addition, the adhesin-toxoid fusion approach used in this study may provide important information for developing effective vaccines against human ETEC diarrhea. PMID:21813665

  20. Novel platform for the detection of Staphylococcus aureus enterotoxin B in foods.

    PubMed

    Tallent, Sandra M; Degrasse, Jeffrey A; Wang, Ningyan; Mattis, Daiva M; Kranz, David M

    2013-03-01

    Staphylococcal contamination of food products and staphylococcal food-borne illnesses continue to be a problem worldwide. Screening of food for the presence of Staphylococcus aureus and/or enterotoxins using traditional methods is laborious. Reliable and rapid multiplex detection methods from a single food extract or culture supernatant would simplify testing. A fluorescence-based cytometric bead array was developed for the detection of staphylococcal enterotoxin B (SEB), using magnetic microspheres coupled with either an engineered, enterotoxin-specific Vβ domain of the T-cell receptor (Vβ-TCR) or polyclonal antibodies. The binding affinity of the Vβ-TCR for SEB has been shown to be in the picomolar range, comparable to the best monoclonal antibodies. The coupled beads were validated with purified enterotoxins and tested in a variety of food matrices spiked with enterotoxins. The Vβ-TCR or antibody was shown to specifically bind SEB in four different food matrices, including milk, mashed potatoes, vanilla pudding, and cooked chicken. The use of traditional polyclonal antibodies and Vβ-TCR provides a redundant system that ensures accurate identification of the enterotoxin, and the use of labeled microspheres permits simultaneous testing of multiple enterotoxins from a single sample.

  1. Clostridium perfringens Enterotoxin: Action, Genetics, and Translational Applications.

    PubMed

    Freedman, John C; Shrestha, Archana; McClane, Bruce A

    2016-03-16

    Clostridium perfringens enterotoxin (CPE) is responsible for causing the gastrointestinal symptoms of several C. perfringens food- and nonfood-borne human gastrointestinal diseases. The enterotoxin gene (cpe) is located on either the chromosome (for most C. perfringens type A food poisoning strains) or large conjugative plasmids (for the remaining type A food poisoning and most, if not all, other CPE-producing strains). In all CPE-positive strains, the cpe gene is strongly associated with insertion sequences that may help to assist its mobilization and spread. During disease, CPE is produced when C. perfringens sporulates in the intestines, a process involving several sporulation-specific alternative sigma factors. The action of CPE starts with its binding to claudin receptors to form a small complex; those small complexes then oligomerize to create a hexameric prepore on the membrane surface. Beta hairpin loops from the CPE molecules in the prepore assemble into a beta barrel that inserts into the membrane to form an active pore that enhances calcium influx, causing cell death. This cell death results in intestinal damage that causes fluid and electrolyte loss. CPE is now being explored for translational applications including cancer therapy/diagnosis, drug delivery, and vaccination.

  2. Effect of Eleutherine americana Merr. extract on enzymatic activity and enterotoxin production of Staphylococcus aureus in broth and cooked pork.

    PubMed

    Ifesan, Beatrice O T; Voravuthikunchai, Supayang P

    2009-01-01

    Crude ethanolic extract from the bulb of Eleutherine americana was investigated for its inhibitory activities against lipase and protease enzymes and enterotoxin production by Staphylococcus aureus. Eleven isolates that demonstrated high enzyme activity with three reference strains were selected to study the effect of extract on enzyme production. Exposure of the isolates to subminimal inhibitory concentrations, (1/2) minimum inhibitory concentration (MIC) (125 microg/mL), and (1/4)MIC (62.5 microg/mL) of the crude extract resulted in both partial and total inhibition of lipase and protease enzymes. About 15% of the 106 isolates were positive for enterotoxin production with staphylococcal enterotoxin A (11.3%), enterotoxin B (3.7%), and enterotoxin C (10.3%), and no enterotoxin D was produced. The production of staphylococcal enterotoxins A-D in the presence or absence of the crude extract was carried out. In the broth system, the extract reduced enterotoxin production at subminimal inhibitory concentrations compared with the control. At MIC, total enterotoxin inhibition was observed for enterotoxin C production, whereas synthesis of enterotoxins A, B, and D was totally eliminated at 2MIC. The food system study revealed that the extract could delay production of enterotoxins A, B, and C compared with the control. The extract at 2 mg/mL delayed production of toxins A and C for 8 and 4 h, while toxin B was not detected in the pork at 48 h. The ability of E. americana extract to inhibit lipase and protease enzymes and to delay enterotoxin production in food could present it as a novel food additive to combat the growth of S. aureus in food.

  3. Detection of classical enterotoxins and identification of enterotoxin genes in Staphylococcus aureus from milk and dairy products.

    PubMed

    Morandi, S; Brasca, M; Lodi, R; Cremonesi, P; Castiglioni, B

    2007-09-20

    Milk and dairy products are frequently contaminated with enterotoxigenic Staphylococcus aureus, which is often involved in staphylococcal food poisoning. The distribution of genes encoding staphylococcal enterotoxins (SE) in S. aureus isolated from bovine, goat, sheep and buffalo milk and dairy products was verified by the presence of the corresponding SE production. A total of 112 strains of S. aureus were tested for SE production by immuno-enzymatic (SEA-SEE) and reversed passive latex agglutination (SEA-SED) methods, while multiplex-PCR was applied for SE genes (sea, sec, sed, seg, seh, sei, sej and sel). Of the total strains studied, 67% were detected to have some SE genes (se), but only 52% produced a detectable amount of the classic antigenic SE types. The bovine isolates frequently had enterotoxin SEA, SED and sej, while SEC and sel predominated in the goat and sheep strains. The results demonstrated (i) marked enterotoxigenic S. aureus strain variations, in accordance with strain origin and (ii) the two methods resulted in different information but concurred on the risk of foodstuff infection by S. aureus.

  4. The Distribution of 18 Enterotoxin and Enterotoxin-Like Genes in Staphylococcus aureus Strains from Different Sources in East China.

    PubMed

    Cheng, Jinghua; Wang, Yan; Cao, Yongzhong; Yan, Wenguang; Niu, Xiaosai; Zhou, Liping; Chen, Jianhao; Sun, Ying; Li, Chenxi; Zhang, Xiaorong; Wu, Yantao

    2016-04-01

    The distribution of 18 staphylococcal enterotoxin (SE) or SE-like (SEl) genes in Staphylococcus aureus strains from different sources in east China was investigated. Among all 496 S. aureus strains, 291 strains carried one or more SE genes. The more frequently occurred genes were sea, seb, seg, selk, sell, selm, selo, and seq; the less frequent occurred genes were sec, selj, and ser. The classic SE genes and the enterotoxin gene cluster (egc) (seg, sei, selm, seln, selo, and/or selu) accounted for 25.67% and 61.68% of all detected genes, respectively. There were three gene clusters (egc, sea-sek-seq, and sed-sej-ser), of which the egc cluster was the important one that could generate novel complexes, and the sea-sek-seq cluster was a close relative to the hospital-acquired methicillin-resistant S. aureus. The SE gene distributions were different among strains of different sources and formed diverse toxin gene profiles. The human- and foodborne-origin strains harbored classic and novel SE and SEl genes, whereas animal-origin strains harbored egc and other novel SE and SEl genes mainly. The foodborne- and human-origin strains were the main dangerous factors of classic staphylococcal foodborne poisoning, whereas the strains (especially from animals) that carried egc and other novel genes mainly should be new potential dangerous factors for food safety.

  5. Colony immunoblot assay for the detection of hemolysin BL enterotoxin producing Bacillus cereus.

    PubMed

    Moravek, Maximilian; Wegscheider, Monika; Schulz, Anja; Dietrich, Richard; Bürk, Christine; Märtlbauer, Erwin

    2004-09-01

    Bacillus cereus strains involved in food poisoning cases of the diarrheal type may produce two different enterotoxin complexes. To facilitate the identification of hemolysin BL-enterotoxin complex (HBL) and/or the nonhemolytic enterotoxin (NHE) producing colonies a colony immunoblot procedure was developed, which allows a fast and easy identification of the respective colonies from blood agar plates. The enterotoxins were transferred from the blood agar medium to a nitrocellulose membrane and the immobilized toxins were probed with monoclonal antibodies. The antibodies 2A3 and 1A8 allowed the specific detection of the B component of HBL and the nheA component of NHE. The assay enabled the reliable identification of HBL expressing colonies and differentiation from NHE producing but HBL negative colonies.

  6. Characterization of a parasporal inclusion body from sporulating, enterotoxin-positive Clostridium perfringens type A.

    PubMed Central

    Löffler, A; Labbé, R

    1986-01-01

    Inclusion bodies (IB) synthesized during sporulation and enterotoxin formation by Clostridium perfringens NCTC 8239 and 8798 were isolated and characterized. IB were isolated by disruption of sporangia by sonication in the presence of tetrasodium EDTA and phenylmethylsulfonyl fluoride. Fractionation was carried out in a linear gradient of sodium bromide, sucrose, or diatrizoate sodium. Denaturing and reducing agents were necessary to solubilize the IB. An alkylating agent was required to prevent reaggregation of the subunits. Molecular weight, compositional, and serological analyses and peptide mapping revealed strong similarities between the IB subunits and the enterotoxin synthesized during sporulation by C. perfringens. IB appear to represent the structural component where overproduced enterotoxin accumulates intracellularly. Enterotoxin-like subunits in the IB appeared to be held together by noncovalent and disulfide bonds, which were generally resistant to the action of intracellular proteases of C. perfringens, trypsin, or trypsin plus bile salts. Images PMID:2867991

  7. Screening, detection, and serotyping methods for toxin genes and enterotoxins in Staphylococcus strains.

    PubMed

    Hait, Jennifer M; Tallent, Sandra M; Bennett, Reginald W

    2014-01-01

    Staphylococcus aureus continues to play a significant role in foodborne outbreak investigations, with numerous individuals sickened each year after ingesting assorted foods contaminated with staphylococcal enterotoxins. The purpose of this study was to evaluate the use of several methods for the screening, detection, and enterotoxin serotyping of staphylococcal bacterial strains for classical staphylococcal enterotoxins (SEs; SEA, SEB, SEC, SED, and SEE) and the newly described SE and SE-like enterotoxin genes (seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, ser, ses, set, and seu). Inclusivity and exclusivity panels of staphylococcal strains were tested using a multiplex PCR method in addition to three polyvalent commercially prepared ELISA systems for the detection of SEA-SEE and one monovalent assay for the identification of classical SE serotypes. The results indicate an overall agreement between serological detection methods with a few exceptions, and molecular characterization identified an abundance of SE and SE-like enterotoxin genes including several potentially enterotoxigenic isolates that would have otherwise been missed by ELISA-based methods. These findings demonstrate the significance of PCR for future screening purposes and the use of ELISA systems for the detection and enterotoxin serotyping of staphylococcal bacterial strains.

  8. Tolerance to staphylococcal enterotoxin B initiated Th1 cell differentiation in mice infected with Candida albicans.

    PubMed Central

    Romani, L; Puccetti, P; Mencacci, A; Spaccapelo, R; Cenci, E; Tonnetti, L; Bistoni, F

    1994-01-01

    Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that specifically activates T cells bearing V beta 8 T-cell receptor domains, which eventually leads to a long-lasting state of clonal anergy accompanied by selective cell death in the targeted CD4+ subset. Because the superantigen is known to promote Th1 cell differentiation in vitro, we have investigated the effect of SEB treatment on the course of Th2-associated progressive disease in mice infected systemically with Candida albicans. On the basis of the kinetics of SEB-induced changes in CD4+ cells and production in sera of interleukin 4 (IL-4), IL-10, and gamma interferon, we obtained evidence that V beta 8+ cell anergy concomitant with infection abolished the early IL-4/IL-10 response of the host to the yeast, ultimately leading to a state of resistance characterized by gamma interferon secretion in vitro by antigen-specific CD4+ cells. In contrast, SEB administered near the time of challenge resulted in accelerated mortality. Significant resistance to infection was also afforded by exposure of mice to a retrovirally encoded endogenous superantigen. These data suggest that CD4+ V beta 8+ T cells play an important role in vivo in the initiation of a Th2 response to C. albicans and that suppression of their activity may alter the qualitative development of the T-cell response and the outcome of infection. PMID:7914883

  9. Detecting staphylococcal enterotoxin B using an automated fiber optic biosensor.

    PubMed

    King, K D; Anderson, G P; Bullock, K E; Regina, M J; Saaski, E W; Ligler, F S

    1999-02-01

    The Man-portable Analyte Identification System (MANTIS), the first fully automated, self-contained, portable fiber optic biosensor, was utilized for the detection of Staphylococcal Enterotoxin B (SEB), a bacterial toxin produced by Staphylococcus aureus that commonly causes food poisoning. Because of its remarkable toxicity and stability, SEB is considered a prime threat as a biological weapon of mass destruction. The assay for SEB was used to evaluate the MANTIS' ability to function in the presence of various environmental interferents. The sensor could reliably detect SEB spiked into liquid samples containing a variety of smoke particles. However, substantial interference occurred when SEB was mixed into matrices capable of adsorbing SEB, such as 1% solutions of clay, topsoil, or pollen. Of equal importance, none of the interferents produced false positives in the MANTIS. The MANTIS demonstrated the capability to perform simultaneous immunoassays rapidly in the field with little or no user intervention.

  10. Effects of staphylococcal enterotoxin A on the rat gastrointestinal tract.

    PubMed Central

    Beery, J T; Taylor, S L; Schlunz, L R; Freed, R C; Bergdoll, M S

    1984-01-01

    Staphylococcal enterotoxin A (SEA) was administered orally (15 micrograms) to two groups of rats. A marked immune reaction was evoked in the stomach and proximal small intestine of the first group. The second group of rats was used to study the absorptive fate and sites of action of orally administered SEA, utilizing immunoperoxidase staining. After oral dosing of the second group of rats. SEA-related immunoperoxidase staining was confined to: (i) neutrophils and macrophages, principally in the duodenum, and (ii) glomerular neutrophils and cells of the proximal convoluted tubules. Peroxidase staining of the kidney was noted within 15 min of exposure, indicating that SEA or some major postabsorption antigenic product can promptly pass through an intact gastrointestinal mucous membrane and become renally localized. Intestinal and renal detoxification and removal was indicated by an absence of detectable antigen in rats 180 min postexposure. Neuronal binding of SEA in the gastrointestinal tract was not demonstrable. Images PMID:6370862

  11. Mutations defining functional regions of the superantigen staphylococcal enterotoxin B

    PubMed Central

    1992-01-01

    Staphylococcal enterotoxin B (SEB) is both a superantigen and toxin. As a superantigen, SEB can bind to major histocompatibility complex (MHC) class II molecules to form a ligand for alpha/beta T cell receptors bearing particular V beta elements. As a toxin, SEB causes rapid weight loss in mice sometimes leading to death. We show here that both of these functions map to the NH2-terminal portion of the toxin. Three regions were identified: one important in MHC class II binding, one in T cell recognition, and one in both functions. These results support the conclusion that the toxicity of SEB is related to massive T cell stimulation and release of cytokine mediators and show that the residues interacting with MHC and the T cell receptor are intertwined. PMID:1370682

  12. Modeling the effect of water activity, pH, and temperature on the probability of enterotoxin production by Staphylococcus aureus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcus aureus is a foodborne pathogen widespread in the environment and found in various food products. This pathogen can produce enterotoxins that cause illnesses in humans. The objectives of this study were to develop a probability model of S. aureus enterotoxin production as affected by w...

  13. CD154 as a potential early molecular biomarker for rapid quantification analysis of active Staphylococcus enterotoxin A

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcus aureus is a major bacterial pathogen producing a group of twenty-one enterotoxins (SEs). These enterotoxins have two separate but related biological activities, They cause gastroenteritis, and they function as a superantigens that activate large numbers of T cells. In the current stud...

  14. Distribution of classical enterotoxin genes in staphylococci from milk of cows with- and without mastitis and the cowshed environment.

    PubMed

    Piechota, M; Kot, B; Zdunek, E; Mitrus, J; Wicha, J; Wolska, M K; Sachanowicz, K

    2014-01-01

    The aim of this study was to analyze by PCR 185 isolates of Staphylococcus from milk of cows with- and without mastitis and from the cowsheds environment for their potential ability to produce five classical staphylococcal enterotoxins. Among S. aureus isolates 8 (32%) carried enterotoxin genes and only 2 of them had more than one gene. The enterotoxin genes were detected in 22 (13.7%) coagulase-negative staphylococci (CNS) isolates, among them in 9 (11.4%) isolates of S. xylosus, 5 (16.7%) S. sciuri, 3 (10.3%) S. epidermidis and in 5 (22.7%) Staphylococcus spp. In some CNS 2 or 3 genes were detected simultaneously. Among the investigated enterotoxin genes, sec was the most prevalent (70%). The genes encoding enterotoxin B and D were detected in 5 (16.7%) and 6 (20%) isolates, respectively. The lowest number of isolates had sea and see genes. The genes encoding enterotoxins were often identified in staphylococci from milk of cows with mastitis (73.4% of detected genes), while only 6 (20%) isolates from milk of cows without mastitis and 2 (6.6%) isolates from cowshed environment were positive for enterotoxin genes. The results showed that CNS from bovine milk, like S. aureus, carried enterotoxin genes and may pose a risk for public health.

  15. Production of Staphylococcal Enterotoxins A, B, and C in Colloidal Dispersions1

    PubMed Central

    Woodburn, Margy; Morita, Toshiko N.; Venn, Sharon Zipperer

    1973-01-01

    Larger amounts of enterotoxin were produced when Staphylococcus aureus S-6 was grown under still (nonshaken) conditions in a medium that was a paste or gel than were produced in a liquid dispersion with the same colloidal ingredient or in control basal broth (4% NZ Amine-NAK containing 50 μg of thiamine per 100 ml and 1 mg of niacin per 100 ml). Four colloidal ingredients were used which had been previously demonstrated to not support enterotoxin production in buffer. The effect of the type of dispersion occurred earlier than that of the colloidal ingredient, but interactions were found. This effect was not observed when the cells were grown with aeration (shaken). Four other strains of S. aureus followed a similar pattern for enterotoxins A, B, and C, although liquid and paste with cornstarch and carrageenan were the only media compared to the control broth. Enterotoxins A and B were produced earlier by S. aureus S-6, and much greater quantities of enterotoxins were produced for all strains when incubated shaken. PMID:4197641

  16. Reduction of reactivity of Escherichia coli enterotoxins by intestinal mucosal components.

    PubMed

    Cole, H D; Staley, T E; Whipp, S C

    1977-04-01

    Incubation studies involving rabbit and piglet small intestinal mucosal components and Escherichia coli and Vibrio cholerae enterotoxins were conducted at 37 and 4 degrees C. Mucosal homogenate cytosol from rabbits did not significantly alter the reactivities of either cholera enterotoxin (CT) or E. coli labile enterotoxin (LT). However, mucosal homogenate cytosol from piglets was capable of neutralizing LT, though it had no effect on E. coli stable enterotoxin. LT became bound to piglet and rabbit microvillous membranes at 4 degrees C in the presence of a protective protein. In rabbits, the binding of LT was not dependent upon an intact glycocalyx or free unbound CT-receptors, although some binding was apparently associated with the glycocalyx and CT-receptors. These results indicated the presence of two different LT-receptors in microvillous membranes one being associated with the membrane proper and the other with the glycocalyx. Stable enterotoxin did not bind to in vitro preparations of piglet mucosal components, which is evidence for a different mechanism for inducing intestinal secretion.

  17. Reduction of reactivity of Escherichia coli enterotoxins by intestinal mucosal components.

    PubMed Central

    Cole, H D; Staley, T E; Whipp, S C

    1977-01-01

    Incubation studies involving rabbit and piglet small intestinal mucosal components and Escherichia coli and Vibrio cholerae enterotoxins were conducted at 37 and 4 degrees C. Mucosal homogenate cytosol from rabbits did not significantly alter the reactivities of either cholera enterotoxin (CT) or E. coli labile enterotoxin (LT). However, mucosal homogenate cytosol from piglets was capable of neutralizing LT, though it had no effect on E. coli stable enterotoxin. LT became bound to piglet and rabbit microvillous membranes at 4 degrees C in the presence of a protective protein. In rabbits, the binding of LT was not dependent upon an intact glycocalyx or free unbound CT-receptors, although some binding was apparently associated with the glycocalyx and CT-receptors. These results indicated the presence of two different LT-receptors in microvillous membranes one being associated with the membrane proper and the other with the glycocalyx. Stable enterotoxin did not bind to in vitro preparations of piglet mucosal components, which is evidence for a different mechanism for inducing intestinal secretion. Images PMID:326677

  18. Expression and production of staphylococcal enterotoxin C is substantially reduced in milk.

    PubMed

    Valihrach, Lukas; Alibayov, Babek; Zdenkova, Kamila; Demnerova, Katerina

    2014-12-01

    Staphylococcal food poisoning is a global problem. The gene encoding enterotoxin C (sec) has been reported several times as the most frequent enterotoxin gene identified in food poisoning cases caused by contaminated milk. In this study, the expression of sec was examined during the growth of Staphylococcus aureus in milk compared to routinely used laboratory media. Additionally, expression of several regulatory genes (sarA, saeS, codY, srrA, rot, hld, agrA, sigB) and other five enterotoxin genes (sea, seg, seh, sek, sel) were observed. It has been well established for that S. aureus is able to grow in milk and we found significantly reduced expression of sec in milk compared to the laboratory medium (P < 0.05). Here, we report the first study providing a comprehensive view on the expression of enterotoxin genes and its regulation in milk. The milk environment dramatically changed the expression profiles of several enterotoxin genes although staphylococcal growth was not affected at all. The mechanism of the reduction may be explained by downregulation of the agr system, although other factors are expected to be involved. The constituent of milk causing the inhibitory effect remains unidentified.

  19. Monoclonal antibody-based sandwich ELISA for the detection of staphylococcal enterotoxin A.

    PubMed

    Kuang, Hua; Wang, Wenbing; Xu, Liguang; Ma, Wei; Liu, Liqiang; Wang, Libing; Xu, Chuanlai

    2013-04-19

    A sensitive and specific monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) was established and validated for the detection of staphylococcal enterotoxin A (SEA). After routine fusion and selection, 10 monoclonal antibodies showed high affinity for SEA. An optimal pair for sandwich ELISA was selected by pairwise interaction analysis. After optimization, the limit of detection (LOD) and linear dynamic range of the method were established, and were found to be 0.0282 ng/mL and 0.06-2 ng/mL, respectively. The recovery in pure milk ranged from 82.67% to 111.95% and the intra- and inter-assay coefficients of variation ranged from 3.16% to 6.05% and from 5.16% to 10.79%, respectively. Cross-reactivity with staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SEC), staphylococcal enterotoxin D (SED), and staphylococcal enterotoxin E (SEE) in this method were insignificant. These results indicate that the sandwich ELISA method developed in our study is effective for routine identification of SEA in food samples.

  20. Two common structural motifs for TCR recognition by staphylococcal enterotoxins

    PubMed Central

    Rödström, Karin E. J.; Regenthal, Paulina; Bahl, Christopher; Ford, Alex; Baker, David; Lindkvist-Petersson, Karin

    2016-01-01

    Superantigens are toxins produced by Staphylococcus aureus, called staphylococcal enterotoxins (abbreviated SEA to SEU). They can cross-link the T cell receptor (TCR) and major histocompatibility complex class II, triggering a massive T cell activation and hence disease. Due to high stability and toxicity, superantigens are potential agents of bioterrorism. Hence, antagonists may not only be useful in the treatment of disease but also serve as countermeasures to biological warfare. Of particular interest are inhibitors against SEA and SEB. SEA is the main cause of food poisoning, while SEB is a common toxin manufactured as a biological weapon. Here, we present the crystal structures of SEA in complex with TCR and SEE in complex with the same TCR, complemented with computational alanine-scanning mutagenesis of SEA, SEB, SEC3, SEE, and SEH. We have identified two common areas that contribute to the general TCR binding for these superantigens. This paves the way for design of single antagonists directed towards multiple toxins. PMID:27180909

  1. The interaction of Clostridium perfringens enterotoxin with receptor claudins.

    PubMed

    Shrestha, Archana; Uzal, Francisco A; McClane, Bruce A

    2016-10-01

    Clostridium perfringens enterotoxin (CPE) has significant medical importance due to its involvement in several common human gastrointestinal diseases. This 35 kDa single polypeptide toxin consists of two domains: a C-terminal domain involved in receptor binding and an N-terminal domain involved in oligomerization, membrane insertion and pore formation. The action of CPE starts with its binding to receptors, which include certain members of the claudin tight junction protein family; bound CPE then forms a series of complexes, one of which is a pore that causes the calcium influx responsible for host cell death. Recent studies have revealed that CPE binding to claudin receptors involves interactions between the C-terminal CPE domain and both the 1st and 2nd extracellular loops (ECL-1 and ECL-2) of claudin receptors. Of particular importance for this binding is the docking of ECL-2 into a pocket present in the C-terminal domain of the toxin. This increased understanding of CPE interactions with claudin receptors is now fostering the development of receptor decoy therapeutics for CPE-mediated gastrointestinal disease, reagents for cancer therapy/diagnoses and enhancers of drug delivery.

  2. Structural basis for abrogated binding between staphylococcal enterotoxin A superantigen vaccine and MHC-IIα

    PubMed Central

    Krupka, Heike I.; Segelke, Brent W.; Ulrich, Robert G.; Ringhofer, Sabine; Knapp, Mark; Rupp, Bernhard

    2002-01-01

    Staphylococcal enterotoxins (SEs) are superantigenic protein toxins responsible for a number of life-threatening diseases. The X-ray structure of a staphylococcal enterotoxin A (SEA) triple-mutant (L48R, D70R, and Y92A) vaccine reveals a cascade of structural rearrangements located in three loop regions essential for binding the α subunit of major histocompatibility complex class II (MHC-II) molecules. A comparison of hypothetical model complexes between SEA and the SEA triple mutant with MHC-II HLA-DR1 clearly shows disruption of key ionic and hydrophobic interactions necessary for forming the complex. Extensive dislocation of the disulfide loop in particular interferes with MHC-IIα binding. The triple-mutant structure provides new insights into the loss of superantigenicity and toxicity of an engineered superantigen and provides a basis for further design of enterotoxin vaccines. PMID:11847286

  3. Detection of enterotoxin genes of Staphylococcus SP isolated from nasal cavities and hands of food handlers

    PubMed Central

    Rall, V.L.M; Sforcin, J.M.; Augustini, V.C.M.; Watanabe, M.T.; Fernandes Jr., A.; Rall, R.; Silva, M.G.; Araújo Jr., J.P.

    2010-01-01

    Food handlers, an important factor in food quality, may contain bacteria that are able to cause foodborne disease. The present study aimed to research coagulase-negative (CNS) and -positive staphylococci (CPS) in 82 food handlers, analyzing nasal and hand swabs, with identification of 62 CNS (75.6%) and 20 CPS strains (24.4%). Staphylococcal enterotoxins genes were investigated by PCR. In 20 CPS strains, 19 were positive for one or more genes. The percentage of CNS presenting genes for enterotoxins was high (46.8%). Despite of the staphylococcal species, the most common gene was sea (35.4%), followed by seh and sej (29.2%). The detection of new staphylococcal enterotoxins (SEs) genes showed a higher pathogenic potential in this genus. The presence of these gene points out the importance of CNS not only as contaminant bacteria but also as a pathogen. PMID:24031464

  4. Simple assay for staphylococcal enterotoxins A, B, and C: modification of enzyme-linked immunosorbent assay.

    PubMed Central

    Stiffler-Rosenberg, G; Fey, H

    1978-01-01

    The enzyme-linked immunosorbent assay (ELISA) introduced for the detection of staphylococcal enterotoxins by Saunders et al., Simon and Terplan, and ourselves has proved to be a simple, reliable, and sensitive test. A new modification is described that uses polystyrene balls (diameter, 6 mm) coated individually with antibody against one of the toxins A, B, or C. In a single tube, 20 ml of the food extract was incubated with the three balls differently stained, which were then each tested for the uptake of enterotoxin by a competitive ELISA. A concentration of 0.1 ng or less of enterotoxin per ml can be measured, making tedious concentration procedures of the extracts superfluous. Culture supernatants and extracts from foods artificially or naturally contaminated with toxin were successfully examined. Cross-reactions did not occur, and nonspecific interfering substances did not create serious problems. PMID:365877

  5. Receptors and cGMP signalling mechanism for E. coli enterotoxin in opossum kidney

    SciTech Connect

    Forte, L.R.; Krause, W.J.; Freeman, R.H. Harry S. Truman Memorial Veterans Medical Center, Columbia, MO )

    1988-11-01

    Receptors for the heat-stable enterotoxin produced by Escherichia coli were found in the kidney and intestine of the North American opossum and in cultured renal cell lines. The enterotoxin markedly increased guanosine 3{prime},5{prime}-cyclic monophosphate (cGMP) production in slices of kidney cortex and medulla, in suspensions of intestinal mucosa, and in the opossum kidney (OK) and rat kangaroo kidney (PtK-2) cell lines. In contrast, atrial natriuretic factor elicited much smaller increases in cGMP levels of kidney, intestine, or cultured kidney cell lines. The enterotoxin receptors in OK cells had a molecular mass of approximately 120 kDa when measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of receptors crosslinked with {sup 125}I-enterotoxin. The occurrence of receptors for the E. coli peptide in OK implies that these receptors may be involved in the regulation of renal tubular function in the opossum. E. coli enterotoxin caused a much larger increase in urine cGMP excretion than did atrial natriuretic factor when these peptides were injected intravenously into opossums. However, atrial natriuretic factor elicited a marked diuresis, natriuresis, and increased urinary excretion of calcium, phosphate, potassium, and magnesium. In contrast, the enterotoxin did not acutely influence OK fluid and electrolyte excretion. Thus the substantial increase in cGMP synthesis produced by the bacterial peptide in OK cortex and medulla in vitro and the increased renal excretion of cGMP in vivo were not associated with changes in electrolyte or water excretion. Whether cGMP represents a second messenger molecule in the kidney is an interesting question that was raised but not answered in this series of experiments.

  6. Enterotoxin-Encoding Genes in Staphylococcus spp. from Food Handlers in a University Restaurant.

    PubMed

    da Silva, Sabina Dos Santos Paulino; Cidral, Thiago André; Soares, Maria José dos Santos; de Melo, Maria Celeste Nunes

    2015-11-01

    Food handlers carrying enterotoxin-producing Staphylococcus are a potential source of food poisoning. The aim of this study was to analyze genes encoding enterotoxins in coagulase-positive Staphylococcus (CoPS) and coagulase-negative Staphylococcus (CoNS) isolated from the anterior nostrils and hands of food handlers at a university restaurant in the city of Natal, Northeast Brazil. Thirty food handlers were screened for the study. The isolates were subjected to Gram staining, a bacitracin sensitivity test, mannitol fermentation, and catalase and coagulase tests. CoNS and CoPS strains were subsequently identified by a Vitek 2 System (BioMerieux, France) and various biochemical tests. Polymerase chain reaction was used to detect genes for enterotoxins A, B, C, D, E, G, H, and I (sea, seb, sec, sed, see, seg, seh, and sei) and a disc-diffusion method was used to determine susceptibility to several classes of antimicrobials. All food handlers presented staphylococci on their hands and/or noses. The study found 58 Staphylococcus spp., of which 20.7% were CoPS and 79.3% were CoNS. S. epidermidis was the most prevalent species. Twenty-nine staphylococci (50%) were positive for one or more enterotoxin genes, and the most prevalent genes were seg and sei, each with a frequency of 29.3%. Indeed, CoNS encoded a high percentage of enterotoxin genes (43.5%). However, S. aureus encoded even more enterotoxin genes (75%). Most isolates showed sensitivity to the antibiotics used for testing, except for penicillin (only 35% sensitive). The results from this study reinforce that coagulase-negative as well as coagulase-positive staphylococci isolated from food handlers are capable of genotypic enterotoxigenicity.

  7. Enterotoxin-encoding genes in Staphylococcus spp. from bulk goat milk.

    PubMed

    Lyra, Daniele G; Sousa, Francisca G C; Borges, Maria F; Givisiez, Patrícia E N; Queiroga, Rita C R E; Souza, Evandro L; Gebreyes, Wondwossen A; Oliveira, Celso J B

    2013-02-01

    Although Staphylococcus aureus has been implicated as the main Staphylococcus species causing human food poisoning, recent studies have shown that coagulase-negative Staphylococcus could also harbor enterotoxin-encoding genes. Such organisms are often present in goat milk and are the most important mastitis-causing agents. Therefore, this study aimed to investigate the occurrence of enterotoxin-encoding genes among coagulase-positive (CoPS) and coagulase-negative (CoNS) staphylococci isolated from raw goat milk produced in the semi-arid region of Paraiba, the most important region for goat milk production in Brazil. Enterotoxin-encoding genes were screened in 74 staphylococci isolates (30 CoPS and 44 CoNS) by polymerase chain reaction targeting the genes sea, seb, sec, sed, see, seg, seh, and sei. Enterotoxin-encoding genes were found in nine (12.2%) isolates, and four different genes (sea, sec, seg, and sei) were identified amongst the isolates. The most frequent genes were seg and sei, which were often found simultaneously in 44.5% of the isolates. The gene sec was the most frequent among the classical genes, and sea was found only in one isolate. All CoPS isolates (n=7) harboring enterotoxigenic genes were identified as S. aureus. The two coagulase-negative isolates were S. haemolyticus and S. hominis subsp. hominis and they harbored sei and sec genes, respectively. A higher frequency of enterotoxin-encoding genes was observed amongst CoPS (23.3%) than CoNS (4.5%) isolates (p<0.05), reinforcing the importance of S. aureus as a potential foodborne agent. However, the potential risk posed by CoNS in goat milk should not be ignored because it has a higher occurrence in goat milk and enterotoxin-encoding genes were detected in some isolates.

  8. Mechanisms mediating enhanced neutralization efficacy of Staphylococcal enterotoxin B by combinations of monoclonal antibodies

    DOE PAGES

    Dutta, Kaushik; Varshney, Avanish K.; Franklin, Matthew C.; ...

    2015-01-08

    Staphylococcal enterotoxin B (SEB) is a superantigen that cross-links the major histocompatibility complex class II and specific V-β chains of the T-cell receptor, thus forming a ternary complex. Developing neutralizing mAb to disrupt the ternary complex and abrogate the resulting toxicity is a major therapeutic challenge because SEB is effective at very low concentrations. We show that combining two SEB-specific mAbs enhances their efficacy, even though one of the two mAbs by itself has no effect on neutralization. Crystallography was employed for fine-mapping conformational epitopes in binary and ternary complexes between SEB and Fab fragments. NMR spectroscopy was used tomore » validate and identify subtle allosteric changes induced by mAbs binding to SEB. The mapping of epitopes established that a combination of different mAbs can enhance efficacy of mAb-mediated protection from SEB induced lethal shock by two different mechanisms: one mAb mixture promoted clearance of the toxin both in vitro and in vivo by FcR-mediated cross-linking and clearance, whereas the other mAb mixture induced subtle allosteric conformational changes in SEB that perturbed formation of the SEB·T-cell receptor·major histocompatibility complex class II trimer. Lastly structural information accurately predicted mAb binding to other superantigens that share conformational epitopes with SEB. Fine mapping of conformational epitopes is a powerful tool to establish the mechanism and optimize the action of synergistic mAb combinations.« less

  9. Mechanisms mediating enhanced neutralization efficacy of Staphylococcal enterotoxin B by combinations of monoclonal antibodies

    SciTech Connect

    Dutta, Kaushik; Varshney, Avanish K.; Franklin, Matthew C.; Goger, Michael; Wang, Xiaobo; Fries, Bettina C.

    2015-01-08

    Staphylococcal enterotoxin B (SEB) is a superantigen that cross-links the major histocompatibility complex class II and specific V-β chains of the T-cell receptor, thus forming a ternary complex. Developing neutralizing mAb to disrupt the ternary complex and abrogate the resulting toxicity is a major therapeutic challenge because SEB is effective at very low concentrations. We show that combining two SEB-specific mAbs enhances their efficacy, even though one of the two mAbs by itself has no effect on neutralization. Crystallography was employed for fine-mapping conformational epitopes in binary and ternary complexes between SEB and Fab fragments. NMR spectroscopy was used to validate and identify subtle allosteric changes induced by mAbs binding to SEB. The mapping of epitopes established that a combination of different mAbs can enhance efficacy of mAb-mediated protection from SEB induced lethal shock by two different mechanisms: one mAb mixture promoted clearance of the toxin both in vitro and in vivo by FcR-mediated cross-linking and clearance, whereas the other mAb mixture induced subtle allosteric conformational changes in SEB that perturbed formation of the SEB·T-cell receptor·major histocompatibility complex class II trimer. Lastly structural information accurately predicted mAb binding to other superantigens that share conformational epitopes with SEB. Fine mapping of conformational epitopes is a powerful tool to establish the mechanism and optimize the action of synergistic mAb combinations.

  10. Mechanisms mediating enhanced neutralization efficacy of staphylococcal enterotoxin B by combinations of monoclonal antibodies.

    PubMed

    Dutta, Kaushik; Varshney, Avanish K; Franklin, Matthew C; Goger, Michael; Wang, Xiaobo; Fries, Bettina C

    2015-03-13

    Staphylococcal enterotoxin B (SEB) is a superantigen that cross-links the major histocompatibility complex class II and specific V-β chains of the T-cell receptor, thus forming a ternary complex. Developing neutralizing mAb to disrupt the ternary complex and abrogate the resulting toxicity is a major therapeutic challenge because SEB is effective at very low concentrations. We show that combining two SEB-specific mAbs enhances their efficacy, even though one of the two mAbs by itself has no effect on neutralization. Crystallography was employed for fine-mapping conformational epitopes in binary and ternary complexes between SEB and Fab fragments. NMR spectroscopy was used to validate and identify subtle allosteric changes induced by mAbs binding to SEB. The mapping of epitopes established that a combination of different mAbs can enhance efficacy of mAb-mediated protection from SEB induced lethal shock by two different mechanisms: one mAb mixture promoted clearance of the toxin both in vitro and in vivo by FcR-mediated cross-linking and clearance, whereas the other mAb mixture induced subtle allosteric conformational changes in SEB that perturbed formation of the SEB·T-cell receptor·major histocompatibility complex class II trimer. Finally structural information accurately predicted mAb binding to other superantigens that share conformational epitopes with SEB. Fine mapping of conformational epitopes is a powerful tool to establish the mechanism and optimize the action of synergistic mAb combinations.

  11. Temporal expression of staphylococcal enterotoxin h in comparison with accessory gene regulator-dependent and -independent enterotoxins.

    PubMed

    Lis, Elżbieta; Podkowik, Magdalena; Bystroń, Jarosław; Stefaniak, Tadeusz; Bania, Jacek

    2012-02-01

    Using sandwich enzyme-linked immunosorbent assay (ELISA), the production of staphylococcal enterotoxin (SE) H was determined in 22 Staphylococcus aureus isolates bearing the seh gene. Samples of supernatants were taken at four time points corresponding to exponential phase (optical density at 600 nm [OD(600)] 0.3 to 0.6), late exponential phase (OD(600) 2 to 4), early stationary phase (OD(600) 4 to 6), and late stationary phase (OD(600) 7 to 12). In four isolates, SEH was detectable at a very low level at the first time point. In 18 isolates, the earliest SEH production was detected in the late exponential phase. For all isolates, there was an increase of SEH concentration with time. Western blot analysis revealed that SEH production, similar to SEA, started in the early exponential phase (OD(600) ∼ 0.5). Isolates with high SEH productivity, as measured by ELISA, demonstrated a higher seh transcription as well. sec transcription was induced in the stationary phase. An induction in the sea transcript was observed during mid- to late exponential phase. Expression profile of seh was similar to that of sea. We showed that the seh expression profile is similar to that of Agr-independent sea and not to that of Agr-dependent sec genes. SEH can be effectively expressed at low bacterial counts, meaning that even in an environment not favorable for S. aureus growth, seh-bearing strains can pose a risk for food safety.

  12. Organ Culture as a Model System for Studies on Enterotoxin Interactions with the Intestinal Epithelium.

    PubMed

    Lorenzen, Ulver Spangsberg; Hansen, Gert H; Danielsen, E Michael

    2016-01-01

    Studies on bacterial enterotoxin-epithelium interactions require model systems capable of mimicking the events occurring at the molecular and cellular levels during intoxication. In this chapter, we describe organ culture as an often neglected alternative to whole-animal experiments or enterocyte-like cell lines. Like cell culture, organ culture is versatile and suitable for studying rapidly occurring events, such as enterotoxin binding and uptake. In addition, it is advantageous in offering an epithelium with more authentic permeability/barrier properties than any cell line, as well as a subepithelial lamina propria, harboring the immune cells of the gut mucosa.

  13. Detection of genes encoding for enterotoxins and determination of the production of enterotoxins by HBL blood plates and immunoassays of psychrotrophic strains of Bacillus cereus isolated from pasteurised milk.

    PubMed

    in't Veld, P H; Ritmeester, W S; Delfgou-van Asch, E H; Dufrenne, J B; Wernars, K; Smit, E; van Leusden, F M

    2001-02-28

    The presence of genes for the production of the three components of the HBL enterotoxin complex and enterotoxin-T in Bacillus cereus was evaluated by PCR tests for strains isolated from milk. In addition enterotoxin production of B. cereus was evaluated by means of the HBL blood agar plate and two commercially available toxin tests. All three genes for the HBL enterotoxin complex were detected in 55% of the 86 strains tested, the enterotoxin-T gene was detected in 62% of the strains. A few strains showed a weak reaction in the PCR tests for the L1 or L2 components of the HBL enterotoxin complex. Many strains that were found to contain the genes for the HBL complex gave negative or doubtful results in the HBL blood agar plate test. All strains that contain the L2 part of the HBL complex showed a titer of at least 8 in the Oxoid RPLA test. Two strains that did not contain the L2 part of the HBL enterotoxin complex gave high titers (= 64) in the RPLA test.

  14. Positive Regulation of Staphylococcal Enterotoxin H by Rot (Repressor of Toxin) Protein and Its Importance in Clonal Complex 81 Subtype 1 Lineage-Related Food Poisoning.

    PubMed

    Sato'o, Yusuke; Hisatsune, Junzo; Nagasako, Yuria; Ono, Hisaya K; Omoe, Katsuhiko; Sugai, Motoyuki

    2015-11-01

    We previously demonstrated the clonal complex 81 (CC81) subtype 1 lineage is the major staphylococcal food poisoning (SFP)-associated lineage in Japan (Y. Sato'o et al., J Clin Microbiol 52:2637-2640, 2014, http://dx.doi.org/10.1128/JCM.00661-14). Strains of this lineage produce staphylococcal enterotoxin H (SEH) in addition to SEA. However, an evaluation of the risk for the recently reported SEH has not been sufficiently conducted. We first searched for staphylococcal enterotoxin (SE) genes and SE proteins in milk samples that caused a large SFP outbreak in Japan. Only SEA and SEH were detected, while there were several SE genes detected in the samples. We next designed an experimental model using a meat product to assess the productivity of SEs and found that only SEA and SEH were detectably produced in situ. Therefore, we investigated the regulation of SEH production using a CC81 subtype 1 isolate. Through mutant analysis of global regulators, we found the repressor of toxin (Rot) functioned oppositely as a stimulator of SEH production. SEA production was not affected by Rot. seh mRNA expression correlated with rot both in media and on the meat product, and the Rot protein was shown to directly bind to the seh promoter. The seh promoter sequence was predicted to form a loop structure and to hide the RNA polymerase binding sequences. We propose Rot binds to the promoter sequence of seh and unfolds the secondary structure that may lead the RNA polymerase to bind the promoter, and then seh mRNA transcription begins. This alternative Rot regulation for SEH may contribute to sufficient toxin production by the CC81 subtype 1 lineage in foods to induce SFP.

  15. Bystander Host Cell Killing Effects of Clostridium perfringens Enterotoxin

    PubMed Central

    Shrestha, Archana; Hendricks, Matthew R.; Bomberger, Jennifer M.

    2016-01-01

    ABSTRACT Clostridium perfringens enterotoxin (CPE) binds to claudin receptors, e.g., claudin-4, and then forms a pore that triggers cell death. Pure cultures of host cells that do not express claudin receptors, e.g., fibroblasts, are unaffected by pathophysiologically relevant CPE concentrations in vitro. However, both CPE-insensitive and CPE-sensitive host cells are present in vivo. Therefore, this study tested whether CPE treatment might affect fibroblasts when cocultured with CPE-sensitive claudin-4 fibroblast transfectants or Caco-2 cells. Under these conditions, immunofluorescence microscopy detected increased death of fibroblasts. This cytotoxic effect involved release of a toxic factor from the dying CPE-sensitive cells, since it could be reproduced using culture supernatants from CPE-treated sensitive cells. Supernatants from CPE-treated sensitive cells, particularly Caco-2 cells, were found to contain high levels of membrane vesicles, often containing a CPE species. However, most cytotoxic activity remained in those supernatants even after membrane vesicle depletion, and CPE was not detected in fibroblasts treated with supernatants from CPE-treated sensitive cells. Instead, characterization studies suggest that a major cytotoxic factor present in supernatants from CPE-treated sensitive cells may be a 10- to 30-kDa host serine protease or require the action of that host serine protease. Induction of caspase-3-mediated apoptosis was found to be important for triggering release of the cytotoxic factor(s) from CPE-treated sensitive host cells. Furthermore, the cytotoxic factor(s) in these supernatants was shown to induce a caspase-3-mediated killing of fibroblasts. This bystander killing effect due to release of cytotoxic factors from CPE-treated sensitive cells could contribute to CPE-mediated disease. PMID:27965452

  16. Evaluation of the VIDAS staph enterotoxin II (SET 2) immunoassay method for the detection of staphylococcal enterotoxins in selected foods: collaborative study.

    PubMed

    Jechorek, Robert P; Johnson, Ronald L

    2008-01-01

    A multilaboratory study was conducted to determine the limit of detection (LOD) of Staphylococcal enterotoxins (SET) in 5 foods. Cooked chicken, ham, potato salad, pasteurized liquid whole milk, and canned mushrooms were each spiked with a different enterotoxin (A, B, C1, D, or E), and tested at 0.25 and 0.5 ng/g SET levels to determine the LOD of the assay for those foods in a collaborative study. Unspiked controls were also included. A total of 19 laboratories representing government and industry participated. In this study, 1674 test portions were analyzed, of which 1638 were used in the statistical analysis. Of the 1638 test portions used in the statistical analysis, 1104 were spiked test portions, of which 1073 were positive by the VIDAS Staph enterotoxin II (SET 2) method. The detection rates at the 0.25 ng/mL level were cooked chicken, 98.2%; ham, 99.0%; potato salad, 99.1%; liquid whole milk, 85.2%; and canned mushrooms, 100%. The detection rates at the 0.5 ng/mL level were cooked chicken, 97.4%; ham, 98.1%; potato salad, 100%; liquid whole milk, 99.0%; and canned mushrooms, 100%. The data indicate that the SET 2 method is capable of detecting SET at 0.25 ng/g in cooked chicken, ham, potato salad, and canned mushrooms and at 0.5 ng/g in pasteurized liquid whole milk.

  17. PCR primers for the detection of staphylococcal enterotoxins K, L, and M and survey of staphylococcal enterotoxin types in Staphylococcus aureus isolates from food poisoning cases in Taiwan.

    PubMed

    Chiang, Yu-Cheng; Chang, Li-Tung; Lin, Chia-Wei; Yang, Chi-Yea; Tsen, Hau-Yang

    2006-05-01

    Staphylococcal enterotoxins (SEs) are important causative agents in gastroenteritidis and food poisoning cases. They are serologically grouped into five major classical types, i.e., SEA, SEB, SEC, SED, and SEE. In addition, new SEs, such as SEG through SEM, have recently been identified and characterized. In an attempt to survey the distribution of classical and new SEs in organisms responsible for staphylococcal infections in Taiwan, we developed PCR primers for the genes that define the SEK, SEL, and SEM types. Bacterial strains other than sek, sel, and sem Staphylococcus aureus, including strains of other Staphylococcus species, did not generate any false-positive results when examined with these primers. The expression potential for the sek, sel, and sem types were also determined by reverse transcription-PCR. Together with the PCR primers specific for the classical SEs and other new SEs, including SEG, SEH, SEI, and SEJ, we surveyed the SE genes in S. aureus strains isolated from food poisoning cases. For 147 S. aureus isolates originating from food poisoning cases, 109 (74.1%) were positive for one or more SE genes. Of them, the major classical enterotoxin type was sea (28.6%), followed by seb (20.4%), sec (8.2%), and sed (2.0%). For the new SE types, sei (30.6%) was detected the most often, followed by sek (18.4%), sem (12.9%), and sel (8.2%). Also, 64 (43.5%) of the total bacterial strains had more than one enterotoxin gene.

  18. From genome to toxicity: a combinatory approach highlights the complexity of enterotoxin production in Bacillus cereus

    PubMed Central

    Jeßberger, Nadja; Krey, Viktoria M.; Rademacher, Corinna; Böhm, Maria-Elisabeth; Mohr, Ann-Katrin; Ehling-Schulz, Monika; Scherer, Siegfried; Märtlbauer, Erwin

    2015-01-01

    In recent years Bacillus cereus has gained increasing importance as a food poisoning pathogen. It is the eponymous member of the B. cereus sensu lato group that consists of eight closely related species showing impressive diversity of their pathogenicity. The high variability of cytotoxicity and the complex regulatory network of enterotoxin expression have complicated efforts to predict the toxic potential of new B. cereus isolates. In this study, comprehensive analyses of enterotoxin gene sequences, transcription, toxin secretion and cytotoxicity were performed. For the first time, these parameters were compared in a whole set of B. cereus strains representing isolates of different origin (food or food poisoning outbreaks) and of different toxic potential (enteropathogenic and apathogenic) to elucidate potential starting points of strain-specific differential toxicity. While toxin gene sequences were highly conserved and did not allow for differentiation between high and low toxicity strains, comparison of nheB and hblD enterotoxin gene transcription and Nhe and Hbl protein titers revealed not only strain-specific differences but also incongruence between toxin gene transcripts and toxin protein levels. With one exception all strains showed comparable capability of protein secretion and so far, no secretion patterns specific for high and low toxicity strains were identified. These results indicate that enterotoxin expression is more complex than expected, possibly involving the orchestrated interplay of different transcriptional regulator proteins, as well as posttranscriptional and posttranslational regulatory mechanisms plus additional influences of environmental conditions. PMID:26113843

  19. Quantitative microbial risk assessment for Staphylococcus aureus and Staphylococcus enterotoxin A in raw milk.

    PubMed

    Heidinger, Joelle C; Winter, Carl K; Cullor, James S

    2009-08-01

    A quantitative microbial risk assessment was constructed to determine consumer risk from Staphylococcus aureus and staphylococcal enterotoxin in raw milk. A Monte Carlo simulation model was developed to assess the risk from raw milk consumption using data on levels of S. aureus in milk collected by the University of California-Davis Dairy Food Safety Laboratory from 2,336 California dairies from 2005 to 2008 and using U.S. milk consumption data from the National Health and Nutrition Examination Survey of 2003 and 2004. Four modules were constructed to simulate pathogen growth and staphylococcal enterotoxin A production scenarios to quantify consumer risk levels under various time and temperature storage conditions. The three growth modules predicted that S. aureus levels could surpass the 10(5) CFU/ml level of concern at the 99.9th or 99.99th percentile of servings and therefore may represent a potential consumer risk. Results obtained from the staphylococcal enterotoxin A production module predicted that exposure at the 99.99th percentile could represent a dose capable of eliciting staphylococcal enterotoxin intoxication in all consumer age groups. This study illustrates the utility of quantitative microbial risk assessments for identifying potential food safety issues.

  20. Development and application of an enzyme linked immunosorbent assay for Clostridium perfringens type A enterotoxin.

    PubMed Central

    Bartholomew, B A; Stringer, M F; Watson, G N; Gilbert, R J

    1985-01-01

    An enzyme linked immunosorbent assay (ELISA) has been developed to quantitate faecal Clostridium perfringens enterotoxin in the investigation of C perfringens food poisoning. The sandwich ELISA could be carried out in 24 h and was sensitive enough to detect as little as 5 ng/g of enterotoxin in faeces. Specificity of the assay was shown by comparing results with those obtained from other standard toxin assays, such as double gel diffusion and counterimmunoelectrophoresis, and by the assay of faecal material from control groups. By means of the ELISA method, 515 faecal samples from 50 separate outbreaks of C perfringens food poisoning were examined, together with 21 food samples from 12 of the outbreaks. A clear distinction was noted between faecal samples collected on the first two days of an outbreak, where 77% were enterotoxin positive, and those specimens collected later than the second day, when only 33% had detectable enterotoxin. The ELISA is recommended as a valuable tool in the investigation of C perfringens foodborne illness. PMID:2857184

  1. Distribution of Toxin Genes and Enterotoxins in Bacillus thuringiensis Isolated from Microbial Insecticide Products.

    PubMed

    Cho, Seung-Hak; Kang, Suk-Ho; Lee, Yea-Eun; Kim, Sung-Jo; Yoo, Young-Bin; Bak, Yeong-Seok; Kim, Jung-Beom

    2015-12-28

    Bacillus thuringiensis microbial insecticide products have been applied worldwide. Although a few cases of B. thuringiensis foodborne illness have been reported, little is known about the toxigenic properties of B. thuringiensis isolates. The aims of this study were to estimate the pathogenic potential of B. thuringiensis selected from microbial insecticide products, based on its possession of toxin genes and production of enterotoxins. Fifty-two B. thuringiensis strains selected from four kinds of microbial insecticide products were analyzed. PCR assay for detection of toxin genes and immunoassay for detection of enterotoxins were performed. The hemolysin BL complex as a major enterotoxin was produced by 17 (32.7%), whereas the nonhemolytic enterotoxin complex was detected in 1 (1.9%) of 52 B. thuringiensis strains. However, cytK, entFM, and ces genes were not detected in any of the tested B. thuringiensis strains. The potential risk of food poisoning by B. thuringiensis along with concerns over B. thuringiensis microbial insecticide products has gained attention recently. Thus, microbial insecticide products based on B. thuringiensis should be carefully controlled.

  2. Staphylococcus epidermidis and Staphylococcus haemolyticus: Molecular Detection of Cytotoxin and Enterotoxin Genes

    PubMed Central

    Pinheiro, Luiza; Ivo Brito, Carla; de Oliveira, Adilson; Yoshida Faccioli Martins, Patrícia; Cataneli Pereira, Valéria; Ribeiro de Souza da Cunha, Maria de Lourdes

    2015-01-01

    Although opportunistic pathogens, coagulase-negative staphylococci (CoNS), including Staphylococcus epidermidis and Staphylococcus haemolyticus, have long been regarded as avirulent organisms. The role of toxins in the development of infections caused by CoNS is still controversial. The objective of this study was to characterize the presence of enterotoxin and cytotoxin genes in S. epidermidis and S. haemolyticus isolates obtained from blood cultures. Cytotoxin genes were detected by PCR using novel species-specific primers. Among the 85 S. epidermidis and 84 S. haemolyticus isolates, 95.3% and 79.8%, respectively, carried at least one enterotoxin gene. The most frequent enterotoxin genes were sea (53.3%), seg (64.5%) and sei (67.5%). The seg gene was positively associated with S. epidermidis (p = 0.02), and this species was more toxigenic than S. haemolyticus. The hla/yidD gene was detected in 92.9% of S. epidermidis and the hla gene in 91.7% of S. haemolyticus isolates; hlb was detected in 92.9% of the S. epidermidis isolates and hld in 95.3%. Nosocomial Staphylococcus epidermidis and S. haemolyticus isolates exhibited a high toxigenic potential, mainly containing the non-classical enterotoxin genes seg and sei. The previously unreported detection of hla/yidD and hlb in S. epidermidis and S. haemolyticus using species-specific primers showed that these hemolysin genes differ between CoNS species and that they are highly frequent in blood culture isolates. PMID:26389954

  3. Staphylococcus epidermidis and Staphylococcus haemolyticus: Molecular Detection of Cytotoxin and Enterotoxin Genes.

    PubMed

    Pinheiro, Luiza; Brito, Carla Ivo; de Oliveira, Adilson; Martins, Patrícia Yoshida Faccioli; Pereira, Valéria Cataneli; da Cunha, Maria de Lourdes Ribeiro de Souza

    2015-09-14

    Although opportunistic pathogens, coagulase-negative staphylococci (CoNS), including Staphylococcus epidermidis and Staphylococcus haemolyticus, have long been regarded as avirulent organisms. The role of toxins in the development of infections caused by CoNS is still controversial. The objective of this study was to characterize the presence of enterotoxin and cytotoxin genes in S. epidermidis and S. haemolyticus isolates obtained from blood cultures. Cytotoxin genes were detected by PCR using novel species-specific primers. Among the 85 S. epidermidis and 84 S. haemolyticus isolates, 95.3% and 79.8%, respectively, carried at least one enterotoxin gene. The most frequent enterotoxin genes were sea (53.3%), seg (64.5%) and sei (67.5%). The seg gene was positively associated with S. epidermidis (p = 0.02), and this species was more toxigenic than S. haemolyticus. The hla/yidD gene was detected in 92.9% of S. epidermidis and the hla gene in 91.7% of S. haemolyticus isolates; hlb was detected in 92.9% of the S. epidermidis isolates and hld in 95.3%. Nosocomial Staphylococcus epidermidis and S. haemolyticus isolates exhibited a high toxigenic potential, mainly producing the non-classical enterotoxins seg and sei. The previously unreported detection of hla/yidD and hlb in S. epidermidis and S. haemolyticus using species-specific primers showed that these hemolysin genes differ between CoNS species and that they are highly frequent in blood culture isolates.

  4. Use of inactivated E.Coli enterotoxins to enhance respiratory mucosal adjuvanticity during vaccination in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to augment responses to respiratory vaccines in swine, various adjuvants were intranasally co-administered with an antigen to pigs. Detoxified E. coli enterotoxins LTK63 and LTR72 enhanced mucosal and systemic immunity to the model peptide, exhibiting their efficacy as mucosal adjuvants for...

  5. Importance of Flagella and Enterotoxins for Aeromonas Virulence in a Mouse Model

    EPA Science Inventory

    A genetic characterization of eight virulence factor genes, elastase, lipase, polar flagella (flaA/flaB, flaG), lateral flagella (lafA), and the enterotoxins alt, act, and ast, was performed using polymerase chain reaction with 55 drinking water and nine clinical isolates. When 1...

  6. In vitro cell based assay for activity analysis of staphylococcal enterotoxin A in food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcal enterotoxins (SEs) are a leading cause of food poisoning. They function both as toxins that cause gastroenteritis after ingestion and as superantigens that non-specifically activate large numbers of T cells. Monkey or kitten bioassays were historically developed for analysis of SE act...

  7. Detection and expression of enterotoxin genes in plant-associated strains of Bacillus cereus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacillus cereus is an environmental microbe that commonly inhabits plants and soil. Twenty five plant-associated B. cereus isolates were obtained from apple, cacao, tomato, and potato. The isolates were screened for the presence and expression of enterotoxin B (BcET) components of the nonhemolytic e...

  8. Nicotinic acid inhibits enterotoxin-induced jejunal secretion in the pig.

    PubMed Central

    Forsyth, G W; Kapitany, R A; Scoot, A

    1981-01-01

    The use of nicotinic acid for preventing intestinal secretion caused by cholera toxin and by the heat-stable enterotoxin of Escherichia coli has been investigated in the weanling pig. Secretory effects were measured in ligated jejunal loops of halothane-anesthetized pigs by dilution of a nonabsorbable marker added to the loop fluid. Different routes of administration and different initial pH values for nicotinate solutions were studied to determine optimal conditions for secretory inhibition. The neutral sodium salt of nicotinic acid had no significant antisecretory activity under any conditions used in these trials. Inhibition of secretion was most effective with partly neutralized nicotinic acid at pH 4.5 added directly to loops containing enterotoxin. Net fluid secretion induced by cholera toxin or heat-stable enterotoxin of E. coli was prevented by this treatment. Reversal of secretion was not accompanied by any measurable changes in cyclic nucleotide concentration in intestinal mucosa. Nicotinic acid antagonism of a secretory step common to cholera toxin and heat-stable enterotoxin of E. coli but subsequent to cyclic nucleotide involvement is indicated by these data. PMID:7020893

  9. The olive compound 4-hydroxytyrosol inactivates Staphyloccoccus aureus bacteria and Staphylococcal enterotoxin A (SEA)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The foodborne pathogen Staphylococcus aureus produces the virulent staphylococcal enterotoxin A (SEA), a single chain protein which consists of 233 amino acid residues with a molecular weight of 27,078 Da. SEA is a superantigen that is reported to contribute to animal (mastitis) and human (emesis, ...

  10. Genotypes and enterotoxin gene profiles of Staphylococcus aureus clinical isolates from China

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A total of 108 S. aureus isolates from 16 hospitals located in 14 different provinces in China were characterized for the profiles of 19 staphylococcal enterotoxin (SE) genes by PCR and genotyped by PFGE and MLST. Of these strains, 88.9% (96/108) harbored SE genes, in which tsst was the most prevale...

  11. Techniques for rapid detection and quantification of active foodborne Staphylococcus Enterotoxin(Abstract)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Staphylococcus aureus is an important bacterial pathogen and causative agent of foodborne illnesses.Staphylococcal enterotoxins(SEs)produced by this organism act upon the gastrointestinal tract and generate a superantigen immune response in low concentrations. Recent S. aureus foodborne ...

  12. Inhibitory effect of totarol on exotoxin proteins hemolysin and enterotoxins secreted by Staphylococcus aureus.

    PubMed

    Shi, Ce; Zhao, Xingchen; Li, Wenli; Meng, Rizeng; Liu, Zonghui; Liu, Mingyuan; Guo, Na; Yu, Lu

    2015-10-01

    Staphylococcus aureus (S. aureus) causes a wide variety of infections, which are of major concern worldwide. S. aureus produces multiple virulence factors, resulting in food infection and poisoning. These virulence factors include hyaluronidases, proteases, coagulases, lipases, deoxyribonucleases and enterotoxins. Among the extracellular proteins produced by S. aureus that contribute to pathogenicity, the exotoxins α-hemolysin, staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin B (SEB) are thought to be of major significance. Totarol, a plant extract, has been revealed to inhibit the proliferation of several pathogens effectively. However, there are no reports on the effects of totarol on the production of α-hemolysin, SEA or SEB secreted by S. aureus. The aim of this study was to evaluate the effects of totarol on these three exotoxins. Hemolysis assay, western blotting and real-time reverse transcriptase-PCR assay were performed to identify the influence of graded subinhibitory concentrations of totarol on the production of α-hemolysin and the two major enterotoxins, SEA and SEB, by S. aureus in a dose-dependent manner. Moreover, an enzyme linked immunosorbent assay showed that the TNF-α production of RAW264.7 cells stimulated by S. aureus supernatants was inhibited by subinhibitory concentrations of totarol. Form the data, we propose that totarol could potentially be used as a promising natural compound in the food and pharmaceutical industries.

  13. The formation of Staphylococcus aureus enterotoxin in food environments and advances in risk assessment

    PubMed Central

    Wallin-Carlquist, Nina; Thorup Cohn, Marianne; Lindqvist, Roland; Barker, Gary C; Rådström, Peter

    2011-01-01

    The recent finding that the formation of staphylococcal enterotoxins in food is very different from that in cultures of pure Staphylococcus aureus sheds new light on, and brings into question, traditional microbial risk assessment methods based on planktonic liquid cultures. In fact, most bacteria in food appear to be associated with surfaces or tissues in various ways, and interaction with other bacteria through molecular signaling is prevalent. Nowadays it is well established that there are significant differences in the behavior of bacteria in the planktonic state and immobilized bacteria found in multicellular communities. Thus, in order to improve the production of high-quality, microbiologically safe food for human consumption, in situ data on enterotoxin formation in food environments are required to complement existing knowledge on the growth and survivability of S. aureus. This review focuses on enterotoxigenic S. aureus and describes recent findings related to enterotoxin formation in food environments, and ways in which risk assessment can take into account virulence behavior. An improved understanding of how environmental factors affect the expression of enterotoxins in foods will enable us to formulate new strategies for improved food safety. PMID:22030860

  14. Influence of starch source on sporulation and enterotoxin production by Clostridium perfringens type A.

    PubMed

    Labbe, R; Somers, E; Duncan, C

    1976-03-01

    Of 16 different starch preparations tested, Clostridium perfringes NCTC 8798 yielded maximum sporulation and enterotoxin formation when ICN-soluble starch was included in Duncan and Strong sporulation medium. In general soluble starches were better than potato, corn, or arrowroot starch with regard to these two parameters.

  15. Effects of frozen storage on survival of Staphylococcus aureus and enterotoxin production in precooked tuna meat.

    PubMed

    Wu, Xulei; Su, Yi-Cheng

    2014-08-01

    This study investigated the survival of Staphylococcus aureus in precooked tuna meat for producing canned products during frozen storage (-20 ± 2 °C) as well as its growth and enterotoxin production at 35 to 37 °C after the storage. Samples (50 ± 5 g) of precooked albacore (loin, chunk, and flake) and skipjack (chunk and flake) tuna were inoculated with 5 enterotoxin-producing strains of S. aureus at a level of approximately 3.5 log CFU/g and individually packed in a vacuum bag after 3 h incubation at 35 to 37 °C. Vacuum-packed samples were stored in a freezer (-20 ± 2 °C) for 4 wk. The frozen samples were then thawed in 37 °C circulating water for 2 h and incubated at 35 to 37 °C for 22 h. Populations of S. aureus in all precooked tuna samples decreased slightly (<0.7 log CFU/g) after 4 wk of storage at -20 ± 2 °C, but increased rapidly once the samples were thawed and held at 35 to 37 °C. Total S. aureus counts in albacore and skipjack samples increased by greater than 3 log CFU/g after 6 and 8 h of exposure to 35 to 37 °C, respectively. All samples became spoiled after 10 h of exposure to 35 to 37 °C, while no enterotoxin was detected in any samples. However, enterotoxins were detected in albacore loin and other samples after 12 and 24 h of incubation at 35 to 37 °C, respectively. Frozen precooked tuna meat should be used for producing canned tuna within 6 to 8 h of thawing to avoid product spoilage and potential enterotoxin production by S. aureus in contaminated precooked tuna meat.

  16. A novel electrochemical immunosensor based on magnetosomes for detection of staphylococcal enterotoxin B in milk.

    PubMed

    Wu, Longyun; Gao, Bo; Zhang, Fang; Sun, Xiulan; Zhang, Yinzhi; Li, Zaijun

    2013-03-15

    In this paper, a novel electrochemical immunosensor to detect staphylococcal enterotoxin B based on bio-magnetosomes, polyaniline nano-gold composite and 1,2-dimethyl-3-butylimidazolium hexafluorophosphate ionic liquid, was developed, and found to exhibit high sensitivity and stability. The specific antibody to staphylococcal enterotoxin B conjugated with the magnetosomes showed rapid immunoreactions and good dispersion, which contributed to the formation of a nanostructurally smooth and dense film on the surface of a gold electrode. Polyaniline nano-gold composite and 1,2-dimethyl-3-butylimidazolium hexafluorophosphate ionic liquid were used to modify the electrode as mediators to improve the electron transfer and offer an excellent biocompatible microenvironment for the antibody to retain its activity to enhance the response of the electrochemical sensor. Under optimal conditions, the developed immunosensor showed a good linear response in the range from 0.05 to 5 ng/mL (R(2)=0.9957) with a detection limit as low as 0.017 ng/mL, compared with the one without magnetosomes (0.05-5 ng/mL, 0.033 ng/mL), this developed immunosensor showed a wider response range and a reduced detection limit. And a good specificity with little adsorption to staphylococcal enterotoxin A, C and Na(+), K(+), Ca(2+) was obtained. Moreover, the immunosensor exhibited a good long-time stability at 4 °C reaching up to 60 days, which showed a relatively long working life. Meanwhile the immunosensor could be regenerated four times using NaOH elution. The sensor also displayed a good repeatability with a relative standard deviation of 5.02% for staphylococcal enterotoxin B detection (1 ng/mL, n=9). Furthermore, high recoveries in milk samples from 81% to 118% were achieved and successfully applied to milk sample detection. The obtained results demonstrate that the developed electrochemical immunosensor is a promising tool for the detection of staphylococcal enterotoxin B in food.

  17. Bacillus cereus enterotoxins act as major virulence factors and exhibit distinct cytotoxicity to different human cell lines.

    PubMed

    Jeßberger, Nadja; Dietrich, Richard; Bock, Stefanie; Didier, Andrea; Märtlbauer, Erwin

    2014-01-01

    A comparative analysis on the relevance of the Bacillus cereus enterotoxins Nhe (nonhemolytic enterotoxin), HBL (haemolysin BL) and CytK (cytotoxin K) was accomplished, concerning their toxic activity towards different target cell lines. Overall, among the components secreted by the reference strains for Nhe and HBL, the enterotoxin complexes accounted for over 90% of the total toxicity. Vero and primary endothelial cells (HUVEC) were highly susceptible to Nhe, whereas Hep-G2, Vero and A549 reacted most sensitive to Nhe plus HBL. For CytK the highest toxicity was observed on CaCo-2 cells. As HBL positive strains always produce Nhe in parallel, the specific contribution of both enterotoxin complexes to the overall observed cytotoxic effects was determined by consecutively removing their single components. While in most cell lines Nhe and HBL contributed more or less equally (40-60%) to cytotoxicity, the relative activity of Nhe was approximately 90% in HUVEC, and that of HBL 75% in A549 cells. With U937, a nearly Nhe resistant cell line was identified for the first time. This distinct susceptibility of cell lines was confirmed by investigating a set of 37 B. cereus strains. Interestingly, whereas Nhe is the enterotoxin mainly responsible for cell death as determined by WST-1 bioassays, more rapid pore formation was observed when HBL was present, pointing to a different mode of action of the two enterotoxin complexes. Furthermore, correlation was observed between cytotoxicity of solely Nhe producing strains and NheB. Cytotoxicity of Nhe/HBL producing isolates correlated with the expression of HBL L1, NheB and HBL B. In conclusion, the observed susceptibilities of target cell lines of different histological origin underline that B. cereus enterotoxins represent major virulence factors and that toxicity is not restricted to gastrointestinal infections. The varying contribution of Nhe and HBL to total cytotoxicity strongly indicates that Nhe as well as HBL specific B

  18. [The significance of some potentially pathogenic microorganisms in occurrence of food toxicosis. Part 1. S. aureus and staphylococcal enterotoxins].

    PubMed

    Efimochkina, N R; Kuvaeva, I B; Fluer, F S

    2011-01-01

    The data on the nomenclature, classification and properties of staphylococci and staphylococcal enterotoxins produced by them are presented. The analysis of cultural and biochemical properties of 137 strains of staphylococci isolated from raw milk and "Russian" cheese was performed. The high degree of correlation between the ability of S. aureus produce enterotoxins and the presence of enzymes coagulase, thermostable DNase, and other factors of pathogenicity is established.

  19. Production of diarrheal enterotoxins and other potential virulence factors by veterinary isolates of bacillus species associated with nongastrointestinal infections.

    PubMed

    Rowan, Neil J; Caldow, George; Gemmell, Curtis G; Hunter, Iain S

    2003-04-01

    With the exceptions of Bacillus cereus and Bacillus anthracis, Bacillus species are generally perceived to be inconsequential. However, the relevance of other Bacillus species as food poisoning organisms and etiological agents in nongastrointestinal infections is being increasingly recognized. Eleven Bacillus species isolated from veterinary samples associated with severe nongastrointestinal infections were assessed for the presence and expression of diarrheagenic enterotoxins and other potential virulence factors. PCR studies revealed the presence of DNA sequences encoding hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T (BceT) in five B. cereus strains and in Bacillus coagulans NB11. Enterotoxin HBL was also harbored by Bacillus polymyxa NB6. After 18 h of growth in brain heart infusion broth, all seven Bacillus isolates carrying genes encoding enterotoxin HBL produced this toxin. Cell-free supernatant fluids from all 11 Bacillus isolates demonstrated cytotoxicity toward human HEp-2 cells; only one Bacillus licheniformis strain adhered to this test cell line, and none of the Bacillus isolates were invasive. This study constitutes the first demonstration that Bacillus spp. associated with serious nongastrointestinal infections in animals may harbor and express diarrheagenic enterotoxins traditionally linked to toxigenic B. cereus.

  20. Enterotoxin genes in coagulase-negative and coagulase-positive staphylococci isolated from bovine milk.

    PubMed

    de Freitas Guimarães, Felipe; Nóbrega, Diego Borin; Richini-Pereira, Virginia Bodelão; Marson, Pâmela Merlo; de Figueiredo Pantoja, José Carlos; Langoni, Helio

    2013-05-01

    The objective of this study was to isolate and identify the main staphylococcal species causing bovine mastitis in 10 Brazilian dairy herds and study their capability to produce enterotoxins. Herds were selected based on size and use of milking technology, and farms were visited once during the study. All mammary glands of all lactating cows were screened using the California Mastitis Test (CMT) and a strip cup. A single aseptic milk sample (20 mL) was collected from all CMT-positive quarters. Identification of Staphylococcus spp. was performed using conventional microbiology, and PCR was used to determine the presence of enterotoxin-encoding genes (sea, seb, sec, and sed). Of the 1,318 CMT-positive milk samples, Staphylococcus spp. were isolated from 263 (19.9%). Of these isolates, 135 (51%) were coagulase-positive staphylococci (CPS) and 128 (49%) were coagulase-negative staphylococci (CNS). Eighteen different species of CNS were isolated, among which S. warneri, S. epidermidis and S. hyicus were the most frequent. The distribution of Staphylococcus species was different among herds: S. epidermidis was found in 8 herds, S. warneri was found in 7 herds, and S. hyicus in 6 herds. Some of the CNS species (S. saprophyticus ssp. saprophyticus, S. auricularis, S. capitis, and S. chromogenes) were isolated in only one of the farms. Genes related to production of enterotoxins were found in 66% (n=85) of all CNS and in 35% of the CPS isolates. For both CNS and CPS isolates, the most frequently identified enterotoxin genes were sea, seb, and sec; the prevalence of sea differed between CPS (9.5%) and CNS (35.1%) isolates. Staphylococcus warneri isolates showed a greater percentage of sea than seb, sec, or sed, whereas S. hyicus isolates showed a greater percentage of sea than sec. Over 60% of CNS belonged to 3 major species, which carried 62.2 to 81.3% of the enterotoxin genes. The high prevalence highlights the potential for food poisoning caused by these species. For

  1. The effect of undissociated lactic acid on Staphylococcus aureus growth and enterotoxin A production.

    PubMed

    Rosengren, Asa; Lindblad, Mats; Lindqvist, Roland

    2013-03-15

    The potential of Staphylococcus aureus cheese isolates to grow and produce staphylococcal enterotoxin A under conditions typical for cheese making was investigated in three broth experiments. The effect of the concentration of undissociated lactic acid (HLac) in conjunction with specific pH values was studied by adjusting pH at a single concentration of lactic acid. First, the time-to-growth of S. aureus was modelled by using survival analysis and absorbance data obtained from an automated turbidity reader. The fitted model describes the time to growth and indicates the growth ⁄ no growth boundary of S. aureus as a function of HLac concentration, temperature and water activity. Second, growth rates and lag times of S. aureus were estimated after two different pre-treatments in skim milk at three HLac concentrations and two temperatures based on optical detection times of serial dilutions of bacterial solutions. Growth rates differed between strains, and increased with increasing temperature and decreasing HLac concentration. Preliminary results indicate that lag times were dependent on pre-treatment suggesting that the growth potential of S. aureus in cheese curd may be greater if milk is used immediately after milking compared to holding at 4°C after milking. Third, growth, inactivation, and enterotoxin A production of S. aureus strains were investigated at twelve combinations of HLac concentration and temperature. Concentrations of enterotoxin A increased linearly during the first four days, with a production rate increasing with increasing temperature and decreasing HLac concentration. Significant amounts of enterotoxin A were produced during extended incubation, up to 14days, but then initial pH had changed. This highlights a potential limitation of modelling based on the initial environmental conditions in batch experiments. In summary, ranges of time-to-growth, growth rates, lag times and enterotoxin A production rates of S. aureus in the presence of HLac

  2. Staphylococcal enterotoxin A regulates bone marrow granulocyte trafficking during pulmonary inflammatory disease in mice

    SciTech Connect

    Takeshita, W.M.; Gushiken, V.O.; Ferreira-Duarte, A.P.; Pinheiro-Torres, A.S.; Roncalho-Buck, I.A.; Squebola-Cola, D.M.; Mello, G.C.; Anhê, G.F.; Antunes, E.; DeSouza, I.A.

    2015-09-15

    Pulmonary neutrophil infiltration produced by Staphylococcal enterotoxin A (SEA) airway exposure is accompanied by marked granulocyte accumulation in bone marrow (BM). Therefore, the aim of this study was to investigate the mechanisms of BM cell accumulation, and trafficking to circulating blood and lung tissue after SEA airway exposure. Male BALB/C mice were intranasally exposed to SEA (1 μg), and at 4, 12 and 24 h thereafter, BM, circulating blood, bronchoalveolar lavage (BAL) fluid and lung tissue were collected. Adhesion of BM granulocytes and flow cytometry for MAC-1, LFA1-α and VLA-4 and cytokine and/or chemokine levels were assayed after SEA-airway exposure. Prior exposure to SEA promoted a marked PMN influx to BAL and lung tissue, which was accompanied by increased counts of immature and/or mature neutrophils and eosinophils in BM, along with blood neutrophilia. Airway exposure to SEA enhanced BM neutrophil MAC-1 expression, and adhesion to VCAM-1 and/or ICAM-1-coated plates. Elevated levels of GM-CSF, G-CSF, INF-γ, TNF-α, KC/CXCL-1 and SDF-1α were detected in BM after SEA exposure. SEA exposure increased production of eosinopoietic cytokines (eotaxin and IL-5) and BM eosinophil VLA-4 expression, but it failed to affect eosinophil adhesion to VCAM-1 and ICAM-1. In conclusion, BM neutrophil accumulation after SEA exposure takes place by integrated action of cytokines and/or chemokines, enhancing the adhesive responses of BM neutrophils and its trafficking to lung tissues, leading to acute lung injury. BM eosinophil accumulation in SEA-induced acute lung injury may occur via increased eosinopoietic cytokines and VLA-4 expression. - Highlights: • Airway exposure to SEA causes acute lung inflammation. • SEA induces accumulation of bone marrow (BM) in immature and mature neutrophils. • SEA increases BM granulocyte or BM PMN adhesion to ICAM-1 and VCAM-1, and MAC-1 expression. • SEA induces BM elevations of CXCL-1, INF-γ, TNF-α, GM-CSF, G-CSF and

  3. Clostridium and Bacillus Binary Enterotoxins: Bad for the Bowels, and Eukaryotic Being

    PubMed Central

    Stiles, Bradley G.; Pradhan, Kisha; Fleming, Jodie M.; Samy, Ramar Perumal; Barth, Holger; Popoff, Michel R.

    2014-01-01

    Some pathogenic spore-forming bacilli employ a binary protein mechanism for intoxicating the intestinal tracts of insects, animals, and humans. These Gram-positive bacteria and their toxins include Clostridium botulinum (C2 toxin), Clostridium difficile (C. difficile toxin or CDT), Clostridium perfringens (ι-toxin and binary enterotoxin, or BEC), Clostridium spiroforme (C. spiroforme toxin or CST), as well as Bacillus cereus (vegetative insecticidal protein or VIP). These gut-acting proteins form an AB complex composed of ADP-ribosyl transferase (A) and cell-binding (B) components that intoxicate cells via receptor-mediated endocytosis and endosomal trafficking. Once inside the cytosol, the A components inhibit normal cell functions by mono-ADP-ribosylation of globular actin, which induces cytoskeletal disarray and death. Important aspects of each bacterium and binary enterotoxin will be highlighted in this review, with particular focus upon the disease process involving the biochemistry and modes of action for each toxin. PMID:25198129

  4. Selection and characterization of DNA aptamers against Staphylococcus aureus enterotoxin C1.

    PubMed

    Huang, Yukun; Chen, Xiujuan; Duan, Nuo; Wu, Shijia; Wang, Zhouping; Wei, Xinlin; Wang, Yuanfeng

    2015-01-01

    Enterotoxins from pathogenic bacteria are known as the main reason that can cause the bacterial foodborne diseases. In this study, aptamers that bound to Staphylococcus aureus enterotoxin C1 (SEC1) with high affinity and selectivity were generated in vitro by twelve rounds of selection based on magnetic separation technology, with a low-level dissociation constant (Kd) value of 65.14 ± 11.64 nmol/L of aptamer C10. Aptamer-based quantification of SEC1 in the food sample by a graphene oxide (GO)-based method was implemented to investigate the potential of the aptamer against SEC1 with a limit of detection of 6 ng/mL. On the basis of this work, biosensors using the selected SEC1 aptamers as new molecular recognition elements could be applied for innovative determinations of SEC1.

  5. Incidence of ketamine-induced emesis in cynomologus monkeys (Macaca fascicularis) used for staphylococcal enterotoxin bioassay.

    PubMed Central

    Adesiyun, A. A.; Tatini, S. R.

    1982-01-01

    Ten (24%) of 41 cynomologus monkeys (Macaca fascicularis) showed emetic response to 2.5-20 mg/Kg of ketamine injected i.m. Reduction of the levels of ketamine to one half or less of the emetic level resulted in faster recovery from sedation yet provided adequate time for intubation and subsequent intragastric feeding of staphylococcal enterotoxin (SE) in only 6 of the 10 monkeys without emesis. The onset of the first emetic episode with ketamine was similar to that induced by staphylococcal enterotoxin A (SEA). Cynomologus monkeys showing emetic response to ketamine could still be used for SE bioassay if an experimentally determined non-emetic dose for individual monkeys is employed for sedation. PMID:7093145

  6. Evaluation of Handheld Assays for the Detection of Ricin and Staphylococcal Enterotoxin B in Disinfected Waters

    DTIC Science & Technology

    2011-01-01

    Staphylococcal Enterotoxin B (SEB) in water. Performance of HHAs was evaluated in formulated tap water with and without chlorine, reverse osmosis water (RO) with...in water. Performance of HHAs was evaluated in formulated tap water with and without chlorine, reverse osmosis water (RO) with chlorine, and RO with...four different water matrices were formulated tap water, formulated tap water with 1 mg/L free available chlorine (FAC), water treated by reverse osmosis

  7. PCR detection of staphylococcal enterotoxin genes in Staphylococcus aureus strains isolated from raw and pasteurized milk.

    PubMed

    Rall, V L M; Vieira, F P; Rall, R; Vieitis, R L; Fernandes, A; Candeias, J M G; Cardoso, K F G; Araújo, J P

    2008-12-10

    Milk is considered a nutritious food because it contains several important nutrients including proteins and vitamins. Conversely, it can be a vehicle for several pathogenic bacteria such as Staphylococcus aureus. This study aimed to analyze the frequency of genes encoding the staphylococcal enterotoxins (SEs) SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI and SEJ in S. aureus strains isolated from raw or pasteurized bovine milk. S. aureus was found in 38 (70.4%) out of 54 raw milk samples at concentrations of up to 8.9 x 10(5) CFU/ml. This microorganism was present in eight samples of pasteurized milk before the expiration date and in 11 samples analyzed on the expiration date. Of the 57 strains studied, 68.4% were positive for one or more genes encoding the enterotoxins, and 12 different genotypes were identified. The gene coding for enterotoxin A, sea, was the most frequent (16 strains, 41%), followed by sec (8 strains, 20.5%), sed (5 strains, 12.8%), seb (3 strains, 7.7%) and see (2 strains, 5.1%). Among the genes encoding the other enterotoxins, seg was the most frequently observed (11 strains, 28.2%), followed by sei (10 strains) and seh and sej (3 strains each). With the recent identification of new SEs, the perceived frequency of enterotoxigenic strains has increased, suggesting that the pathogenic potential of staphylococci may be higher than previously thought; however, further studies are required to assess the expression of these new SEs by S. aureus, and their impact in foodborne disease. The quality of Brazilian milk is still low, and efforts from the government and the entire productive chain are required to attain consumer safety.

  8. (Staphylococcal Aureus Enterotoxin A): Partial Characterization of an N-Terminal Peptide

    DTIC Science & Technology

    1989-05-01

    monitored with the ninhydrin test (Kaiser, et al., 1970). The protecting groups used were tert-butyl(t-Bu) for Glu and Ser, and tert-butyloxycarbonyl(t...sequence of staphylococcal enterotoxin A. J. Biol. Chem. 262: 7006-7013. Kaiser, E., Colescott, R.L., Bossinger, C.D., and Cook, P.Y. 1970. Color test ...location may also be included. If Possible keywords should be selected from a published thesaurus. e.g. Thesaurus of Engineering and Scientific Terms ( TEST

  9. BEC, a novel enterotoxin of Clostridium perfringens found in human clinical isolates from acute gastroenteritis outbreaks.

    PubMed

    Yonogi, Shinya; Matsuda, Shigeaki; Kawai, Takao; Yoda, Tomoko; Harada, Tetsuya; Kumeda, Yuko; Gotoh, Kazuyoshi; Hiyoshi, Hirotaka; Nakamura, Shota; Kodama, Toshio; Iida, Tetsuya

    2014-06-01

    Clostridium perfringens is a causative agent of food-borne gastroenteritis for which C. perfringens enterotoxin (CPE) has been considered an essential factor. Recently, we experienced two outbreaks of food-borne gastroenteritis in which non-CPE producers of C. perfringens were strongly suspected to be the cause. Here, we report a novel enterotoxin produced by C. perfringens isolates, BEC (binary enterotoxin of C. perfringens). Culture supernatants of the C. perfringens strains showed fluid-accumulating activity in rabbit ileal loop and suckling mouse assays. Purification of the enterotoxic substance in the supernatants and high-throughput sequencing of genomic DNA of the strains revealed BEC, composed of BECa and BECb. BECa and BECb displayed limited amino acid sequence similarity to other binary toxin family members, such as the C. perfringens iota toxin. The becAB genes were located on 54.5-kb pCP13-like plasmids. Recombinant BECb (rBECb) alone had fluid-accumulating activity in the suckling mouse assay. Although rBECa alone did not show enterotoxic activity, rBECa enhanced the enterotoxicity of rBECb when simultaneously administered in suckling mice. The entertoxicity of the mutant in which the becB gene was disrupted was dramatically decreased compared to that of the parental strain. rBECa showed an ADP-ribosylating activity on purified actin. Although we have not directly evaluated whether BECb delivers BECa into cells, rounding of Vero cells occurred only when cells were treated with both rBECa and rBECb. These results suggest that BEC is a novel enterotoxin of C. perfringens distinct from CPE, and that BEC-producing C. perfringens strains can be causative agents of acute gastroenteritis in humans. Additionally, the presence of becAB on nearly identical plasmids in distinct lineages of C. perfringens isolates suggests the involvement of horizontal gene transfer in the acquisition of the toxin genes.

  10. Effect of cholera enterotoxin on carbohydrate metabolism in the liver and small intestinal mucosa of rabbits

    SciTech Connect

    Vengrov, P.R.; Cherkasova, T.D.; Yurkiv, V.A.; Pokrovskii, V.I.

    1987-09-01

    The effect of cholera enterotoxin injected in vivo on glucose formation from alanine, and also on glucose-6-phosphatase activity in the liver and mucosa of the small intestine was studied. L-(2,3-/sup 3/H)-alanine was added to the incubation medium. Chromatograms were developed with 5% AgNO/sub 3/ with the addition of an aqueous solution of ammonia. The quantity of radioactive glucose was determined in a scintillation counter.

  11. BEC, a Novel Enterotoxin of Clostridium perfringens Found in Human Clinical Isolates from Acute Gastroenteritis Outbreaks

    PubMed Central

    Yonogi, Shinya; Matsuda, Shigeaki; Kawai, Takao; Yoda, Tomoko; Harada, Tetsuya; Kumeda, Yuko; Gotoh, Kazuyoshi; Hiyoshi, Hirotaka; Nakamura, Shota; Kodama, Toshio

    2014-01-01

    Clostridium perfringens is a causative agent of food-borne gastroenteritis for which C. perfringens enterotoxin (CPE) has been considered an essential factor. Recently, we experienced two outbreaks of food-borne gastroenteritis in which non-CPE producers of C. perfringens were strongly suspected to be the cause. Here, we report a novel enterotoxin produced by C. perfringens isolates, BEC (binary enterotoxin of C. perfringens). Culture supernatants of the C. perfringens strains showed fluid-accumulating activity in rabbit ileal loop and suckling mouse assays. Purification of the enterotoxic substance in the supernatants and high-throughput sequencing of genomic DNA of the strains revealed BEC, composed of BECa and BECb. BECa and BECb displayed limited amino acid sequence similarity to other binary toxin family members, such as the C. perfringens iota toxin. The becAB genes were located on 54.5-kb pCP13-like plasmids. Recombinant BECb (rBECb) alone had fluid-accumulating activity in the suckling mouse assay. Although rBECa alone did not show enterotoxic activity, rBECa enhanced the enterotoxicity of rBECb when simultaneously administered in suckling mice. The entertoxicity of the mutant in which the becB gene was disrupted was dramatically decreased compared to that of the parental strain. rBECa showed an ADP-ribosylating activity on purified actin. Although we have not directly evaluated whether BECb delivers BECa into cells, rounding of Vero cells occurred only when cells were treated with both rBECa and rBECb. These results suggest that BEC is a novel enterotoxin of C. perfringens distinct from CPE, and that BEC-producing C. perfringens strains can be causative agents of acute gastroenteritis in humans. Additionally, the presence of becAB on nearly identical plasmids in distinct lineages of C. perfringens isolates suggests the involvement of horizontal gene transfer in the acquisition of the toxin genes. PMID:24664508

  12. The Molecular Basis for Control of ETEC Enterotoxin Expression in Response to Environment and Host

    PubMed Central

    Haycocks, James R. J.; Sharma, Prateek; Stringer, Anne M.; Wade, Joseph T.; Grainger, David C.

    2015-01-01

    Enterotoxigenic Escherichia coli (ETEC) cause severe diarrhoea in humans and neonatal farm animals. Annually, 380,000 human deaths, and multi-million dollar losses in the farming industry, can be attributed to ETEC infections. Illness results from the action of enterotoxins, which disrupt signalling pathways that manage water and electrolyte homeostasis in the mammalian gut. The resulting fluid loss is treated by oral rehydration. Hence, aqueous solutions of glucose and salt are ingested by the patient. Given the central role of enterotoxins in disease, we have characterised the regulatory trigger that controls toxin production. We show that, at the molecular level, the trigger is comprised of two gene regulatory proteins, CRP and H-NS. Strikingly, this renders toxin expression sensitive to both conditions encountered on host cell attachment and the components of oral rehydration therapy. For example, enterotoxin expression is induced by salt in an H-NS dependent manner. Furthermore, depending on the toxin gene, expression is activated or repressed by glucose. The precise sensitivity of the regulatory trigger to glucose differs because of variations in the regulatory setup for each toxin encoding gene. PMID:25569153

  13. Enterotoxin production in natural isolates of Bacillaceae outside the Bacillus cereus group.

    PubMed

    Phelps, Rebecca J; McKillip, John L

    2002-06-01

    Thirty-nine Bacillus strains obtained from a variety of environmental and food sources were screened by PCR for the presence of five gene targets (hblC, hblD, hblA, nheA, and nheB) in two enterotoxin operons (HBL and NHE) traditionally harbored by Bacillus cereus. Seven isolates exhibited a positive signal for at least three of the five possible targets, including Bacillus amyloliquefaciens, B. cereus, Bacillus circulans, Bacillus lentimorbis, Bacillus pasteurii, and Bacillus thuringiensis subsp. kurstaki. PCR amplicons were confirmed by restriction enzyme digest patterns compared to a positive control strain. Enterotoxin gene expression of each strain grown in a model food system (skim milk) was monitored by gene-specific reverse transcription-PCR and confirmed with the Oxoid RPLA and Tecra BDE commercial kits. Lecithinase production was noted on egg yolk-polymyxin B agar for all strains except B. lentimorbis, whereas discontinuous beta hemolysis was exhibited by all seven isolates grown on 5% sheep blood agar plates. The results of this study confirm the presence of enterotoxin genes in natural isolates of Bacillus spp. outside the B. cereus group and the ability of these strains to produce toxins in a model food system under aerated conditions at 32 degrees C.

  14. Differential RNA regulation by staphylococcal enterotoxins A and B in murine macrophages

    NASA Technical Reports Server (NTRS)

    Chapes, S. K.; Beharka, A. A.; Hart, M. E.; Smeltzer, M. S.; Iandolo, J. J.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Staphylococcal enterotoxin A (SEA) is significantly better than enterotoxin B (SEB) in activating tumor necrosis factor (TNF) secretion by B6MP102 cells. Both toxins bound to B6MP102 cells; however, SEB competed less effectively with SEA than SEA competed with SEB. This suggested that receptors unique to SEA were present on B6MP102 cells. Signal transduction occurred in response to both toxins. Within 30 s after addition, SEA and SEB significantly increased the F-actin concentration in B6MP102 cells. However, only SEA induced increased TNF mRNA levels. B6MP102 cells incubated with interferon-gamma and SEB secreted TNF. However, enhanced mRNA expression was delayed and the concentration of TNF secreted was less than that of B6MP102 cells stimulated with SEA. Although these data suggest that receptors unique to SEA are present on B6MP102 cells, they also indicate that staphylococcal enterotoxins differentially regulate TNF at the RNA level, perhaps because of differences in binding to the plasma membrane.

  15. Staphylococcal enterotoxin H induced apoptosis of bovine mammary epithelial cells in vitro.

    PubMed

    Liu, Yongxia; Chen, Wei; Ali, Tariq; Alkasir, Rashad; Yin, Jinhua; Liu, Gang; Han, Bo

    2014-12-19

    Staphylococcal enterotoxins (SEs) are powerful superantigenic toxins produced by Staphylococcus aureus (S. aureus). They can cause food poisoning and toxic shock. However, their impact on bovine mammary epithelial cells (bMECs) is still unknown. In this study, the distribution of SE genes was evaluated in 116 S. aureus isolates from bovine mastitis, and the most prevalent genes were seh (36.2%), followed by sei (12.1%), seg (11.2%), ser (4.3%), sec (3.4%), sea (2.6%) and sed (1.7%). To better understand the effect of staphylococcal enterotoxin H (SEH) on bMECs, the seh gene was cloned and inserted into the prokaryotic expression vector, pET28a, and transformed into Escherichia coli BL21 (DE3). The recombinant staphylococcal enterotoxin H (rSEH) was expressed and purified as soluble protein. Bioactivity analysis showed that rSEH possessed the activity of stimulating lymphocytes proliferation. The XTT assay showed that 100 μg/mL of rSEH produced the cytotoxic effect on bMECs, and fluorescence microscopy and flow cytometry analysis revealed that a certain dose of rSEH is effective at inducing bMECs apoptosis in vitro. This indicates that SEs can directly lead to cellular apoptosis of bMECs in bovine mastitis associated with S. aureus.

  16. Prosequence switching: an effective strategy to produce biologically active E. coli heat-stable enterotoxin STh.

    PubMed

    Weiglmeier, Philipp R; Berkner, Hanna; Seebahn, Angela; Vogel, Nico; Schreiber, Rainer; Wöhrl, Birgitta M; Schwarzinger, Stephan; Rösch, Paul

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC) infections account for the majority of cases of acute secretory diarrhea. The causative agents are enterotoxins secreted by ETEC, among them is the heat-stable enterotoxin, STh. STh is a 19-amino acid peptide containing three disulfide bonds that stimulates fluid secretion in the bowel by binding to the receptor domain of intestinal guanylyl cyclase C (GC-C). Since GC-C agonists have pharmacologic potential for diagnosis and treatment of disorders such as constipation-predominant irritable bowel syndrome (IBS-C), chronic constipation, and colorectal carcinoma, it is crucial to develop methods for the large-scale production of STh and related peptides. Here, we present a strategy for recombinant expression of STh that relies on the use of the prosequence of human uroguanylin to support proper folding and disulfide bond formation. The chimeric protein CysCys-STh consisting of the propeptide of uroguanylin as N-terminus and the STh peptide as C-terminus was expressed in E. coli, and an efficient purification protocol was developed. Trypsin digestion of this protein released the enterotoxin which could be obtained in high purity. NMR and mass spectrometry confirmed the identity and homogeneity of the toxin, and its biological activity was confirmed by a cell-based in vivo assay. The expression scheme introduced here represents a cost-efficient and scalable way of STh production.

  17. Various Enterotoxin and Other Virulence Factor Genes Widespread Among Bacillus cereus and Bacillus thuringiensis Strains.

    PubMed

    Kim, Min-Ju; Han, Jae-Kwang; Park, Jong-Su; Lee, Jin-Sung; Lee, Soon-Ho; Cho, Joon-Il; Kim, Keun-Sung

    2015-06-01

    Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen Bacillus thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal diseases are attributed to enterotoxins. Sixteen reference strains of B. cereus and nine commercial and 12 reference strains of B. thuringiensis were screened by PCR for the presence of 10 enterotoxigenic genes (hblA, hblC, hblD, nheA, nheB, nheC, cytK, bceT, entFM, and entS), one emetogenic gene (ces), seven hemolytic genes (hlyA, hlyII, hlyIII, plcA, cerA, cerB, and cerO), and a pleiotropic transcriptional activator gene (plcR). These genes encode various enterotoxins and other virulence factors thought to play a role in infections of mammals. Amplicons were successfully generated from the strains of B. cereus and B. thuringiensis for each of these sequences, except the ces gene. Intriguingly, the majority of these B. cereus enterotoxin genes and other virulence factor genes appeared to be widespread among B. thuringiensis strains as well as B. cereus strains.

  18. Identification of enterotoxigenic Escherichia coli isolates with enzyme-labeled synthetic oligonucleotide probes.

    PubMed Central

    Medon, P P; Lanser, J A; Monckton, P R; Li, P; Symons, R H

    1988-01-01

    Commercially available kits containing alkaline phosphatase-labeled oligonucleotide probes for Escherichia coli heat-stable enterotoxins (STI-H, STI-P, and STII) and the heat-labile enterotoxin were compared with bioassays and radiolabeled recombinant DNA probes to identify enterotoxigenic E. coli from 100 clinical isolates. There was very good agreement between the three methods. PMID:3053766

  19. A probability model for enterotoxin production of Bacillus cereus as a function of pH and temperature.

    PubMed

    Ding, Tian; Wang, Jun; Park, Myoung-Su; Hwang, Cheng-An; Oh, Deog-Hwan

    2013-02-01

    Bacillus cereus is frequently isolated from a variety of foods, including vegetables, dairy products, meats, and other raw and processed foods. The bacterium is capable of producing an enterotoxin and emetic toxin that can cause severe nausea, vomiting, and diarrhea. The objectives of this study were to assess and model the probability of enterotoxin production of B. cereus in a broth model as affected by the broth pH and storage temperature. A three-strain mixture of B. cereus was inoculated in tryptic soy broth adjusted to pH 5.0, 6.0, 7.2, 8.0, and 8.5, and the samples were stored at 15, 20, 25, 30, and 35°C for 24 h. A total of 25 combinations of pH and temperature, each with 10 samples, were tested. The presence of enterotoxin in broth was assayed using a commercial test kit. The probabilities of positive enterotoxin production in 25 treatments were fitted with a logistic regression to develop a probability model to describe the probability of toxin production as a function of pH and temperature. The resulting model showed that the probabilities of enterotoxin production of B. cereus in broth increased as the temperature increased and/or as the broth pH approached 7.0. The model described the experimental data satisfactorily and identified the boundary of pH and temperature for the production of enterotoxin. The model could provide information for assessing the food poisoning risk associated with enterotoxins of B. cereus and for the selection of product pH and storage temperature for foods to reduce the hazards associated with B. cereus.

  20. Antagonistic effects of the staphylococcal enterotoxin a mutant, SEA(F47A/D227A), on psoriasis in the SCID-hu xenogeneic transplantation model.

    PubMed

    Boehncke, W H; Hardt-Weinelt, K; Nilsson, H; Wolter, M; Dohlsten, M; Ochsendorf, F R; Kaufmann, R; Antonsson, P

    2001-04-01

    Psoriasis is a T-cell-mediated immune dermatosis probably triggered by bacterial superantigens. This pathomechanism has been experimentally reproduced in a SCID-hu xenogeneic transplantation model. We analyzed the effects of different bacterial superantigens on the induction of psoriasis in this model. Staphylococcal enterotoxin B and exfoliative toxin triggered the onset of psoriasis when administered repetitively intracutaneously over a period of 2 wk, whereas staphylococcal enterotoxin A representing a distinct subfamily of staphylococcal enterotoxins only mimicked certain aspects of psoriasis. The biologic effects of staphylococcal enterotoxin A were more pronounced when a mutated form, SEA(H187A), of this superantigen with reduced affinity to major histocompatibility complex class II was coinjected. Another mutated variant, SEA(F47A/D227A), exhibiting no measurable major histocompatibility complex class II affinity blocked the effects triggered by wild-type staphylococcal enterotoxin A when injected in a 10-fold higher dose. Inhibition was specific as induction of psoriasiform epidermal changes by staphylococcal enterotoxin B could not be blocked. As staphylococcal enterotoxin A, in contrast to the other superantigens tested, is capable of inducing epidermal thickening but not the typical appearance of psoriasis, we conclude that bacterial superantigens may differ with regard to their effects on human nonlesional psoriatic skin. Staphylococcal-enterotoxin-A-mediated effects were blocked by a genetically engineered superantigen highlighting the potential therapeutic use of mutated superantigens.

  1. High incidence of enterotoxin D producing Staphylococcus spp. in Brazilian cow's raw milk and its relation with coagulase and thermonuclease enzymes.

    PubMed

    Oliveira, Ana Maria; Padovani, Carlos Roberto; Miya, Norma Teruko Nagô; Sant'ana, Anderson S; Pereira, José Luiz

    2011-01-01

    In this study, the enterotoxigenic potential of Staphylococcus strains (n = 574) isolated from raw milk samples (n = 140) was determined for their capacity to produce staphylococcal enterotoxins. In addition, the relationship between the presence of enterotoxins, coagulase, and thermonuclease (Tnase) was assessed. The results showed that 19% of Staphylococcus was enterotoxigenic, being able to produce at least one of the staphylococcal enterotoxins (A, B, C, and D). Most of the strains were able to produce enterotoxin D (68.8%), whereas 12.8% of the Staphylococcus strains were able to produce staphylococcal enterotoxin A. Besides, the production of more than one type of enterotoxins by the same strain was observed. Tnase was considered the best marker for enterotoxigenic potential of isolates, although some of them were negative for coagulase and Tnase but positive for enterotoxin production. Therefore, either the use of Tnase to assess Staphylococcus enterotoxigenic potential or the use of simple and easy screening tests for enterotoxin production should receive more attention when evaluating the pathogenic potential of foodborne Staphylococcus strains. Due to the association of both coagulase positive Staphylococcus and coagulase negative Staphylococcus with foodborne disease outbreaks, regulators and industries should pay more attention to enterotoxigenic Staphylococcus rather than focusing only on S. aureus or coagulase positive Staphylococcus. Finally, data found here suggest a high risk of staphylococcal intoxication with the consumption of raw milk or dairy products made from raw milk.

  2. Diversity of Staphylococcus species and prevalence of enterotoxin genes isolated from milk of healthy cows and cows with subclinical mastitis.

    PubMed

    Rall, V L M; Miranda, E S; Castilho, I G; Camargo, C H; Langoni, H; Guimarães, F F; Araújo Júnior, J P; Fernandes Júnior, A

    2014-02-01

    The objectives of this study were to determine the occurrence and diversity of Staphylococcus spp. in milk from healthy cows and cows with subclinical mastitis in Brazil and to examine the profile of enterotoxin genes and some enterotoxins produced by Staphylococcus spp. A total of 280 individual mammary quarter milk samples from 70 healthy cows and 292 samples from 73 cows with subclinical mastitis were collected from 11 farms in the state of São Paulo, Brazil. Staphylococcus spp. were recovered from 63 (22.5%) samples from healthy cows and from 80 samples (27.4%) from cows with mastitis. The presence of Staphylococcus aureus was significantly different between these 2 groups and was more prevalent in the cows with mastitis. The presence of Staphylococcus saprophyticus was also significantly different between these 2 groups, but this organism was more prevalent in healthy cows. No statistically significant differences were observed in the numbers of other staphylococci in milk samples from the 2 groups. The sea gene was the most prevalent enterotoxin gene in both groups. Eight of 15 (53.3%) Staph. aureus carried this gene and all produced the SEA toxin. In the coagulase-negative staphylococci (CNS) group, 61 of 128 (47.5%) had the same gene and just 1 (1.6%) Staphylococcus epidermidis strain produced the enterotoxin in vitro. Because CNS were isolated from both groups of cows and most CNS contained enterotoxin genes but did not produce toxins, the role of CNS in mastitis should be carefully defined.

  3. Comparative Bioinformatics and Experimental Analysis of the Intergenic Regulatory Regions of Bacillus cereus hbl and nhe Enterotoxin Operons and the Impact of CodY on Virulence Heterogeneity

    PubMed Central

    Böhm, Maria-Elisabeth; Krey, Viktoria M.; Jeßberger, Nadja; Frenzel, Elrike; Scherer, Siegfried

    2016-01-01

    Bacillus cereus is a food contaminant with greatly varying enteropathogenic potential. Almost all known strains harbor the genes for at least one of the three enterotoxins Nhe, Hbl, and CytK. While some strains show no cytotoxicity, others have caused outbreaks, in rare cases even with lethal outcome. The reason for these differences in cytotoxicity is unknown. To gain insight into the origin of enterotoxin expression heterogeneity in different strains, the architecture and role of 5′ intergenic regions (5′ IGRs) upstream of the nhe and hbl operons was investigated. In silico comparison of 142 strains of all seven phylogenetic groups of B. cereus sensu lato proved the presence of long 5′ IGRs upstream of the nheABC and hblCDAB operons, which harbor recognition sites for several transcriptional regulators, including the virulence regulator PlcR, redox regulators ResD and Fnr, the nutrient-sensitive regulator CodY as well as the master regulator for biofilm formation SinR. By determining transcription start sites, unusually long 5′ untranslated regions (5′ UTRs) upstream of the nhe and hbl start codons were identified, which are not present upstream of cytK-1 and cytK-2. Promoter fusions lacking various parts of the nhe and hbl 5′ UTR in B. cereus INRA C3 showed that the entire 331 bp 5′ UTR of nhe is necessary for full promoter activity, while the presence of the complete 606 bp hbl 5′ UTR lowers promoter activity. Repression was caused by a 268 bp sequence directly upstream of the hbl transcription start. Luciferase activity of reporter strains containing nhe and hbl 5′ IGR lux fusions provided evidence that toxin gene transcription is upregulated by the depletion of free amino acids. Electrophoretic mobility shift assays showed that the branched-chain amino acid sensing regulator CodY binds to both nhe and hbl 5′ UTR downstream of the promoter, potentially acting as a nutrient-responsive roadblock repressor of toxin gene transcription. Plc

  4. Isolation, purification, and partial characterization of an enterotoxin from extracts of Entamoeba histolytica trophozoites.

    PubMed Central

    Feingold, C; Bracha, R; Wexler, A; Mirelman, D

    1985-01-01

    Soluble cell-free extracts of pathogenic Entamoeba histolytica, as well as serum-free minimal media in which trophozoites are incubated, contain substances that cause the rapid rounding up and detachment of tissue-cultured monolayers of mammalian cells (cytopathic activity) and induce fluid secretion in ligated intestinal loops of indomethacin-pretreated rats (enterotoxic activity). A semiquantitative assay for the determination of the cytopathic activity based on the rate of detachment of tissue-cultured baby hamster kidney cells was developed. Two peaks containing cytopathic activity were obtained upon gel filtration of the soluble extracts: peak I, with over 60% of the activity, emerged in the 30,000 to 50,000 molecular weight region, and peak II, containing the remaining activity, was in the 15,000 to 25,000 molecular weight region. The activity of peak I was found to be heat labile and inhibited by sialoglycoproteins such as fetuin and mucin (5 mg/ml), as well as by sialic acid. Protease inhibitors such as antitrypsin, pepstatin, phenylmethylsulfonyl fluoride, metaloprotease inhibitors, and bacitracin had no effect on the cytopathic activity. Marked inhibition of cytopathic activity was observed, however, with iodoacetamide and p-chloromercuribenzoate, which affect sulfhydryl groups. The toxic material in peak II was found to have ionophoric activity and was not inhibited by sialic acid-containing compounds. The materials from both peaks had enterotoxic activity in intestinal ligated loops. The active substance from peak I was further purified (200X) on an agarose-fetuin affinity column, yielding one major protein band with an apparent molecular weight of ca. 30,000 on sodium dodecyl sulfate. Amino acid analysis revealed that the protein was very poor in sulfur amino acids. The sialic acid-sensitive toxic activity was higher in known virulent strains such as HM-1:IMSS and could be markedly augmented after preincubation of the trophozoites with certain

  5. Staphylococcal enterotoxin A gene-carrying Staphylococcus aureus isolated from foods and its control by crude alkaloid from papaya leaves.

    PubMed

    Handayani, Lita; Faridah, Didah Nur; Kusumaningrum, Harsi D

    2014-11-01

    Staphylococcus aureus is a known pathogen causing intoxication by producing enterotoxins in food. Staphylococcal enterotoxin A is one of the enterotoxins commonly implicated in staphylococcal food poisoning. The ability of crude alkaloid extract from papaya leaves to inhibit the growth of S. aureus and staphylococcal enterotoxin A synthesis was investigated. Staphylococcal enterotoxin A gene-carrying S. aureus was isolated from raw milk and ready-to-eat foods. Crude alkaloid was extracted from ground, dried papaya leaves using ultrasonic-assisted extraction, and a MIC of the alkaloid was determined by the broth macrodilution method. Furthermore, S. aureus isolate was exposed to the crude alkaloid extract at one- and twofold MIC, and the expression of sea was subsequently analyzed using a quantitative reverse transcription real-time PCR. Ten isolates of S. aureus were obtained, and nine of those isolates were sea carriers. The yield of crude alkaloid extract was 0.48 to 1.82% per dry weight of papaya leaves. A MIC of crude alkaloid to S. aureus was 0.25 mg/ml. After exposure to the alkaloid at 0.25 and 0.5 mg/ml for 2 h, a significant increase in cycle threshold values of sea was observed. The sea was expressed 29 and 41 times less when S. aureus was exposed to crude alkaloid at one- and twofold MIC, respectively. This study revealed that crude alkaloid of papaya leaves could control staphylococcal enterotoxin A gene-carrying S. aureus by suppressing the expression of sea, in addition to the ability to inhibit the growth of S. aureus. The expression of sea was successfully quantified.

  6. Development of IgY based sandwich ELISA for the detection of staphylococcal enterotoxin G (SEG), an egc toxin.

    PubMed

    Nagaraj, Sowmya; Ramlal, Shylaja; Kingston, Joseph; Batra, Harsh Vardhan

    2016-11-21

    Staphylococcal food poisoning (SFP) is a major foodborne illness caused by staphylococcal enterotoxins (SEs). It is a well known fact that foodborne outbreak investigations are solely characterized by commercially available immunoassay kits. However, these assays encompass only few enterotoxins such as SEA-SEE which are renowned as "classical" enterotoxins and unable to detect any other novel enterotoxins even though their involvement is predicted. In this context, the present study involved development of a sandwich ELISA immunoassay for the specific detection of "non-classical" enterotoxin G (SEG). The toxin belongs to enterotoxin gene cluster (egc) which comprises a bunch of five toxin genes that are known to co-express. Thus, the developed assay might indirectly speculate the presence of other toxins in the cluster. The efficiency of ELISA was compared with PCR analysis where all strains possessing seg were found positive for toxin production. Additionally, analogous to other studies which reported the co-occurrence of seg and sei, the PCR analysis accomplished in the study evinced the same. The sandwich format allowed sensitive detection with a detection limit of 1ng/mL. High specificity was achieved in presence of non-target protein as well as bacteria. Likewise, staphylococcal protein A (SpA) interference that is inevitably associated with immunoassays was eliminated by implementation of anti-SEG IgY in our study. Consequently, chicken IgY were used to capture target antigen in developed sandwich ELISA. Further, spiking studies and analysis on natural samples emphasized the robustness as well as applicability of developed method. Altogether, the established assay could be a reliable detection tool for the routine investigation of SEG as well as to predict other egc toxins in samples from food and clinical sources.

  7. Uncoupling of T cell receptor zeta chain function during the induction of anergy by the superantigen, staphylococcal enterotoxin A.

    PubMed

    Cornwell, William D; Rogers, Thomas J

    2010-07-01

    Staphylococcus aureus enterotoxins have immunomodulatory properties. In this study, we show that Staphylococcal enterotoxin A (SEA) induces a strong proliferative response in a murine T cell clone independent of MHC class II bearing cells. SEA stimulation also induces a state of hypo-responsiveness (anergy). We characterized the components of the T cell receptor (TCR) during induction of anergy by SEA. Most interestingly, TCR zeta chain phosphorylation was absent under SEA anergizing conditions, which suggests an uncoupling of zeta chain function. We characterize here a model system for studying anergy in the absence of confounding costimulatory signals.

  8. Rapid Cell-Based Assay for Detection and Quantification of Active Staphylococcal Enterotoxin Type D.

    PubMed

    Rasooly, Reuven; Do, Paula M; Hernlem, Bradley J

    2017-03-01

    Food poisoning by Staphylococcus aureus is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by this bacterium and is a major source of foodborne illness. Staphylococcal enterotoxin D (SED) is one of the predominant enterotoxins recovered in Staphylococcal food poisoning incidences, including a recent outbreak in Guam affecting 300 children. Current immunology methods for SED detection cannot distinguish between the biologically active form of the toxin, which poses a threat, from the inactive form, which poses no threat. In vivo bioassays that measure emetic activity in kitten and monkeys have been used, but these methods rely upon expensive procedures using live animals and raising ethical concerns. A rapid (5 h) quantitative bioluminescence assay, using a genetically engineered T-cell Jurkat cell line expressing luciferase under regulation of nuclear factor of activated T cells response elements, in combination with the lymphoblastoid B-cell line Raji for antigen presentation, was developed. In this assay, the detection limit of biologically active SED is 100 ng/mL, which is 10 times more sensitive than the splenocyte proliferation assay, and 10(5) times more sensitive than monkey or kitten bioassay. Pasteurization or repeated freeze-thaw cycles had no effect on SED activity, but reduction in SED activity was shown with heat treatment at 100°C for 5 min. It was also shown that milk exhibits a protective effect on SED. This bioluminescence assay may also be used to rapidly evaluate antibodies to SED for potential therapeutic application as a measurement of neutralizing biological effects of SED.

  9. Prevalence of staphylococcal enterotoxins in Staphylococcus pseudintermedius isolates from dogs with pyoderma and healthy dogs.

    PubMed

    Tanabe, Taishi; Toyoguchi, Midori; Hirano, Fumitaka; Chiba, Mei; Onuma, Kenta; Sato, Hisaaki

    2013-09-01

    To investigate the role of staphylococcal enterotoxins (SEs) produced by Staphylococcus pseudintermedius in the pathogenesis of pyoderma, isolates from dogs with pyoderma and healthy dogs were analyzed. According to reverse passive latex agglutination, 14/184 isolates (7.6%) from dogs with pyoderma and 9/87 (10.3%) from healthy dogs produced SEs (SEA, SEC or SED). According to multiplex PCR, 99 isolates (53.7%) from dogs with pyoderma and 97 (90.8%) from healthy dogs possessed one or more se genes. There was no significant difference regarding ses between dogs with pyoderma and healthy dogs. Therefore, SEs may not be a direct virulence factor in pyoderma.

  10. The Clostridium perfringens enterotoxin from equine isolates; its characterization, sequence and role in foal diarrhoea.

    PubMed Central

    Netherwood, T.; Binns, M.; Townsend, H.; Wood, J. L.; Mumford, J. A.; Chanter, N.

    1998-01-01

    During a survey of foal diarrhoea between 1991 and 1994, Clostridium perfringens was significantly associated with disease with 56% of cases infected [1]. The contribution of enterotoxigenic C. perfringens to this association, was assessed by use of the reverse passive latex agglutination test for enterotoxin (RPLA; Oxoid Unipath) and vero cell toxicity neutralized by antitoxin on stored faecal samples and sporulated faecal isolates of C. perfringens. Polymerase chain reaction (PCR1) based on the DNA sequence for the whole enterotoxin gene [2] yielded a fragment from an equine isolate of the anticipated size which, cloned into plasmid M13 phage, had a sequence essentially identical to the published sequence. Consequently, all faecal isolates were also tested by PCR1 and for a part of the enterotoxin gene (PCR2). Significant association with diarrhoea (controls not in contact with cases) was found with positive RPLA tests on faeces (OR = 13, P = 0.002) and isolates (OR = 4.57, P = < 0.0001), vero cell toxicity of isolates (OR = 1.78, P = 0.026), and PCR1 (OR = nd, P = 0.029) but not PCR2 or vero cell toxicity of faeces. Significant association with diarrhoea was also found for isolates negative by RPLA (OR = 3.91; CI 2.05-7.57; P < 0.0001) or PCR1 (OR = 4.81; CI 2.84-8.20; P < 0.0001). Many of the isolates from RPLA positive faeces and verotoxic isolates were PCR negative and no evidence could be found for the presence of the enterotoxin gene in a random selection of RPLA positive/PCR negative isolates by gene probe on chromosomal DNA and PCR reaction product or vero cell toxicity neutralized by specific antiserum. Failure of the vero cell toxicity on faeces to be associated with diarrhoea or for cytotoxicity of cultures and RPLA on cultures to agree with the PCRs was believed to be related to the presence of other cytotoxins, the inherent cytotoxicity of equine faeces and to the poor specificity of the commercial antiserum used in the test. Enterotoxigenic C

  11. Assay of Blood and Synovial Fluid of Patients With Rheumatoid Arthritis for Staphylococcus aureus Enterotoxin D: Absence of Bacteria But Presence of Its Toxin

    PubMed Central

    Ataee, Ramezan Ali; Kashefi, Reyhane; Alishiri, Gholam Hossein; Esmaieli, Davoud

    2015-01-01

    Background: Rheumatoid arthritis (RA) is the most common chronic inflammatory disease. The staphylococcal superantigens are considered as the causative agent of RA disease. Objectives: This study aimed to assess the presence of staphylococcal enterotoxin D in synovial fluid and blood of patients with RA. Patients and Methods: A total of 120 blood and SF samples of patients with RA were studied. Bacterial culture, primer pairs design, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) methods have been used to assess of the staphylococcal enterotoxin D. The data were analyzed through descriptive statistics. Results: During this study and after sequential subcultures, only 5 bacterial strains were isolated. The results of PCR showed the presence of staphylococcal enterotoxin D gene in almost 50% of SF and also in 48.4% of blood samples of patients with RA. Similarly, the ELISA method detected staphylococcal enterotoxin D in 36.16% of SF and in 33.33% of blood of patients with RA. Conclusions: The result of this study showed that a high percentage of patients with RA have shown staphylococcal enterotoxin D (superantigen D) or entD gene in SF and in blood. However, the origin of this superantigen was not clarified and no Staphylococcus aureus enterotoxin D producer was isolated. This finding indicates other role of this superantigen besides its intoxication. Therefore, staphylococcal enterotoxin D as a biomarker may provide a good model for the diagnosis and treatment of patients with RA. PMID:26870313

  12. Molecular homogeneity of heat-stable enterotoxins produced by bovine enterotoxigenic Escherichia coli.

    PubMed Central

    Saeed, A M; Magnuson, N S; Sriranganathan, N; Burger, D; Cosand, W

    1984-01-01

    Heat-stable enterotoxins (STs) from four strains of bovine enterotoxigenic Escherichia coli representing four serogroups were purified to homogeneity by utilizing previously published purification schemata. Biochemical characterization of the purified STs showed that they met the basic criteria for the heat-stable enterotoxins of E. coli. Amino acid analysis of the purified STs revealed that they were peptides of identical amino acid composition. This composition consisted of 18 residues of 10 different amino acids, 6 of which were cysteine. The amino acid composition of the four ST peptides was identical to that reported for the STs of human and porcine E. coli. In addition, complete sequence analysis of two of the ST peptides and partial sequencing of several others revealed strong homology to the sequences of STs from human and porcine E. coli and to the sequence predicted from the last 18 codons of the transposon Tn1681. There was also substantial homology to the sequence predicted from the ST-coding genetic element of human E. coli, which may indicate the existence of identical bioactive configuration among ST peptides of E. coli strains of various host origins. These data support the hypothesis that STs produced by human, bovine, and porcine E. coli are coded by a closely related genetic element which may have originated from a single, widely disseminated transposon. Images PMID:6376355

  13. Impedance Analysis of Ovarian Cancer Cells upon Challenge with C-terminal Clostridium Perfringens Enterotoxin

    NASA Astrophysics Data System (ADS)

    Gordon, Geoffrey; Lo, Chun-Min

    2007-03-01

    Both in vitro and animal studies in breast, prostate, and ovarian cancers have shown that clostridium perfringens enterotoxin (CPE), which binds to CLDN4, may have an important therapeutic benefit, as it is rapidly cytotoxic in tissues overexpressing CLDN4. This study sought to evaluate the ability of C-terminal clostridium perfringens enterotoxin (C-CPE), a CLDN4-targetting molecule, to disrupt tight junction barrier function. Electric cell-substrate impedance sensing (ECIS) was used to measure both junctional resistance and average cell-substrate separation of ovarian cancer cell lines after exposure to C-CPE. A total of 14 ovarian cancer cell lines were used, and included cell lines derived from serous, mucinous, and clear cells. Our results showed that junctional resistance increases as CLDN4 expression increases. In addition, C-CPE is non-cytotoxic in ovarian cancer cells expressing CLDN4. However, exposure to C-CPE results in a significant (p<0.05) dose- and CLDN4-dependent decrease in junctional resistance and an increase in cell-substrate separation. Treatment of ovarian cancer cell lines with C-CPE disrupts tight junction barrier function.

  14. Frequency and cytokine phenotype of blood T cells from premature infants responding to staphylococcal enterotoxin B.

    PubMed

    Hayward, A R; Cosyns, M; Zhang, Y

    1995-04-01

    The responder cell frequency (RCF) of premature (< 1900 g birth weight) infants' blood lymphocytes, which proliferate in cultures stimulated by staphylococcal enterotoxin B, falls from 1:3400 to about 1:8000 during the first 2 wk of life. Term infants, in contrast, show no fall in RCF. The reduced RCF in the premature infants affected cells that make interferon-gamma more than cells making IL-4. The reduced RCF was accompanied by a fall in the fraction of V beta 3+ T cells that entered cell cycle in stimulated cultures. The RCF of premature infants' T cells was increased in cultures supplemented with irradiated monocytes from adults. Addition of IL-4 (but not IL-2, IL-6, or indomethacin) increased the RCF and fraction of cells entering cell cycle of the premature infants. The data suggest that postnatal environmental factors limit the ability of premature infants' monocytes to support a T-cell response to staphylococcal enterotoxin B in vitro and that this limitation is overcome by adding IL-4.

  15. Crystal structure of the ADP-ribosylating component of BEC, the binary enterotoxin of Clostridium perfringens.

    PubMed

    Kawahara, Kazuki; Yonogi, Shinya; Munetomo, Ryota; Oki, Hiroya; Yoshida, Takuya; Kumeda, Yuko; Matsuda, Shigeaki; Kodama, Toshio; Ohkubo, Tadayasu; Iida, Tetsuya; Nakamura, Shota

    2016-11-11

    Binary enterotoxin of Clostridium perfringens (BEC), consisting of the components BECa and BECb, was recently identified as a novel enterotoxin produced by C. perfringens that causes acute gastroenteritis in humans. Although the detailed mechanism of cell intoxication by BEC remains to be defined, BECa shows both NAD(+)-glycohydrolase and actin ADP-ribosyltransferase activities in the presence of NAD(+). In this study, we determined the first crystal structure of BECa in its apo-state and in complex with NADH. The structure of BECa shows striking resemblance with other binary actin ADP-ribosylating toxins (ADPRTs), especially in terms of its overall protein fold and mechanisms of substrate recognition. We present a detailed picture of interactions between BECa and NADH, including bound water molecules located near the C1'-N glycosidic bond of NADH and the catalytically important ADP-ribosylating turn-turn (ARTT) loop. We observed that the conformational rearrangement of the ARTT loop, possibly triggered by a conformational change involving a conserved tyrosine residue coupled with substrate binding, plays a crucial role in catalysis by properly positioning a catalytic glutamate residue in the E-X-E motif of the ARTT loop in contact with the nucleophile. Our results for BECa provide insight into the common catalytic mechanism of the family of binary actin ADPRTs.

  16. [Staphylococcus aureus enterotoxin A detection using the polymerase chain reaction (PCR) and its correlation with coagulase and thermonuclease tests].

    PubMed

    Suarez, María José; Arias, María Laura; del Mar Gamboa, María

    2008-03-01

    Staphylococcus aureus is a pathogenic bacterium, widely distributed on nature and associated to general infection and food borne outbreaks. The relationship between this bacterium and food borne outbreaks has been done, historically, using several tests, including coagulase, thermonuclease and actually, PCR for the genes codifying for the enterotoxin responsible of clinical symptoms. The objective of this work is to detect enterotoxin A codifying gene through PCR in a group of S. aureus strains isolated from food samples, and also to correlate the presence of this gene with the production of coagulase and thermonuclease enzymes. A total of 69 staphylococcal strains were analyzed, 58 obtained from non pasteurized milk samples from the Estación Experimental Alfredo Volio Mata and 11 from the Food and Water Microbiology Laboratory collection, Universidad de Costa Rica. Coagulase, thermonuclease and enterotoxin A were analyzed in all the strains, and a statistical correlation was performed in order to verify possible associations. Results show that there is no correlation between the three variables, nevertheless, all coagulase positive strains were thermonuclease positive, and all enterotoxin positive strains were coagulase and thermonuclease positive, but not inversely. These results show that the use of presumptive or indirect tests for establishing entorotoxigenity of S. aureus strains is not truthful, more sensible and specific analysis, as PCR, shall be performed.

  17. A probability model for enterotoxin production of Bacillus cereus as a function of pH and temperature

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacillus cereus is frequently isolated from a variety of foods including vegetables, dairy products, meat, and other raw and processed foods. The bacterium is capable of producing enterotoxin and emetic toxin that can cause severe nausea, vomiting and diarrhea. The objectives of this study were to a...

  18. Ocurrence of Staphylococcus aureus and multiplex pcr detection of classic enterotoxin genes in cheese and meat products

    PubMed Central

    Pelisser, Marcia Regina; Klein, Cátia Silene; Ascoli, Kelen Regina; Zotti, Thaís Regina; Arisi, Ana Carolina Maisonnave

    2009-01-01

    Multiplex PCR was used to investigate the presence of enterotoxins genes (sea, seb, sec, sed and see) and femA gene (specific for Staphylococcus aureus) in coagulase-positive staphylococci (CPS) isolated from cheese and meat products. From 102 CPS isolates, 91 were positive for femA, 10 for sea, 12 for sed and four for see. PMID:24031334

  19. Peracetic acid: a practical agent for sterilizing heat-labile polymeric tissue-engineering scaffolds.

    PubMed

    Yoganarasimha, Suyog; Trahan, William R; Best, Al M; Bowlin, Gary L; Kitten, Todd O; Moon, Peter C; Madurantakam, Parthasarathy A

    2014-09-01

    Advanced biomaterials and sophisticated processing technologies aim at fabricating tissue-engineering scaffolds that can predictably interact within a biological environment at the cellular level. Sterilization of such scaffolds is at the core of patient safety and is an important regulatory issue that needs to be addressed before clinical translation. In addition, it is crucial that meticulously engineered micro- and nano- structures are preserved after sterilization. Conventional sterilization methods involving heat, steam, and radiation are not compatible with engineered polymeric systems because of scaffold degradation and loss of architecture. Using electrospun scaffolds made from polycaprolactone, a low melting polymer, and employing spores of Bacillus atrophaeus as biological indicators, we compared ethylene oxide, autoclaving and 80% ethanol to a known chemical sterilant, peracetic acid (PAA), for their ability to sterilize as well as their effects on scaffold properties. PAA diluted in 20% ethanol to 1000 ppm or above sterilized electrospun scaffolds in 15 min at room temperature while maintaining nano-architecture and mechanical properties. Scaffolds treated with PAA at 5000 ppm were rendered hydrophilic, with contact angles reduced to 0°. Therefore, PAA can provide economical, rapid, and effective sterilization of heat-sensitive polymeric electrospun scaffolds that are used in tissue engineering.

  20. Inhibition of Cronobacter sakazakii by heat labile bacteriocins produced by probiotic LAB isolated from healthy infants.

    PubMed

    Awaisheh, Saddam S; Al-Nabulsi, Anas A; Osaili, Tareq M; Ibrahim, Salam; Holley, Richard

    2013-09-01

    Cronobacter sakazakii is an opportunistic pathogen that can cause bacteremia, meningitis, and necrotizing enterocolitis, most often in neonates with case-fatality rates that may reach 80%. The antimicrobial activity of lactic acid bacteria against a wide range of foodborne pathogens is well-established in different types of food products. The objective of the current study was to investigate the antibacterial activity of Lactobacillus acidophilus and L. casei isolated from feces of healthy infants against different strains of C. sakazakii in agar and a rehydrated infant milk formula (RIMF) model. The inhibition zones of C. sakazakii around L. acidophilus or L. casei ranged from 22 to 32 mm on eMan Rogosa Sharpe (MRS) agar under aerobic conditions, while a slight reduction in antibacterial activity was noted on modified MRS (0.2% glucose) under anaerobic conditions. It was observed that pH-neutralized cell-free supernatant (CFS) of L. acidophilus or L. casei was inhibitory against tested C. sakazakii strains. The inhibition zones of neutralized CFS were lower than the antibacterial activities of live cultures. The antibacterial activity of CFS was abolished when CFS from L. acidophilus or L. casei was heated at 60 or 80 °C for either 10 min or 2 h, or treated with trypsin or pepsin. This was considered strong evidence that the inhibition was due to the production of bacteriocins by L. casei and L. acidophilus. Both the CFS and active growing cells of L. casei and L. acidophilus were able to reduce the viability of C. sakazakii in the RIMF model. The results may extend the use of natural antimicrobials instead of conventional preservation methods to improve the safety of RIMF.

  1. ANIMAL ENTEROTOXIGENIC ESCHERICHIA COLI

    PubMed Central

    Dubreuil, J. Daniel; Isaacson, Richard E.; Schifferli, Dieter M.

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC) is the most common cause of E. coli diarrhea in farm animals. ETEC are characterized by the ability to produce two types of virulence factors; adhesins that promote binding to specific enterocyte receptors for intestinal colonization and enterotoxins responsible for fluid secretion. The best-characterized adhesins are expressed in the context of fimbriae, such as the F4 (also designated K88), F5 (K99), F6 (987P), F17 and F18 fimbriae. Once established in the animal small intestine, ETEC produces enterotoxin(s) that lead to diarrhea. The enterotoxins belong to two major classes; heat-labile toxin that consist of one active and five binding subunits (LT), and heat-stable toxins that are small polypeptides (STa, STb, and EAST1). This chapter describes the disease and pathogenesis of animal ETEC, the corresponding virulence genes and protein products of these bacteria, their regulation and targets in animal hosts, as well as mechanisms of action. Furthermore, vaccines, inhibitors, probiotics and the identification of potential new targets identified by genomics are presented in the context of animal ETEC. PMID:27735786

  2. A Review of the Methods for Detection of Staphylococcus aureus Enterotoxins

    PubMed Central

    Wu, Shijia; Duan, Nuo; Gu, Huajie; Hao, Liling; Ye, Hua; Gong, Wenhui; Wang, Zhouping

    2016-01-01

    Food safety has attracted extensive attention around the world, and food-borne diseases have become one of the major threats to health. Staphylococcus aureus is a major food-borne pathogen worldwide and a frequent contaminant of foodstuffs. Staphylococcal enterotoxins (SEs) produced by some S. aureus strains will lead to staphylococcal food poisoning (SFP) outbreaks. The most common symptoms caused by ingestion of SEs within food are nausea, vomiting, diarrhea and cramps. Children will suffer SFP by ingesting as little as 100 ng of SEs, and only a few micrograms of SEs are enough to cause SPF in vulnerable populations. Therefore, it is a great challenge and of urgent need to detect and identify SEs rapidly and accurately for governmental and non-governmental agencies, including the military, public health departments, and health care facilities. Herein, an overview of SE detection has been provided through a comprehensive literature survey. PMID:27348003

  3. Cure and curse: E. coli heat-stable enterotoxin and its receptor guanylyl cyclase C.

    PubMed

    Weiglmeier, Philipp R; Rösch, Paul; Berkner, Hanna

    2010-09-01

    Enterotoxigenic Escherichia coli (ETEC) associated diarrhea is responsible for roughly half a million deaths per year, the majority taking place in developing countries. The main agent responsible for these diseases is the bacterial heat-stable enterotoxin STa. STa is secreted by ETEC and after secretion binds to the intestinal receptor guanylyl cyclase C (GC-C), thus triggering a signaling cascade that eventually leads to the release of electrolytes and water in the intestine. Additionally, GC-C is a specific marker for colorectal carcinoma and STa is suggested to have an inhibitory effect on intestinal carcinogenesis. To understand the conformational events involved in ligand binding to GC-C and to devise therapeutic strategies to treat both diarrheal diseases and colorectal cancer, it is paramount to obtain structural information on the receptor ligand system. Here we summarize the currently available structural data and report on physiological consequences of STa binding to GC-C in intestinal epithelia and colorectal carcinoma cells.

  4. Toward single molecule detection of staphylococcal enterotoxin B: mobile sandwich immunoassay on gliding microtubules.

    PubMed

    Soto, Carissa M; Martin, Brett D; Sapsford, Kim E; Blum, Amy Szuchmacher; Ratna, Banahalli R

    2008-07-15

    An immunoassay based on gliding microtubules (MTs) is described for the detection of staphylococcal enterotoxin B. Detection is performed in a sandwich immunoassay format. Gliding microtubules carry the antigen-specific "capture" antibody, and bound analyte is detected using a fluorescent viral scaffold as the tracer. A detailed modification scheme for the MTs postpolymerization is described along with corresponding quantification by fluorescence spectroscopy. The resultant antibody-MTs maintain their morphology and gliding capabilities. We report a limit of detection down to 0.5 ng/mL during active transport in a 30 min assay time and down to 1 ng/mL on static surfaces. This study demonstrates the kinesin/MT-mediated capture, transport, and detection of the biowarfare agent SEB in a microfluidic format.

  5. Radiolabeled Escherichia coli heat-stable enterotoxin analogs for in vivo imaging of colorectal cancer

    NASA Astrophysics Data System (ADS)

    Giblin, M. F.; Sieckman, G. L.; Owen, N. K.; Hoffman, T. J.; Forte, L. R.; Volkert, W. A.

    2005-12-01

    The human Escherichia coli heat-stable enterotoxin (STh, amino acid sequence N1SSNYCCELCCNPACTGCY19) binds specifically to the guanylate cyclase C (GC-C) receptor, which is present in high density on the apical surface of normal intestinal epithelial cells as well as on the surface of human colon cancer cells. In the current study, two STh analogs were synthesized and evaluated in vitro and in vivo. Both analogs shared identical 6-19 core sequences, and had N-terminal pendant DOTA moieties. The analogs differed in the identity of a 6 amino acid peptide sequence intervening between DOTA and the 6-19 core. In one analog, the peptide was an RGD-containing sequence found in human fibronectin (GRGDSP), while in the other this peptide sequence was randomly scrambled (GRDSGP). The results indicated that the presence of the human fibronectin sequence in the hybrid peptide did not affect tumor localization in vivo.

  6. Staphylococcus aureus enterotoxins A and B inhibit human and mice eosinophil chemotaxis and adhesion in vitro.

    PubMed

    Squebola-Cola, Dalize M; De Mello, Glaucia C; Anhê, Gabriel F; Condino-Neto, Antonio; DeSouza, Ivani A; Antunes, Edson

    2014-12-01

    Staphylococcus aureus aggravates the allergic eosinophilic inflammation. We hypothesized that Staphylococcus aureus-derived enterotoxins directly affect eosinophil functions. Therefore, this study investigated the effects of Staphylococcal enterotoxins A and B (SEA and SEB) on human and mice eosinophil chemotaxis and adhesion in vitro, focusing on p38 MAPK phosphorylation and intracellular Ca(2+) mobilization. Eosinophil chemotaxis was evaluated using a microchemotaxis chamber, whereas adhesion was performed in VCAM-1 and ICAM-1-coated plates. Measurement of p38 MAPK phosphorylation and intracellular Ca(2+) levels were monitored by flow cytometry and fluorogenic calcium-binding dye, respectively. Prior incubation (30 to 240 min) of human blood eosinophils with SEA (0.5 to 3 ng/ml) significantly reduced eotaxin-, PAF- and RANTES-induced chemotaxis (P<0.05). Likewise, SEB (1 ng/ml, 30 min) significantly reduced eotaxin-induced human eosinophil chemotaxis (P<0.05). The reduction of eotaxin-induced human eosinophil chemotaxis by SEA and SEB was prevented by anti-MHC monoclonal antibody (1 μg/ml). In addition, SEA and SEB nearly suppressed the eotaxin-induced human eosinophil adhesion in ICAM-1- and VCAM-1-coated plates. SEA and SEB prevented the increases of p38 MAPK phosphorylation and Ca(2+) levels in eotaxin-activated human eosinophils. In separate protocols, we evaluated the effects of SEA on chemotaxis and adhesion of eosinophils obtained from mice bone marrow. SEA (10 ng/ml) significantly reduced the eotaxin-induced chemotaxis along with cell adhesion to both ICAM-1 and VCAM-1-coated plates (P<0.05). In conclusion, the inhibition by SEA and SEB of eosinophil functions (chemotaxis and adhesion) are associated with reductions of p38 MAPK phosphorylation and intracellular Ca(2+) mobilization.

  7. Identification and Characterization of a New Enterotoxin Produced by Clostridium perfringens Isolated from Food Poisoning Outbreaks

    PubMed Central

    Suzuki, Yasunori; Nakama, Akiko; Kai, Akemi; Fukui-Miyazaki, Aya; Horiguchi, Yasuhiko; Yoshinari, Tomoya; Sugita-Konishi, Yoshiko; Kamata, Yoichi

    2015-01-01

    There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins’ gene(s) among the Genus Clostridium. PMID:26584048

  8. Short communication: Pasteurization as a means of inactivating staphylococcal enterotoxins A, B, and C in milk.

    PubMed

    Necidova, Lenka; Bogdanovicova, Katerina; Harustiakova, Danka; Bartova, Katerina

    2016-11-01

    Our aim was to assess the effect of pasteurization temperature on inactivation of staphylococcal enterotoxins (SE). Milk samples were inoculated with log 4.38 to 5.18cfu/mL of 40 different Staphylococcus aureus strains having the ability to produce types A, B, or C SE and incubated at 37°C for 24h to develop SE. This incubation was followed by heat treatment for 15 s at 72, 85, and 92°C. Samples were analyzed for Staph. aureus count by plate method and, specifically, for SE presence. An enzyme-linked immunofluorescent assay on a MiniVIDAS analyzer (bioMérieux, Marcy l'Étoile, France) was used to detect SE, which were determined semiquantitatively based on test values. The Staph. aureus count in milk before pasteurization did not affect the amount of SE. Before pasteurization, SEB was detected in the lowest amount compared with other SE types. Staphylococcal enterotoxins were markedly reduced with pasteurization and inactivated at pasteurization temperatures to an extent depending on the amount in the sample before pasteurization. After pasteurization at 72°C, SE were detected in 87.5% of samples (35/40), after pasteurization at 85°C in 52.5% of samples (21/40), and after pasteurization at 92°C in 45.0% of samples (18/40). We determined that SE may still persist in milk even when Staph. aureus bacteria are inactivated through pasteurization. Although pasteurization may partially inactivate SE in milk, a key measure in the prevention of staphylococcal enterotoxicosis linked to pasteurized milk consumption is to avoid any cold chain disruption during milk production and processing.

  9. Acute pulmonary inflammation induced by exposure of the airways to staphylococcal enterotoxin type B in rats

    SciTech Connect

    Desouza, Ivani A. . E-mail: ivanidesouza@fcm.unicamp.br; Franco-Penteado, Carla F.; Camargo, Enilton A.; Lima, Carmen S.P.; Teixeira, Simone A.; Muscara, Marcelo N.; De Nucci, Gilberto; Antunes, Edson

    2006-11-15

    Staphylocococcus aureus is a gram-positive bacterium that produces several enterotoxins, which are responsible for most part of pathological conditions associated to staphylococcal infections, including lung inflammation. This study aimed to investigate the underlying inflammatory mechanisms involved in leukocyte recruitment in rats exposed to staphylococcal enterotoxin B (SEB). Rats were anesthetized with pentobarbital sodium and intratracheally injected with either SEB or sterile phosphate-buffered saline (PBS, 0.4 ml). Airways exposition to SEB (7.5-250 ng/trachea) caused a dose- and time-dependent neutrophil accumulation in BAL fluid, the maximal effects of which were observed at 4 h post-SEB exposure (250 ng/trachea). Eosinophils were virtually absent in BAL fluid, whereas mononuclear cell counts increased only at 24 h post-SEB. Significant elevations of granulocytes in bone marrow (mature and immature forms) and peripheral blood have also been detected. In BAL fluid, marked elevations in the levels of lipid mediators (LTB{sub 4} and PGE{sub 2}) and cytokines (TNF-{alpha}, IL-6 and IL-10) were observed after SEB instillation. The SEB-induced neutrophil accumulation in BAL fluid was reduced by pretreatment with dexamethasone (0.5 mg/kg), the COX-2 inhibitor celecoxib (3 mg/kg), the selective iNOS inhibitor compound 1400 W (5 mg/kg) and the lipoxygenase inhibitor AA-861 (200 {mu}g/kg). In separate experiments carried out with rat isolated peripheral neutrophils, SEB failed to induce neutrophil adhesion to serum-coated plates and chemotaxis. In conclusion, rat airways exposition to SEB causes a neutrophil-dependent lung inflammation at 4 h as result of the release of proinflammatory (NO, PGE{sub 2}, LTB{sub 4}, TNF-{alpha}, IL-6) and anti-inflammatory mediators (IL-10)

  10. Gold nanoparticle-based enhanced chemiluminescence immunosensor for detection of Staphylococcal Enterotoxin B (SEB) in food

    PubMed Central

    Yang, Minghui; Kostov, Yordan; Bruck, Hugh A.; Rasooly, Avraham

    2010-01-01

    Staphylococcal enterotoxins (SEs) are major cause of foodborne diseases, so sensitive detection (<1 ng/ml) methods are needed for SE detection in food. The surface area, geometric and physical properties of gold nanoparticles make them well-suited for enhancing interactions with biological molecules in assays. To take advantage of the properties of gold nanoparticles for immunodetection, we have developed a gold nanoparticle-based enhanced chemiluminescence (ECL) immunosensor for detection of Staphylococcal Enterotoxin B (SEB) in food. Anti-SEB primary antibodies were immobilized onto a gold nanoparticle surface through physical adsorption and then the antibody–gold nanoparticle mixture was immobilized onto a polycarbonate surface. SEB was detected by a “sandwich-type” ELISA assay on the polycarbonate surface with a secondary antibody and ECL detection. The signal from ECL was read using a point-of-care detector based on a cooled charge-coupled device (CCD) sensor or a plate reader. The system was used to test for SEB in buffer and various foods (mushrooms, tomatoes, and baby food meat). The limit of detection was found to be ~0.01 ng/mL, which is ~10 times more sensitive than traditional ELISA. The gold nanoparticles were relatively easy to use for antibody immobilization because of their physical adsorption mechanism; no other reagents were required for immobilization. The use of our simple and inexpensive detector combined with the gold nanoparticle-based ECL method described here is adaptable to simplify and increase sensitivity of any immunological assay and for point-of-care diagnostics. PMID:19540011

  11. Identification and Characterization of a New Enterotoxin Produced by Clostridium perfringens Isolated from Food Poisoning Outbreaks.

    PubMed

    Irikura, Daisuke; Monma, Chie; Suzuki, Yasunori; Nakama, Akiko; Kai, Akemi; Fukui-Miyazaki, Aya; Horiguchi, Yasuhiko; Yoshinari, Tomoya; Sugita-Konishi, Yoshiko; Kamata, Yoichi

    2015-01-01

    There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins' gene(s) among the Genus Clostridium.

  12. Development of a reference material for Staphylococcus aureus enterotoxin A in cheese: feasibility study, processing, homogeneity and stability assessment.

    PubMed

    Zeleny, R; Emteborg, H; Charoud-Got, J; Schimmel, H; Nia, Y; Mutel, I; Ostyn, A; Herbin, S; Hennekinne, J-A

    2015-02-01

    Staphylococcal food poisoning is caused by enterotoxins excreted into foods by strains of staphylococci. Commission Regulation 1441/2007 specifies thresholds for the presence of these toxins in foods. In this article we report on the progress towards reference materials (RMs) for Staphylococcal enterotoxin A (SEA) in cheese. RMs are crucial to enforce legislation and to implement and safeguard reliable measurements. First, a feasibility study revealed a suitable processing procedure for cheese powders: the blank material was prepared by cutting, grinding, freeze-drying and milling. For the spiked material, a cheese-water slurry was spiked with SEA solution, freeze-dried and diluted with blank material to the desired SEA concentration. Thereafter, batches of three materials (blank; two SEA concentrations) were processed. The materials were shown to be sufficiently homogeneous, and storage at ambient temperature for 4weeks did not indicate degradation. These results provide the basis for the development of a RM for SEA in cheese.

  13. Human Leukocyte Antigen-DQ8 Transgenic Mice: A Model to Examine the Toxicity of Aerosolized Staphylococcal Enterotoxin B

    DTIC Science & Technology

    2004-11-30

    enterotoxins: a novel model for superantigen vaccines. J. Infect. Dis. 185:1754–1760. 12. Edling, A. E., S. Choksi, Z . Huang, and R. Korngold. 2001. Effect of...D. L. Hoover, W. R. Bryne , J. A. Pavlin, G. W. Christopher, and E. M. Eitzen, Jr. 1997. Clinical recognition and management of patients exposed to...Gao, T. Satoh, T. M. Friedman, A. E. Edling, U. Koch, S. Choksi, X. Han, R. Korngold, and Z . Huang. 1997. A computer screening approach to

  14. Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity.

    PubMed

    Sharma, P; Postel, S; Sundberg, E J; Kranz, D M

    2013-12-01

    Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection.

  15. Survival and Germination of Bacillus cereus Spores without Outgrowth or Enterotoxin Production during In Vitro Simulation of Gastrointestinal Transit

    PubMed Central

    Ceuppens, Siele; Uyttendaele, Mieke; Drieskens, Katrien; Heyndrickx, Marc; Rajkovic, Andreja; Boon, Nico

    2012-01-01

    To study the gastrointestinal survival and enterotoxin production of the food-borne pathogen Bacillus cereus, an in vitro simulation experiment was developed to mimic gastrointestinal passage in 5 phases: (i) the mouth, (ii) the stomach, with gradual pH decrease and fractional emptying, (iii) the duodenum, with high concentrations of bile and digestive enzymes, (iv) dialysis to ensure bile reabsorption, and (v) the ileum, with competing human intestinal bacteria. Four different B. cereus strains were cultivated and sporulated in mashed potato medium to obtain an inoculum of 7.0 log spores/ml. The spores showed survival and germination during the in vitro simulation of gastrointestinal passage, but vegetative outgrowth of the spores was suppressed by the intestinal bacteria during the final ileum phase. No bacterial proliferation or enterotoxin production was observed, despite the high inoculum levels. Little strain variability was observed: except for the psychrotrophic food isolate, the spores of all strains survived well throughout the gastrointestinal passage. The in vitro simulation experiments investigated the survival and enterotoxin production of B. cereus in the gastrointestinal lumen. The results obtained support the hypothesis that localized interaction of B. cereus with the host's epithelium is required for diarrheal food poisoning. PMID:22923409

  16. Bacillus cereus NVH 0500/00 Can Adhere to Mucin but Cannot Produce Enterotoxins during Gastrointestinal Simulation

    PubMed Central

    Tsilia, Varvara; Kerckhof, Frederiek-Maarten; Heyndrickx, Marc

    2015-01-01

    Adhesion to the intestinal epithelium could constitute an essential mechanism of Bacillus cereus pathogenesis. However, the enterocytes are protected by mucus, a secretion composed mainly of mucin glycoproteins. These may serve as nutrients and sites of adhesion for intestinal bacteria. In this study, the food poisoning bacterium B. cereus NVH 0500/00 was exposed in vitro to gastrointestinal hurdles prior to evaluation of its attachment to mucin microcosms and its ability to produce nonhemolytic enterotoxin (Nhe). The persistence of mucin-adherent B. cereus after simulated gut emptying was determined using a mucin adhesion assay. The stability of Nhe toward bile and pancreatin (intestinal components) in the presence of mucin agar was also investigated. B. cereus could grow and simultaneously adhere to mucin during in vitro ileal incubation, despite the adverse effect of prior exposure to a low pH or intestinal components. The final concentration of B. cereus in the simulated lumen at 8 h of incubation was 6.62 ± 0.87 log CFU ml−1. At that point, the percentage of adhesion was approximately 6%. No enterotoxin was detected in the ileum, due to either insufficient bacterial concentrations or Nhe degradation. Nevertheless, mucin appears to retain B. cereus and to supply it to the small intestine after simulated gut emptying. Additionally, mucin may play a role in the protection of enterotoxins from degradation by intestinal components. PMID:26497468

  17. Bacillus cereus NVH 0500/00 Can Adhere to Mucin but Cannot Produce Enterotoxins during Gastrointestinal Simulation.

    PubMed

    Tsilia, Varvara; Kerckhof, Frederiek-Maarten; Rajkovic, Andreja; Heyndrickx, Marc; Van de Wiele, Tom

    2015-10-23

    Adhesion to the intestinal epithelium could constitute an essential mechanism of Bacillus cereus pathogenesis. However, the enterocytes are protected by mucus, a secretion composed mainly of mucin glycoproteins. These may serve as nutrients and sites of adhesion for intestinal bacteria. In this study, the food poisoning bacterium B. cereus NVH 0500/00 was exposed in vitro to gastrointestinal hurdles prior to evaluation of its attachment to mucin microcosms and its ability to produce nonhemolytic enterotoxin (Nhe). The persistence of mucin-adherent B. cereus after simulated gut emptying was determined using a mucin adhesion assay. The stability of Nhe toward bile and pancreatin (intestinal components) in the presence of mucin agar was also investigated. B. cereus could grow and simultaneously adhere to mucin during in vitro ileal incubation, despite the adverse effect of prior exposure to a low pH or intestinal components. The final concentration of B. cereus in the simulated lumen at 8 h of incubation was 6.62 ± 0.87 log CFU ml(-1). At that point, the percentage of adhesion was approximately 6%. No enterotoxin was detected in the ileum, due to either insufficient bacterial concentrations or Nhe degradation. Nevertheless, mucin appears to retain B. cereus and to supply it to the small intestine after simulated gut emptying. Additionally, mucin may play a role in the protection of enterotoxins from degradation by intestinal components.

  18. Prevalence of enterotoxin genes and antimicrobial resistance of coagulase-positive staphylococci recovered from raw cow milk.

    PubMed

    Rola, J G; Korpysa-Dzirba, W; Czubkowska, A; Osek, J

    2015-07-01

    Raw milk may be contaminated by enterotoxigenic coagulase-positive staphylococci (CPS). Several of these microorganisms show antimicrobial resistance, which poses a potential risk for consumers. The aim of this study was to determine the occurrence of enterotoxin genes and antimicrobial resistance of CPS isolated from cow milk. A total of 115 samples were analyzed for the presence of CPS according to the International Organization for Standardization standard (ISO 6888-2). The genes were identified using 2multiplex PCR assays. Resistance of the isolates to 10 antimicrobials was determined using the minimum inhibitory concentration method. Overall, 71 samples (62%) were contaminated with CPS and 69 isolates were further analyzed. Among them, 20 (29%) strains harbored the enterotoxin genes. The most commonly detected staphylococcal enterotoxin markers were sed, sej, and ser, whereas none of the analyzed isolates possessed the seb and see genes. Almost one-half of the tested strains (43%) were resistant to one or more antimicrobial agents. Resistance to penicillin was the most common, followed by sulfamethoxazole and chloramphenicol. On the other hand, all strains were susceptible to ciprofloxacin, erythromycin, gentamicin, cefoxitin, and streptomycin. None of the strains was positive for the mecA and mecC (methicillin-resistant Staphylococcus aureus) genes. These results indicate that enterotoxigenic and antimicrobial-resistant CPS strains are present in raw milk, which may be a potential risk for public health.

  19. Enterotoxin/guanylin receptor type guanylyl cyclases in non-mammalian vertebrates.

    PubMed

    Nakauchi, Mina; Suzuki, Norio

    2005-05-01

    Cyclic GMP is a ubiquitous intracellular second messenger produced by guanylyl cyclases (GCs). The enterotoxin/guanylin receptor type membrane GC (designated as GC-C in mammals) is activated by exogenous ligands such as heat-stable enterotoxins (STa), small peptides secreted by some pathogenic strains of Escherichia coli which cause severe secretory diarrhea and also activated by endogenous ligands such as guanylin and uroguanylin. The STa/guanylin receptor type membrane GC, as well as other type membrane GCs, is composed of an extracellular domain, a single transmembrane domain, and an intracellular region comprising a kinase-like domain and a catalytic domain. The STa/guanylin receptor type membrane GC is identified in various vertebrates including fishes, amphibians, reptiles, and birds, implying that it serves some important and undefined physiological roles in the intestine of non-mammalian vertebrates, e.g. the regulation of water and salt absorption. In mammals, only a single membrane GC (GC-C) is known to be the STa/guanylin receptor. On the contrary, two membrane GC cDNAs are cloned from the intestine of the European eel Anguilla anguilla (GC-C1 and GC-C2) and the medaka fish Oryzias latipes (OlGC6 and OlGC9). OlGC6 and OlGC9 are structurally distinct and show different ligand responsibility. Accumulated evidences indicate that the transcriptional regulatory mechanism of the human GC-C gene is different from that of the corresponding medaka fish GC gene; the human GC-C gene is regulated by Cdx2 and/or HNF-4, and the medaka fish OlGC6 gene is regulated by OlPC4, which is a medaka fish homologue of the mammalian transcriptional positive co-factor 4 (PC4). Furthermore, the transcriptional regulatory mechanism of the OlGC9 gene is different from those of both the OlGC6 and human GC-C genes, indicating that the study on these two medaka fish GCs will be useful for further understanding of the STa/guanylin receptor type membrane GC in the vertebrates.

  20. Staphylococcus aureus enterotoxins induce IL-8 secretion by human nasal epithelial cells

    PubMed Central

    O'Brien, Garrett J; Riddell, Gareth; Elborn, J Stuart; Ennis, Madeleine; Skibinski, Grzegorz

    2006-01-01

    Background Staphylococcus aureus produces a set of proteins which act both as superantigens and toxins. Although their mode of action as superantigens is well understood, little is known about their effects on airway epithelial cells. Methods To investigate this problem, primary nasal epithelial cells derived from normal and asthmatic subjects were stimulated with staphylococcal enterotoxin A and B (SEA and SEB) and secreted (supernatants) and cell-associated (cell lysates) IL-8, TNF-α, RANTES and eotaxin were determined by specific ELISAs. Results Non-toxic concentrations of SEA and SEB (0.01 μg/ml and 1.0 μg/ml) induced IL-8 secretion after 24 h of culture. Pre-treatment of the cells with IFN-γ (50 IU/ml) resulted in a further increase of IL-8 secretion. In cells from healthy donors pretreated with IFN-γ, SEA at 1.0 μg/ml induced release of 1009 pg/ml IL-8 (733.0–1216 pg/ml, median (range)) while in cells from asthmatic donors the same treatment induced significantly higher IL-8 secretion – 1550 pg/ml (1168.0–2000.0 pg/ml p = 0.04). Normal cells pre-treated with IFN-γ and then cultured with SEB at 1.0 μg/ml released 904.6 pg/ml IL-8 (666.5–1169.0 pg/ml). Cells from asthmatics treated in the same way produced significantly higher amounts of IL-8 – 1665.0 pg/ml (1168.0–2000.0 pg/ml, p = 0.01). Blocking antibodies to MHC class II molecules added to cultures stimulated with SEA and SEB, reduced IL-8 secretion by about 40% in IFN-γ unstimulated cultures and 75% in IFN-γ stimulated cultures. No secretion of TNF-α, RANTES and eotaxin was noted. Conclusion Staphylococcal enterotoxins may have a role in the pathogenesis of asthma. PMID:16952309

  1. [Poisoning by enterotoxin from Staphylococcus aureus associated with mocha pastry. Microbiology and epidemiology].

    PubMed

    Escartín, E F; Saldaña-Lozano, J; Montiel-Falcón, A

    1998-01-01

    A brief description of a foodborne outbreak due to S. aureus enterotoxin associated with the consumption of mocha cake in the city of Guadalajara is presented. The cake was prepared in a bakery and affected nearly 100 persons. S. aureus was isolated from the nose and skin of one of the pastry cooks. A S. aureus strain isolated from the cake involved in the outbreak was not only unable to grow in the mocha cream, but it actually decreased in numbers by 2 log after 72 h of storage at 30 degrees C. The pH of mocha cream ranged from 6.2 to 6.6, and water activity from 0.833 to 0.859, with a media of 0.841. In preparing mocha cake at the shop, one half of the dough used to be sprayed with a sucrose solution in water (20% w/v); mocha cream was spread on the other half of the dough before overlapping the two halves. When mocha cake was prepared in this manner, and stored at 30 degrees C, S. aureus increased in number by more than 4 log after 48 h. S. aureus did not grow in the cake stored at 4-7 degrees C. Contributory factors in this outbreak were an increase of water activity in the interphase of the mocha and the cake dough, storage of the cake in an unrefrigerated area, and an unusually high ambient temperature (28-32 degrees C) at that time.

  2. Amygdaloid signature of peripheral immune activation by bacterial lipopolysaccharide or staphylococcal enterotoxin B.

    PubMed

    Prager, Geraldine; Hadamitzky, Martin; Engler, Andrea; Doenlen, Raphael; Wirth, Timo; Pacheco-López, Gustavo; Krügel, Ute; Schedlowski, Manfred; Engler, Harald

    2013-03-01

    Activated immune cells produce soluble mediators that not only coordinate local and systemic immune responses but also act on the brain to initiate behavioral, neuroendocrine and metabolic adaptations. Earlier studies have shown that the amygdala, a group of nuclei located in the medial temporal lobe, is engaged in the central processing of afferent signals from the peripheral immune system. Here, we compared amygdaloid responses to lipopolysaccharide (LPS) and staphylococcal enterotoxin B (SEB), two prototypic bacterial products that elicit distinct immune responses. Intraperitoneal administration of LPS (0.1 mg/kg) or SEB (1 mg/kg) in adult rats induced substantial increases in amygdaloid neuronal activity as measured by intracerebral electroencephalography and c-fos gene expression. Amygdaloid neuronal activation was accompanied by an increase in anxiety-related behavior in the elevated plus-maze test. However, only treatment with LPS, but not SEB, enhanced amygdaloid IL-1β and TNF-α mRNA expression. This supports the view of the immune system as a sensory organ that recognizes invading pathogens and rapidly relays this information to the brain, independent of the nature of the immune response induced. The observation that neuronal and behavioral responses to peripheral immune challenges are not necessarily accompanied by increased brain cytokine expression suggests that cytokines are not the only factors driving sickness-related responses in the CNS.

  3. [Isolation of Vibrio cholerae in imported frozen seafood and their cholera-enterotoxin production].

    PubMed

    Shiraishi, S; Takeda, K; Taga, K; Hirata, K; Hayashi, K; Honda, T

    1996-02-01

    A survey study for Vibrio cholerae in imported seafood was conducted during January 1991 to December 1994. A total of 7,439 specimens (approximately 20% of all imported food) were randomly picked up and examined for contamination of V. cholerae. Among these, V. cholerae O1 were isolated from 9 specimens, but they were all cholerae enterotoxin (CT)-negative. In terms of V. cholerae non-O1, a total of 2,803 specimens (37.4%) were contaminated with this vibrio. Shrimp, especially the ones still in their shells and imported from Asian countries such as India and Indonesia, were highly contaminated with V. cholerae. Although no strains of V. cholerae O1 isolated in this study produced CT, 2 strains of V. cholerae non-O1 were proved to be CT-producers. Taking together the high contamination of V. cholerae in imported seafood and a part of those strains producing CT, we believe that careful survey for the possible contamination of V. choleare in imported seafood is necessary.

  4. An Automated Point-of-Care System for Immunodetection of Staphylococcal Enterotoxin B

    PubMed Central

    Yang, Minghui; Sun, Steven; Kostov, Yordan; Rasooly, Avraham

    2011-01-01

    An automated point-of-care (POC) immunodetection system for immunological detection of Staphylococcal enterotoxin B (SEB) was designed, fabricated, and tested. The system combines several elements: (1) ELISA-Lab-on-a-chip (ELISA-LOC) with fluidics, (2) a CCD camera detector, (3) pumps and valves for fluid delivery to the ELISA-LOC, (4) a computer interface board, and (5) a computer for controlling the fluidics, logging and data analysis of the CCD data. The ELISA-LOC integrates a simple microfluidics system into a miniature ninety-six well sample plate, allowing the user to carry out immunological assays without a laboratory. The analyte is measured in a sandwich ELISA assay format combined with a sensitive Electrochemiluminescence (ECL) detection method. Using the POC system, SEB, a major foodborne toxin, was detected at concentrations as low as 0.1 ng/ml. This is similar to the reported sensitivity of conventional ELISA. The open platform with simple modular fluid delivery automation design described here is interchangeable between detection systems and because of its versatility it can be also used to automate many other LOC systems, simplifying LOC development. This new point-of-care system is useful for carrying out various immunological and other complex medical assays without a laboratory and can easily be adapted for high throughput biological screening in remote and resource poor areas. PMID:21640067

  5. Detection of genes for heat-stable enterotoxin I in Escherichia coli strains isolated in Brazil.

    PubMed Central

    Maas, R; Silva, R M; Gomes, T A; Trabulsi, L R; Maas, W K

    1985-01-01

    Heat-stable enterotoxin I (STI) can be assayed in intestinal loops of pigs and rabbits and in the gut of infant mice. To produce a simpler and more discriminating assay procedure, we used three gene probes corresponding to three forms of STI called STIa, STIb, and STIc. We tested 159 Brazilian isolates, of which 40 were positive in the infant mouse assay. The STIb and STIc probes are similar (93% DNA homology) and are both different from the STIa probe (70% DNA homology). Of 33 strains that were still active for STI 3 years after their isolation, 25 reacted with both the STIb and STIc probes, 4 reacted with the STIc probe only, and 7 reacted strongly with the STIa probe and weakly or not at all with the other probes. Two strains reacted with all three probes. Further analysis showed that each of these two strains contains a small plasmid that reacts with the STIa probe and a large plasmid that reacts with the STIc probe in one strain and weakly with both the STIa and STIc probes in the other strain. It was also shown that the STIa probe reacts with the cloning vehicle pACYC184 used for the cloning of STIc. We conclude that the gene probes used can identify most STI-producing strains and that in cases of positive responses with several probes careful scrutiny is necessary for analysis. Images PMID:3891629

  6. Application of LC-MS/MS MRM to Determine Staphylococcal Enterotoxins (SEB and SEA) in Milk.

    PubMed

    Andjelkovic, Mirjana; Tsilia, Varvara; Rajkovic, Andreja; De Cremer, Koen; Van Loco, Joris

    2016-04-20

    Staphylococcus aureus is one of the important aetiological agents of food intoxications in Europe and can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Due to their stability and ease of production and dissemination, some SEs have also been studied as potential agents for bioterrorism. Therefore, specific and accurate analytical tools are required to detect and quantify SEs. Online solid-phase extraction liquid chromatography electrospray ionization tandem mass spectrometry (online SPE-LC-ESI-MS/MS) based on multiple reaction monitoring (MRM) was used to detect and quantify two types of SE (A and B) spiked in milk and buffer solution. SE extraction and concentration was performed according to the European Screening Method developed by the European Reference Laboratory for Coagulase Positive Staphylococci. Trypsin digests were screened for the presence of SEs using selected proteotypic heavy-labeled peptides as internal standards. SEA and SEB were successfully detected in milk samples using LC-MS/MS in MRM mode. The selected SE peptides were proteotypic for each toxin, allowing the discrimination of SEA and SEB in a single run. The detection limit of SEA and SEB was approximately 8 and 4 ng/g, respectively.

  7. Further clonal expansion of T cells upon rechallenge of superantigen staphylococcal enterotoxin A.

    PubMed

    Aoki, Y; Yoshikai, Y

    1997-01-01

    Superantigens are known to induce clonal anergy and/or deletion in reactive T cells peripherally. This study was undertaken to investigate the T-cell status early after exposure to staphylococcal enterotoxin A (SEA) in vivo and in vitro. At the peak of clonal expansion following the administration of 5 microg SEA (i.e., 2 days after the injection), C57BL/6 mice were rechallenged with the same dose of SEA in vivo. The secondary stimulation augmented clonal expansion of the T cells bearing Vbeta3 and Vbeta11 in both CD4+ and CD8+ populations. In vitro restimulation of the spleen cells taken from the SEA-primed mice also induced further expansion of the Vbeta3+ T cells during 2 days of culturing, whereas without restimulation, a marked reduction of Vbeta3+ T cells occurred. The spleen cells from the SEA-primed mice were hyper-reactive to in vitro restimulation with SEA as measured by 3H-TdR uptake on day 1 of culturing, but augmented proliferation leveled off thereafter. By day 3, the values of 3H-TdR uptake were less than 20% of those of the controls in which spleen cells from native mice were stimulated with SEA in vitro. These results suggest that T cells exposed to SEA in vivo are still capable of proliferating upon SEA rechallenge, but subsequently, the proliferation starts to wane.

  8. spa typing and enterotoxin gene profile of Staphylococcus aureus isolated from bovine raw milk in Korea

    PubMed Central

    Hwang, Sun Young; Park, Young Kyung

    2010-01-01

    Staphylococcus aureus is a major etiological pathogen of bovine mastitis, which triggers significant economic losses in dairy herds worldwide. In this study, S. aureus strains isolated from the milk of cows suffering from mastitis in Korea were investigated by spa typing and staphylococcal enterotoxin (SE) gene profiling. Forty-four S. aureus strains were isolated from 26 farms in five provinces. All isolates grouped into five clusters and two singletons based on 14 spa types. Cluster 1 and 2 isolates comprised 38.6% and 36.4% of total isolates, respectively, which were distributed in more than four provinces. SE and SE-like toxin genes were detected in 34 (77.3%) isolates and the most frequently detected SE gene profile was seg, sei, selm, seln, and selo genes (16 isolates, 36.3%), which was comparable to one of the genomic islands, Type I νSaβ. This is a first report of spa types and the prevalence of the recently described SE and SE-like toxin genes among S. aureus isolates from bovine raw milk in Korea. Two predominant spa groups were distributed widely and recently described SE and SE-like toxin genes were detected frequently. PMID:20458153

  9. Staphylococcus aureus enterotoxin C2 mutants: biological activity assay in vitro.

    PubMed

    Hui, Jing; Cao, Yan; Xiao, Fang; Zhang, Jin; Li, Hui; Hu, Fengqing

    2008-09-01

    Staphylococcal enterotoxin C2 (SEC2) is one member of bacterial superantigens produced by Staphylococcus aureus. It can be attributed to its superantigenic activity to cross-link major histocompatibility complex class II molecules with T-cell receptors and activate a large number of resting T cells resulting in release of massive cytokines, which will produce significant tumor inhibition in vivo and in vitro. However, it could be not broadly applied to cure malignant tumors in clinic because of emetic activity of SEC2. The aim of this study was to inactivate emetic activity of SEC2 through site-directed mutagenesis. Cys93, Cys110 and His118 were selected as substitutional sites based on the functional sites responsible for emesis. The mutated proteins were used to determine Peripheral blood mononuclear cell proliferation activity and anti-tumor activity in vitro. Results showed that these mutated proteins efficiently stimulated T cell and exhibited the same tumor-inhibition effect as SEC2. It is possible to inactivate emetic activity of SEC2 through site-directed mutagenesis and provide satisfying agents for tumor treatment in clinic.

  10. Evaluation of handheld assays for the detection of ricin and staphylococcal enterotoxin B in disinfected waters.

    PubMed

    Wade, Mary Margaret; Biggs, Tracey D; Insalaco, Joseph M; Neuendorff, Lisa K; Bevilacqua, Vicky L H; Schenning, Amanda M; Reilly, Lisa M; Shah, Saumil S; Conley, Edward K; Emanuel, Peter A; Zulich, Alan W

    2011-01-01

    Development of a rapid field test is needed capable of determining if field supplies of water are safe to drink by the warfighter during a military operation. The present study sought to assess the effectiveness of handheld assays (HHAs) in detecting ricin and Staphylococcal Enterotoxin B (SEB) in water. Performance of HHAs was evaluated in formulated tap water with and without chlorine, reverse osmosis water (RO) with chlorine, and RO with bromine. Each matrix was prepared, spiked with ricin or SEB at multiple concentrations, and then loaded onto HHAs. HHAs were allowed to develop and then read visually. Limits of detection (LOD) were determined for all HHAs in each water type. Both ricin and SEB were detected by HHAs in formulated tap water at or below the suggested health effect levels of 455 ng/mL and 4.55 ng/mL, respectively. However, in brominated or chlorinated waters, LODs for SEB increased to approximately 2,500 ng/mL. LODs for ricin increased in chlorinated water, but still remained below the suggested health effect level. In brominated water, the LOD for ricin increased to approximately 2,500 ng/mL. In conclusion, the HHAs tested were less effective at detecting ricin and SEB in disinfected water, as currently configured.

  11. Stress-caused anergy of leukocytes towards Staphylococcal enterotoxin B and exposure transcriptome signatures.

    PubMed

    Muhie, S; Hammamieh, R; Cummings, C; Yang, D; Jett, M

    2015-01-01

    Leucocytes from soldiers exposed to battlefield-like stress (RASP: Rangers Assessment and Selection Program) were exposed in vitro to Staphylococcal enterotoxin B (SEB). We assayed SEB-induced regulation of gene expression, both in the presence and absence of severe stress, to generate two sets of gene profiles. One set of transcripts and microRNAs were specific to post-RASP SEB exposure, and another set were signatures of SEB exposure common to both the pre- and post-RASP leucocytes. Pathways and upstream regulatory analyses indicated that the post-RASP SEB-signature transcripts were manifestation of the anergic state of post-RASP leucocytes. These were further verified using expression-based predictions of cellular processes and literature searches. Specificity of the second set of transcripts to SEB exposure was verified using machine-learning algorithms on our and four other (Gene Expression Omnibus) data sets. Cell adhesion, coagulation, hypoxia and vascular endothelial growth factor-mediated vascular leakage were SEB-specific pathways even under the background of severe stress. Hsa-miR-155-3p was the top SEB exposure predictor in our data set, and C-X-C motif chemokine ligand 9 was SEB specific in all the analyzed data sets. The SEB-signature transcripts (which also showed distinct expression signatures from Yersinia pestis and dengue virus) may serve as potential biomarkers of SEB exposure even under the background of stress.

  12. Application of LC-MS/MS MRM to Determine Staphylococcal Enterotoxins (SEB and SEA) in Milk

    PubMed Central

    Andjelkovic, Mirjana; Tsilia, Varvara; Rajkovic, Andreja; De Cremer, Koen; Van Loco, Joris

    2016-01-01

    Staphylococcus aureus is one of the important aetiological agents of food intoxications in Europe and can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Due to their stability and ease of production and dissemination, some SEs have also been studied as potential agents for bioterrorism. Therefore, specific and accurate analytical tools are required to detect and quantify SEs. Online solid-phase extraction liquid chromatography electrospray ionization tandem mass spectrometry (online SPE-LC-ESI-MS/MS) based on multiple reaction monitoring (MRM) was used to detect and quantify two types of SE (A and B) spiked in milk and buffer solution. SE extraction and concentration was performed according to the European Screening Method developed by the European Reference Laboratory for Coagulase Positive Staphylococci. Trypsin digests were screened for the presence of SEs using selected proteotypic heavy-labeled peptides as internal standards. SEA and SEB were successfully detected in milk samples using LC-MS/MS in MRM mode. The selected SE peptides were proteotypic for each toxin, allowing the discrimination of SEA and SEB in a single run. The detection limit of SEA and SEB was approximately 8 and 4 ng/g, respectively. PMID:27104569

  13. Humans as Reservoir for Enterotoxin Gene–carrying Clostridium perfringens Type A

    PubMed Central

    Lindström, Miia; Granum, Per Einar; Korkeala, Hannu

    2006-01-01

    We found a prevalence of 18% for enterotoxin gene–carrying (cpe+) Clostridium perfringens in the feces of healthy food handlers by PCR and isolated the organism from 11 of 23 PCR-positive persons by using hydrophobic grid membrane filter-colony hybridization. Several different cpe genotypes were recovered. The prevalence was 3.7% for plasmidial IS1151-cpe, 2.9% for plasmidial IS1470-like-cpe, 0.7% for chromosomal IS1470-cpe, and 1.5% for unknown cpe genotype. Lateral spread of cpe between C. perfringens strains was evident because strains from the same person carried IS1470-like cpe but shared no genetic relatedness according to pulsed-field gel electrophoresis analysis. Our findings suggest that healthy humans serve as a rich reservoir for cpe+ C. perfringens type A and may play a role in the etiology of gastrointestinal diseases caused by this organism. The results also indicate that humans should be considered a risk factor for spread of C. perfringens type A food poisoning and that they are a possible source of contamination for C. perfringens type A food poisoning. PMID:17283623

  14. Synthetic human monoclonal antibodies toward staphylococcal enterotoxin B (SEB) protective against toxic shock syndrome.

    PubMed

    Karauzum, Hatice; Chen, Gang; Abaandou, Laura; Mahmoudieh, Mahta; Boroun, Atefeh R; Shulenin, Sergey; Devi, V Sathya; Stavale, Eric; Warfield, Kelly L; Zeitlin, Larry; Roy, Chad J; Sidhu, Sachdev S; Aman, M Javad

    2012-07-20

    Staphylococcal enterotoxin B (SEB) is a potent toxin that can cause toxic shock syndrome and act as a lethal and incapacitating agent when used as a bioweapon. There are currently no vaccines or immunotherapeutics available against this toxin. Using phage display technology, human antigen-binding fragments (Fabs) were selected against SEB, and proteins were produced in Escherichia coli cells and characterized for their binding affinity and their toxin neutralizing activity in vitro and in vivo. Highly protective Fabs were converted into full-length IgGs and produced in mammalian cells. Additionally, the production of anti-SEB antibodies was explored in the Nicotiana benthamiana plant expression system. Affinity maturation was performed to produce optimized lead anti-SEB antibody candidates with subnanomolar affinities. IgGs produced in N. benthamiana showed characteristics comparable with those of counterparts produced in mammalian cells. IgGs were tested for their therapeutic efficacy in the mouse toxic shock model using different challenge doses of SEB and a treatment with 200 μg of IgGs 1 h after SEB challenge. The lead candidates displayed full protection from lethal challenge over a wide range of SEB challenge doses. Furthermore, mice that were treated with anti-SEB IgG had significantly lower IFNγ and IL-2 levels in serum compared with mock-treated mice. In summary, these anti-SEB monoclonal antibodies represent excellent therapeutic candidates for further preclinical and clinical development.

  15. Sandwich electrochemical immunoassay for the detection of Staphylococcal enterotoxin B based on immobilized thiolated antibodies.

    PubMed

    Chatrathi, Madhu Prakash; Wang, Joseph; Collins, Greg E

    2007-06-15

    A new approach for the sensitive detection of Staphylococcal enterotoxin B (SEB) is presented based upon an electrochemical enzymatic immunoassay that utilizes thiolated antibodies immobilized on a gold surface. This method relies on the use of amine- or sulfhydryl-reactive heterobifunctional cross-linkers for the introduction of 2-pyridyl-disulfide groups to the antibody. The disulfide-containing linkages are subsequently cleaved with a suitable reducing agent, such as dithiothreitol (DTT), and the thiolated antibody-gold bond is covalently formed on a gold working electrode. Various cross-linking agents for immobilization of the capture antibody onto the gold electrode were investigated and compared. Factors influencing the thiolation and immobilization were investigated and optimized. The feasibility of such antibody immobilization and the subsequent sandwich enzyme immunoassay is demonstrated for the sensitive detection of SEB. The detection limit estimated from a representative dose-response curve is 1 ng/mL, corresponding to 5 pg in a 5-microL sample. Coupling the specificity of immunoassays with the sensitivity and low detection limits of electrochemical detection shows real promise for future sensing technology in enabling the development of single-use disposable devices.

  16. Detection of staphylococcal enterotoxin A (SEA) at picogram level by a capacitive immunosensor.

    PubMed

    Jantra, Jongjit; Kanatharana, Proespichaya; Asawatreratanakul, Punnee; Wongkittisuksa, Booncharoen; Limsakul, Chusak; Thavarungkul, Panote

    2011-01-01

    This work presents the use of a flow injection capacitive immunosensor to detect staphylococcal enterotoxin A (SEA). The study was based on the direct detection of a capacitance change due to the binding between SEA and anti-SEA immobilized on a gold electrode. The optimal regeneration solution, flow rate, sample volume and buffer conditions were studied. Under the optimum conditions, this label-free biosensor provided linearity between 1 × 10(-12) g L(-1) and 1 × 10(-8) g L(-1) of SEA and the limit of detection was 1 × 10(-12) g L(-1) which was much lower than the infectious dose (0.5 × 10(-6) - 1 × 10(-6) g L(-1)). Using the regeneration solution of, 15.0 mM glycine-HCl pH 2.20, to break the binding between SEA and the immobilized anti-SEA enabled the electrode to be reused up to 39 times. This technique was applied to analyze SEA in liquid and solid food samples. Any matrix effect can be eliminated by simple dilution. SEA contamination was found in three samples, iced tea with milk (28 ± 1 ng L(-1)), orange juice (113 ± 6 ng L(-1)) and fried chicken (1.1 ± 0.2 ng g(-1)); however, the concentrations were much lower than the infectious dose. The proposed method would be useful for rapid screening of SEA in various matrices.

  17. Synthetic Human Monoclonal Antibodies toward Staphylococcal Enterotoxin B (SEB) Protective against Toxic Shock Syndrome*

    PubMed Central

    Karauzum, Hatice; Chen, Gang; Abaandou, Laura; Mahmoudieh, Mahta; Boroun, Atefeh R.; Shulenin, Sergey; Devi, V. Sathya; Stavale, Eric; Warfield, Kelly L.; Zeitlin, Larry; Roy, Chad J.; Sidhu, Sachdev S.; Aman, M. Javad

    2012-01-01

    Staphylococcal enterotoxin B (SEB) is a potent toxin that can cause toxic shock syndrome and act as a lethal and incapacitating agent when used as a bioweapon. There are currently no vaccines or immunotherapeutics available against this toxin. Using phage display technology, human antigen-binding fragments (Fabs) were selected against SEB, and proteins were produced in Escherichia coli cells and characterized for their binding affinity and their toxin neutralizing activity in vitro and in vivo. Highly protective Fabs were converted into full-length IgGs and produced in mammalian cells. Additionally, the production of anti-SEB antibodies was explored in the Nicotiana benthamiana plant expression system. Affinity maturation was performed to produce optimized lead anti-SEB antibody candidates with subnanomolar affinities. IgGs produced in N. benthamiana showed characteristics comparable with those of counterparts produced in mammalian cells. IgGs were tested for their therapeutic efficacy in the mouse toxic shock model using different challenge doses of SEB and a treatment with 200 μg of IgGs 1 h after SEB challenge. The lead candidates displayed full protection from lethal challenge over a wide range of SEB challenge doses. Furthermore, mice that were treated with anti-SEB IgG had significantly lower IFNγ and IL-2 levels in serum compared with mock-treated mice. In summary, these anti-SEB monoclonal antibodies represent excellent therapeutic candidates for further preclinical and clinical development. PMID:22645125

  18. Genetics of staphylococcal enterotoxin B in methicillin-resistant isolates of Staphylococcus aureus.

    PubMed Central

    Shafer, W M; Iandolo, J J

    1979-01-01

    Biophysical and genetic analysis of staphylococcal enterotoxin B (SEB) synthesis in 16 methicillin-resistant (Mecr) Staphylococcus aureus isolates demonstrated that the toxin gene (entB) can occupy either a plasmid or chromosomal locus. Biophysical analysis of the plasmid deoxyribonucleic acid content of these strains by agarose gel electrophoresis revealed the presence of a 1.15-megadalton plasmid in six isolates that appears to contain the entB gene. Genetic manipulation of SEB synthesis by transduction and elimination procedures demonstrated that this plasmid is critical for enterotoxigenesis. Nevertheless, the majority of the Mecr SEB+ isolates (62.5%) analyzed in this investigation were found to lack the 1.15-megadalton plasmid. In at least two of these strains (COL and 57-dk), transduction and elimination procedures showed that entB was chromosomal. Genetic studies involving strains harboring either a plasmid or chromosomal entB gene demonstrated that toxin synthesis was coeliminated with mec. However, analysis of the entB and mec loci by transformation or transduction showed that the genes are not closely linked. On the other hand, transduction of entB, regardless of the donor, was observed when both mec and the Tcr plasmid were jointly cotransduced. This finding suggests that, during transduction, a transient association between entB, mec, and the Tcr plasmid may exist. Images PMID:259057

  19. Characterization of novel type C staphylococcal enterotoxins: biological and evolutionary implications.

    PubMed Central

    Marr, J C; Lyon, J D; Roberson, J R; Lupher, M; Davis, W C; Bohach, G A

    1993-01-01

    The type C staphylococcal enterotoxins (SEC) are a group of highly conserved proteins with significant immunological cross-reactivity. Although three antigenically distinct SEC subtypes (SEC1, SEC2, and SEC3) have been reported in the literature, we observed that the isoelectric points of SEC from several Staphylococcus aureus isolates are different from those of any of these three subtypes. This observation led us to propose that additional SEC molecular variants exist. For assessment of this possibility, the sec genes from representative human, animal, and food isolates were cloned and sequenced. The toxins encoded by the 18 isolates used in this study included five unique SEC proteins in addition to SEC1, SEC2, and SEC3. Six of the SEC proteins (including SEC1, SEC2, and SEC3) were produced by human and food isolates. Analysis of seven bovine and ovine isolates showed that isolates from each animal species produced a unique host-specific SEC. All of the SEC caused lymphocyte proliferation, although some of the toxins differed in their ability to stimulate cells from several animal species. An explanation for these results, which is supported by our phenotypic analysis of Sec+ staphylococcal isolates, is that toxin heterogeneity is due to selection for modified SEC sequences that facilitate the survival of S. aureus isolates in their respective hosts. Images PMID:8406814

  20. Plasmid-chromosomal transition of genes important in staphylococcal enterotoxin B expression.

    PubMed Central

    Dyer, D W; Iandolo, J J

    1981-01-01

    Experiments were performed to further elucidate the genetic mechanisms underlying the synthesis of staphylococcal enterotoxin B (SEB). Our laboratory has previously shown that, in strains of Staphylococcus aureus which harbor a 1.15-megadalton plasmid (pentB or pSN2), the plasmid appears to be required for SEB synthesis; in other S. aureus strains, designated chromosomal SEB producers, this 1.15-megadalton plasmid is conspicuously absent. We report here than in both Bacillus subtilis minicells and a coupled translational assay, pSN2 codes for a polypeptide of 18,000 daltons. This product is not immunologically reactive with purified anti-SEB globulin. Nevertheless, pSN2 is necessary but not sufficient for SEB synthesis in strains which harbor the plasmid. Further, the data provide a reasonable link between plasmid-bearing and chromosomal SEB producers: transformational analysis indicates that both require functions specified (in plasmid-bearing strains) by pSN2 for SEB synthesis. The combined genetic and biochemical data suggest that pSN2 is not the reservoir for the SEB structural gene, but that the pSN2-specific functions required for SEB synthesis are regulatory in nature. Images PMID:6792077

  1. Staphylococcal Enterotoxin P Predicts Bacteremia in Hospitalized Patients Colonized With Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Calderwood, Michael S.; Desjardins, Christopher A.; Sakoulas, George; Nicol, Robert; DuBois, Andrea; Delaney, Mary L.; Kleinman, Ken; Cosimi, Lisa A.; Feldgarden, Michael; Onderdonk, Andrew B.; Birren, Bruce W.; Platt, Richard; Huang, Susan S.

    2014-01-01

    Background. Methicillin-resistant Staphylococcus aureus (MRSA) colonization predicts later infection, with both host and pathogen determinants of invasive disease. Methods. This nested case-control study evaluates predictors of MRSA bacteremia in an 8–intensive care unit (ICU) prospective adult cohort from 1 September 2003 through 30 April 2005 with active MRSA surveillance and collection of ICU, post-ICU, and readmission MRSA isolates. We selected MRSA carriers who did (cases) and those who did not (controls) develop MRSA bacteremia. Generating assembled genome sequences, we evaluated 30 MRSA genes potentially associated with virulence and invasion. Using multivariable Cox proportional hazards regression, we assessed the association of these genes with MRSA bacteremia, controlling for host risk factors. Results. We collected 1578 MRSA isolates from 520 patients. We analyzed host and pathogen factors for 33 cases and 121 controls. Predictors of MRSA bacteremia included a diagnosis of cancer, presence of a central venous catheter, hyperglycemia (glucose level, >200 mg/dL), and infection with a MRSA strain carrying the gene for staphylococcal enterotoxin P (sep). Receipt of an anti-MRSA medication had a significant protective effect. Conclusions. In an analysis controlling for host factors, colonization with MRSA carrying sep increased the risk of MRSA bacteremia. Identification of risk-adjusted genetic determinants of virulence may help to improve prediction of invasive disease and suggest new targets for therapeutic intervention. PMID:24041793

  2. Intestinal Enteroids Model Guanylate Cyclase C-Dependent Secretion Induced by Heat-Stable Enterotoxins

    PubMed Central

    Pattison, Amanda M.; Blomain, Erik S.; Merlino, Dante J.; Wang, Fang; Crissey, Mary Ann S.; Kraft, Crystal L.; Rappaport, Jeff A.; Snook, Adam E.; Lynch, John P.

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC) causes ∼20% of the acute infectious diarrhea (AID) episodes worldwide, often by producing heat-stable enterotoxins (STs), which are peptides structurally homologous to paracrine hormones of the intestinal guanylate cyclase C (GUCY2C) receptor. While molecular mechanisms mediating ST-induced intestinal secretion have been defined, advancements in therapeutics have been hampered for decades by the paucity of disease models that integrate molecular and functional endpoints amenable to high-throughput screening. Here, we reveal that mouse and human intestinal enteroids in three-dimensional ex vivo cultures express the components of the GUCY2C secretory signaling axis. ST and its structural analog, linaclotide, an FDA-approved oral secretagog, induced fluid accumulation quantified simultaneously in scores of enteroid lumens, recapitulating ETEC-induced intestinal secretion. Enteroid secretion depended on canonical molecular signaling events responsible for ETEC-induced diarrhea, including cyclic GMP (cGMP) produced by GUCY2C, activation of cGMP-dependent protein kinase (PKG), and opening of the cystic fibrosis transmembrane conductance regulator (CFTR). Importantly, pharmacological inhibition of CFTR abrogated enteroid fluid secretion, providing proof of concept for the utility of this model to screen antidiarrheal agents. Intestinal enteroids offer a unique model, integrating the GUCY2C signaling axis and luminal fluid secretion, to explore the pathophysiology of, and develop platforms for, high-throughput drug screening to identify novel compounds to prevent and treat ETEC diarrheal disease. PMID:27481254

  3. High sensitivity chemiluminescence enzyme immunoassay for detecting staphylococcal enterotoxin A in multi-matrices.

    PubMed

    Zhang, Chunmei; Liu, Zhijia; Li, Yongming; Li, Qi; Song, Chaojun; Xu, Zhuwei; Zhang, Yun; Zhang, Yusi; Ma, Ying; Sun, Yuanjie; Chen, Lihua; Fang, Liang; Yang, Angang; Yang, Kun; Jin, Boquan

    2013-09-24

    In this study, detection of staphylococcal enterotoxin A (SEA) in multi-matrices using a highly sensitive and specific microplate chemiluminescence enzyme immunoassay (CLEIA) has been established. A pair of monoclonal antibodies (mAbs) was selected from 37 anti-SEA mAbs by pairwise analysis, and the experimental conditions of the CLEIA were optimized. This CLEIA exhibited high performance with a wide dynamic range from 6.4 pg mL(-1) to 1600 pg mL(-1), and the measured low limit of detection (LOD) was 3.2 pg mL(-1). No cross-reactivity was observed when this method was applied to test SEB, SEC1, and SED. It has also been successfully applied for analyzing SEA in a variety of environmental, biological, and clinical matrices, such as sewage, tap water, river water, roast beef, peanut butter, cured ham, 10% nonfat dry milk, milk, orange juice, human urine, and serum. Thus, the highly sensitive and SEA-specific CLEIA should make it attractive for quantifying SEA in public health and diagnosis in near future.

  4. Superantigen staphylococcal enterotoxin C1 mutant can reduce paraquat pulmonary fibrosis.

    PubMed

    Li, Tiegang; Xu, Mingkai; Wang, Nana; Zhao, Min

    2015-01-01

    A network of inflammation factors is related to pulmonary fibrosis induced by paraquat (PQ) poisoning. At high doses, the superantigen staphylococcal enterotoxin C1 (SEC1) can induce immunological unresponsiveness and inhibit release of inflammation factors. In this study, site-directed mutagenesis was performed at the H118 and H122 amino acid residues of SEC1 to reduce SEC1 toxicity. The SEC1 mutant showed significantly decreased pyrogenic toxicity, but retained clonal anergy at high dosages in vitro. Pretreatment with the SEC1 mutant prior to PQ poisoning in mice reduced symptom duration and severity, prolonged survival time, and decreased the splenocyte response to ConA induction. The SEC1 mutant also down-regulated several important cytokines related to fibrosis in the plasma after PQ poisoning. SEC1 decreased the expression of genes related to pulmonary fibrosis, including α-SMA, COL1a1, COL3 and TGF-β1, in PQ poisoned mice. Histomorphological observation indicated alleviation of pathological changes in the lungs after SEC1 pretreatment compared to mice in the PQ group. In conclusion, the SEC1 mutant reduced pulmonary interstitial fibrosis induced by PQ poisoning.

  5. Piezoelectric immunosensor for direct and rapid detection of staphylococcal enterotoxin A (SEA) at the ng level.

    PubMed

    Salmain, Michèle; Ghasemi, Mahsa; Boujday, Souhir; Spadavecchia, Jolanda; Técher, Clarisse; Val, Florence; Le Moigne, Vincent; Gautier, Michel; Briandet, Romain; Pradier, Claire-Marie

    2011-11-15

    A direct, label-free immunosensor was designed for the rapid detection and quantification of staphylococcal enterotoxin A (SEA) in buffered solutions using quartz crystal microbalance with dissipation (QCM-D) as transduction method. The sensing layer including the anti-SEA antibody was constructed by chemisorption of a self-assembled monolayer of cysteamine on the gold electrodes placed over the quartz crystal sensor followed by activation of the surface amino groups with the rigid homobifunctional cross-linker 1,4-phenylene diisothiocyanate (PDITC) and covalent linking of binding protein (protein A or protein G). Four anti-SEA antibodies (two of which from commercial source) have been selected to set up the most sensitive detection device. With the optimized sensing layer, a standard curve for the direct assay of SEA was established from QCM-D responses within a working range of 50-1000 or 2000 ngml(-1) with a detection limit of 20 ngml(-1). The total time for analysis was 15 min. Using a sandwich type assay, the response was ca. twice higher and consequently the lowest measurable concentration dropped down to 7 ngml(-1) for a total assay time of 25 min.

  6. Effect of thermal processing during yogurt production upon the detection of staphylococcal enterotoxin B.

    PubMed

    Principato, Maryann; Boyle, Thomas; Njoroge, Joyce; Jones, Robert L; O'Donnell, Michael

    2009-10-01

    This research was conducted to examine the inherent properties of yogurt contaminated with staphylococcal enterotoxin B (SEB). Two types of yogurts were produced for this study. Type I yogurts were produced by adding SEB at the start of yogurt production, and type II yogurts were produced by adding SEB after the milk base had been boiled. Biochemical characteristics inherent to yogurt, including pH, lactic acid and acetaldehyde concentrations, were analyzed weekly for each batch beginning at a time just after production and throughout a storage period of at least 4 weeks. The presence of toxin during yogurt production did not result in any significant biochemical or physical changes in yogurt. However, we were unable to detect SEB toxin in type I yogurt using a commercially available enzyme-linked immunosorbent assay (ELISA). In contrast, SEB was easily detectable by our ELISA in type II yogurt samples. Higher levels of SEB were recovered from type II yogurt that had been stored for 1 week than from type II yogurt that had been stored for any other length of time. These results indicate that the biochemical characteristics of yogurt did not change significantly (relative to control yogurt) in the presence of either thermally processed SEB or native SEB. However, the ability to detect SEB by ELISA was dependent on whether the toxin had been processed.

  7. [Presence of enterotoxin C and toxic shock syndrome toxin--1 (TSST-1) genes in population of Staphylococcus aureus phage type 187].

    PubMed

    Garbacz, Katarzyna; Piechowicz, Lidia; Galiński, Janusz

    2006-01-01

    The aim of this study was to examine whether Staphylococcus aureus of phage type 187 possess the genes of enterotoxins and toxic shock syndrom toxin. Sixteen phage type 187 strains were isolated from the hospital patients (12) and the carriers (4) in twelve medical centres in Poland during 1991 and 2005. Biotyping, phage typing, antibiotic susceptibility, detection of the genes of enterotoxins (sea--sed) and toxic shock syndrome toxin (tst) was tested. The results of this study showed that all staphylococci of phage type 187 belonged to the human biotype (A) and appeared to be sensitive to all of the tested antibiotics, including methicillin (MSSA). Almost all of them (93.8%) had the enterotoxin C gene and TSST-1 gene. This fact allows to consider them the strains of potentially high virulence.

  8. Detection of Staphylococcus aureus enterotoxin production genes from patient samples using an automated extraction platform and multiplex real-time PCR.

    PubMed

    Chiefari, Amy K; Perry, Michael J; Kelly-Cirino, Cassandra; Egan, Christina T

    2015-12-01

    To minimize specimen volume, handling and testing time, we have developed two TaqMan(®) multiplex real-time PCR (rtPCR) assays to detect staphylococcal enterotoxins A-E and Toxic Shock Syndrome Toxin production genes directly from clinical patient stool specimens utilizing a novel lysis extraction process in parallel with the Roche MagNA Pure Compact. These assays are specific, sensitive and reliable for the detection of the staphylococcal enterotoxin encoding genes and the tst1 gene from known toxin producing strains of Staphylococcus aureus. Specificity was determined by testing a total of 47 microorganism strains, including 8 previously characterized staphylococcal enterotoxin producing strains against each rtPCR target. Sensitivity for these assays range from 1 to 25 cfu per rtPCR reaction for cultured isolates and 8-20 cfu per rtPCR for the clinical stool matrix.

  9. Enterotoxins and emetic toxins production by Bacillus cereus and other species of Bacillus isolated from Soumbala and Bikalga, African alkaline fermented food condiments.

    PubMed

    Ouoba, Labia Irene I; Thorsen, Line; Varnam, Alan H

    2008-06-10

    The ability of various species of Bacillus from fermented seeds of Parkia biglobosa known as African locust bean (Soumbala) and fermented seeds of Hibiscus sabdariffa (Bikalga) was investigated. The study included screening of the isolates by haemolysis on blood agar, detection of toxins in broth and during the fermentation of African locust bean using the Bacillus cereus Enterotoxin Reverse Passive Latex Agglutination test kit (BCET-RPLA) and the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (BDEVIA). Detection of genes encoding cytotoxin K (CytK), haemolysin BL (Hbl A, Hbl C, Hbl D), non-hemolytic enterotoxin (NheA, NheB, NheC) and EM1 specific of emetic toxin producers was also investigated using PCR with single pair and multiplex primers. Of 41 isolates, 29 Bacillus belonging to the species of B. cereus, Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus showed haemolysis on blood agar. Using RPLA, enterotoxin production was detected for three isolates of B. cereus in broth and all B. cereus (9) in fermented seeds. Using BDEVIA, enterotoxin production was detected in broth as well as in fermented seeds for all B. cereus isolates. None of the isolates belonging to the other Bacillus species was able to produce enterotoxins either by RPLA or BDEVIA. Nhe genes were detected in all B. cereus while Hbl and CytK genes were detected respectively in five and six B. cereus strains. A weak presence of Hbl (A, D) and CytK genes was detected in two isolates of B. subtilis and one of B. licheniformis but results were inconsistent, especially for Hbl genes. The emetic specific gene fragment EM1 was not detected in any of the isolates studied.

  10. Detection of viable enterotoxin-producing Bacillus cereus and analysis of toxigenicity from ready-to-eat foods and infant formula milk powder by multiplex PCR.

    PubMed

    Zhang, Zhihong; Feng, Lixia; Xu, Hengyi; Liu, Chengwei; Shah, Nagendra P; Wei, Hua

    2016-02-01

    Bacillus cereus is responsible for several outbreaks of foodborne diseases due to its emetic toxin and enterotoxin. Enterotoxins, cytotoxin K (CytK), nonhemolytic enterotoxin (Nhe), and hemolysin BL (Hbl), have been recorded in several diarrheal cases due to food poisoning from B. cereus. The objective of this study was to develop a rapid and accurate method that combines multiplex PCR with propidium monoazide to selectively detect viable cells of enterotoxin-producing B. cereus in milk powder, noodles, and rice, and investigate the distribution of enterotoxins in 62 strains of B. cereus in Jiangxi province, China. The specificity of primers of 3 enterotoxins (i.e., cytK, nheA, and hblD) of B. cereus was verified by inclusivity and exclusivity tests using single PCR. Upon optimization of multiplex PCR conditions, it was found that the detection limit of viable cells was 10(2) cfu/mL of B. cereus in pure culture. By enrichment for 3 or 4 h and propidium monoazide pretreatment, a protocol for detection of viable cells as low as 2.2×10(1) cfu/g in spiked food (e.g., milk powder, noodles, and rice) was established and proved valid even under the interference of non-Bacillus cereus at as high as 10(5) cfu/g. Moreover, the protocol based on multiplex PCR for detection was applied for the analysis of distribution of toxin gene of B. cereus, and the results showed a regional feature for toxin gene distribution, indicating that potential toxigenicity of B. cereus should be evaluated further.

  11. Comparison of antibiogram, staphylococcal enterotoxin productivity, and coagulase genotypes among Staphylococcus aureus isolated from animal and vegetable sources in Korea.

    PubMed

    Moon, Jin San; Lee, Ae Ri; Jaw, Seung Hyeup; Kang, Hyun Mi; Joo, Yi Seok; Park, Yong Ho; Kim, Mal Nam; Koo, Hye Cheong

    2007-11-01

    Staphylococcal food poisoning is caused by enterotoxin-producing Staphylococcus aureus. We investigated the prevalence of such organisms in samples of bovine mastitic milk (n = 714), raw meat (n = 139), and vegetables (n = 616). We determined the degrees of relatedness of isolates as indicated by antibiogram, staphylococcal enterotoxin (SE) productivity, and coagulase gene restriction fragment length polymorphism analysis. We examined 297 S. aureus isolates and found SE production in 57 (31.8%), 4 (7.8%), and 49 (73.1%) isolates from raw milk, raw meat, and vegetables, respectively. A high proportion of the isolates obtained from milk produced more than two types of toxins (mainly SEA, SEB, and/or SEC), whereas isolates from raw meat and vegetables primarily produced SEA alone. Most isolates were sensitive to cephalothin (97.6%), gentamicin (80.8%), erythromycin (79.5%), and tetracycline (72.7%), but were resistant to penicillin (90.2%) and ampicillin (88.9%). The proportion of antibiotic-resistant isolates differed according the source of the bacteria; the milk and vegetable isolates were more resistant to penicillin and ampicillin than were the meat isolates (P < 0.05), whereas tetracycline resistance was limited to the milk and vegetables isolates. The coagulase genotypes (I to XII) varied with the source of the organism, and only a few genotypes prevailed in each source: II (42.4%) and IV (24%) types in isolates from milk, IX (35.3%) and XI (45%) from raw meat, and III (40.3%) and XII (32.8%) from vegetables. These findings suggest that remarkable differences exist in antibiogram, SE productivity, and coagulase genotypes, resulting in limited clonal transmission of S. aureus into various food sources. As enterotoxin production only occurs when S. aureus grows to high numbers, staphylococcal food poisoning can be prevented by proper refrigeration.

  12. Sensitive, Rapid, Quantitative and in Vitro Method for the Detection of Biologically Active Staphylococcal Enterotoxin Type E

    PubMed Central

    Rasooly, Reuven; Do, Paula; Hernlem, Bradley

    2016-01-01

    Staphylococcus aureus is a major bacterial cause of clinical infections and foodborne illnesses through its production of a group of enterotoxins (SEs) which cause gastroenteritis and also function as superantigens to massively activate T cells. In the present study, we tested Staphylococcal enterotoxin type E (SEE), which was detected in 17 of the 38 suspected staphylococcal food poisoning incidents in a British study and was the causative agent in outbreaks in France, UK and USA. The current method for detection of enterotoxin activity is an in vivo monkey or kitten bioassay; however, this expensive procedure has low sensitivity and poor reproducibility, requires many animals, is impractical to test on a large number of samples, and raises ethical concerns with regard to the use of experimental animals. The purpose of this study is to develop rapid sensitive and quantitative bioassays for detection of active SEE. We apply a genetically engineered T cell-line expressing the luciferase reporter gene under the regulation of nuclear factor of activated T-cells response element (NFAT-RE), combined with a Raji B-cell line that presents the SEE-MHC (major histocompatibility complex) class II to the engineered T cell line. Exposure of the above mixed culture to SEE induces differential expression of the luciferase gene and bioluminescence is read out in a dose dependent manner over a 6-log range. The limit of detection of biologically active SEE is 1 fg/mL which is 109 times more sensitive than the monkey and kitten bioassay. PMID:27187474

  13. Lactobacillus zeae Protects Caenorhabditis elegans from Enterotoxigenic Escherichia coli-Caused Death by Inhibiting Enterotoxin Gene Expression of the Pathogen

    PubMed Central

    Zhou, Mengzhou; Yu, Hai; Yin, Xianhua; Sabour, Parviz M.; Chen, Wei; Gong, Joshua

    2014-01-01

    Background The nematode Caenorhabditis elegans has become increasingly used for screening antimicrobials and probiotics for pathogen control. It also provides a useful tool for studying microbe-host interactions. This study has established a C. elegans life-span assay to preselect probiotic bacteria for controlling K88+ enterotoxigenic Escherichia coli (ETEC), a pathogen causing pig diarrhea, and has determined a potential mechanism underlying the protection provided by Lactobacillus. Methodology/Principal Findings Life-span of C. elegans was used to measure the response of worms to ETEC infection and protection provided by lactic acid-producing bacteria (LAB). Among 13 LAB isolates that varied in their ability to protect C. elegans from death induced by ETEC strain JG280, Lactobacillus zeae LB1 offered the highest level of protection (86%). The treatment with Lactobacillus did not reduce ETEC JG280 colonization in the nematode intestine. Feeding E. coli strain JFF4 (K88+ but lacking enterotoxin genes of estA, estB, and elt) did not cause death of worms. There was a significant increase in gene expression of estA, estB, and elt during ETEC JG280 infection, which was remarkably inhibited by isolate LB1. The clone with either estA or estB expressed in E. coli DH5α was as effective as ETEC JG280 in killing the nematode. However, the elt clone killed only approximately 40% of worms. The killing by the clones could also be prevented by isolate LB1. The same isolate only partially inhibited the gene expression of enterotoxins in both ETEC JG280 and E. coli DH5α in-vitro. Conclusions/Significance The established life-span assay can be used for studies of probiotics to control ETEC (for effective selection and mechanistic studies). Heat-stable enterotoxins appeared to be the main factors responsible for the death of C. elegans. Inhibition of ETEC enterotoxin production, rather than interference of its intestinal colonization, appears to be the mechanism of protection

  14. 40 CFR 725.421 - Introduced genetic material.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... perfringens Beta-toxin; Delta-toxin Escherichia coli & other Enterobacteriaceae spp. Heat-labile enterotoxins... difficile Cytotoxin (toxin B) Clostridium perfringens Beta-toxin; Epsilon-toxin; Kappa-toxin Escherichia coli & other Enterobacteriaceae spp. Cytotoxin (Shiga-like toxin, Vero cell toxin)...

  15. 40 CFR 725.421 - Introduced genetic material.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... perfringens Beta-toxin; Delta-toxin Escherichia coli & other Enterobacteriaceae spp. Heat-labile enterotoxins... difficile Cytotoxin (toxin B) Clostridium perfringens Beta-toxin; Epsilon-toxin; Kappa-toxin Escherichia coli & other Enterobacteriaceae spp. Cytotoxin (Shiga-like toxin, Vero cell toxin)...

  16. 40 CFR 725.421 - Introduced genetic material.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... perfringens Beta-toxin; Delta-toxin Escherichia coli & other Enterobacteriaceae spp. Heat-labile enterotoxins... difficile Cytotoxin (toxin B) Clostridium perfringens Beta-toxin; Epsilon-toxin; Kappa-toxin Escherichia coli & other Enterobacteriaceae spp. Cytotoxin (Shiga-like toxin, Vero cell toxin)...

  17. 40 CFR 725.421 - Introduced genetic material.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... perfringens Beta-toxin; Delta-toxin Escherichia coli & other Enterobacteriaceae spp. Heat-labile enterotoxins... difficile Cytotoxin (toxin B) Clostridium perfringens Beta-toxin; Epsilon-toxin; Kappa-toxin Escherichia coli & other Enterobacteriaceae spp. Cytotoxin (Shiga-like toxin, Vero cell toxin)...

  18. Membrane interactions of a novel viral enterotoxin: rotavirus nonstructural glycoprotein NSP4.

    PubMed

    Huang, H; Schroeder, F; Zeng, C; Estes, M K; Schoer, J K; Ball, J M

    2001-04-03

    The rotavirus enterotoxin, NSP4, is a novel secretory agonist that also plays a role in the unique rotavirus morphogenesis that involves a transient budding of newly made immature viral particles into the endoplasmic reticulum. NSP4 and an active peptide corresponding to NSP4 residues 114 to 135 (NSP4(114-135)) mobilize intracellular calcium and induce secretory chloride currents when added exogenously to intestinal cells or mucosa. Membrane-NSP4 interactions may contribute to these alterations; however, details of a lipid-binding domain are unresolved. Therefore, circular dichroism was used to determine (i) the interaction(s) of NSP4 and NSP4(114-135) with model membranes, (ii) the conformational changes elicited in NSP4 upon interacting with membranes, (iii) if NSP4(114-135) is a membrane interacting domain, and (iv) the molar dissociation constant (K(d)) of NSP4(114-135) with defined lipid vesicles. Circular dichroism revealed for the first time that NSP4 and NSP4(114-135) undergo secondary structural changes upon interaction with membrane vesicles. This interaction was highly dependent on both the membrane surface curvature and the lipid composition. NSP4 and NSP4(114-135) preferentially interacted with highly curved, small unilamellar vesicle membranes (SUV), but significantly less with low-curvature, large unilamellar vesicle membranes (LUV). Binding to SUV, but not LUV, was greatly enhanced by negatively charged phospholipids. Increasing the SUV cholesterol content, concomitant with the presence of negatively charged phospholipids, further potentiated the interaction of NSP4(114-135) with the SUV membrane. The K(d) of NSP4(114-135) was determined as well as partitioning of NSP4(114-135) with SUVs in a filtration-binding assay. These data confirmed NSP4 and its active peptide interact with model membranes that mimic caveolae.

  19. Prevalence of Clostridium perfringens, Clostridium perfringens enterotoxin and dysbiosis in fecal samples of dogs with diarrhea.

    PubMed

    Minamoto, Yasushi; Dhanani, Naila; Markel, Melissa E; Steiner, Jörg M; Suchodolski, Jan S

    2014-12-05

    Clostridium perfringens has been suspected as an enteropathogen in dogs. However, its exact role in gastrointestinal (GI) disorders in dogs remains unknown. Recent studies suggest the importance of an altered intestinal microbiota in the activation of virulence factors of enteropathogens. The aim of this study was to evaluate the relationship between diarrhea, dysbiosis, and the presence of C. perfringens and its enterotoxin (CPE). Fecal samples were collected prospectively from 95 healthy control dogs and 104 dogs with GI disease and assessed for bacterial abundances and the presence of CPE using quantitative PCR and ELISA, respectively. C. perfringens was detected in all dogs. Potentially enterotoxigenic C. perfringens were detected in 33.7% (32/95) of healthy control dogs and 48.1% (50/104) diseased dogs, respectively. CPE was detected by ELISA in 1.0% (1/95) of control dogs and 16.3% (17/104) of diseased dogs. Abundances of Fusobacteria, Ruminococcaceae, Blautia, and Faecalibacterium were significantly decreased in diseased dogs, while abundances of Bifidobacterium, Lactobacillus, and Escherichia coli were significantly increased compared to control dogs. The microbial dysbiosis was independent of the presence of the enterotoxigenic C. perfringens or CPE. In conclusion, the presence of CPE as well as fecal dysbiosis was associated with GI disease. However, the presence of C. perfringens was not indicative of GI disease in all cases of diarrhea, and the observed increased abundance of enterotoxigenic C. perfringens may be part of intestinal dysbiosis occurring in GI disease. The significance of an intestinal dysbiosis in dogs with GI disease deserves further attention.

  20. Synergistic Effects of Clostridium perfringens Enterotoxin and Beta Toxin in Rabbit Small Intestinal Loops

    PubMed Central

    Ma, Menglin; Gurjar, Abhijit; Theoret, James R.; Garcia, Jorge P.; Beingesser, Juliann; Freedman, John C.; Fisher, Derek J.; McClane, Bruce A.

    2014-01-01

    The ability of Clostridium perfringens type C to cause human enteritis necroticans (EN) is attributed to beta toxin (CPB). However, many EN strains also express C. perfringens enterotoxin (CPE), suggesting that CPE could be another contributor to EN. Supporting this possibility, lysate supernatants from modified Duncan-Strong sporulation (MDS) medium cultures of three CPE-positive type C EN strains caused enteropathogenic effects in rabbit small intestinal loops, which is significant since CPE is produced only during sporulation and since C. perfringens can sporulate in the intestines. Consequently, CPE and CPB contributions to the enteropathogenic effects of MDS lysate supernatants of CPE-positive type C EN strain CN3758 were evaluated using isogenic cpb and cpe null mutants. While supernatants of wild-type CN3758 MDS lysates induced significant hemorrhagic lesions and luminal fluid accumulation, MDS lysate supernatants of the cpb and cpe mutants caused neither significant damage nor fluid accumulation. This attenuation was attributable to inactivating these toxin genes since complementing the cpe mutant or reversing the cpb mutation restored the enteropathogenic effects of MDS lysate supernatants. Confirming that both CPB and CPE are needed for the enteropathogenic effects of CN3758 MDS lysate supernatants, purified CPB and CPE at the same concentrations found in CN3758 MDS lysates also acted together synergistically in rabbit small intestinal loops; however, only higher doses of either purified toxin independently caused enteropathogenic effects. These findings provide the first evidence for potential synergistic toxin interactions during C. perfringens intestinal infections and support a possible role for CPE, as well as CPB, in some EN cases. PMID:24778117

  1. Different roles of Staphylococcus aureus enterotoxin in different subtypes of nasal polyps

    PubMed Central

    Cheng, Ke-Jia; Wang, Shen-Qing; Xu, Ying-Ying

    2017-01-01

    Chronic rhinosinusitis with nasal polyps (CRSwNP) is a multifactorial disease. The pathogenesis of CRSwNP remains unclear. This study was designed to investigate the role of inflammation and Staphylococcus aureus enterotoxin (SE) in this disease. The study included a total of 74 patients with CRSwNP and 6 controls. A serum Phadiatop assay was conducted to detect atopy status, and serum eosinophil cationic protein (ECP) and total immunoglobulin (Ig)E levels were determined using ELISA. SEA, SEB, total IgE, ECP and myeloperoxidase (MPO) levels in nasal tissue supernatant were measured using ELISA. The results indicated that 15 (22.1%) patients had systemic allergies. On the basis of the ECP/MPO ratio, the patients were divided into an eosinophilic CRSwNP group (n=18) and a non-eosinophilic CRSwNP group (n=56). The total ECP/MPO ratio was 0.572, with a notable bias toward neutrophilic inflammation. The supernatant ECP and MPO levels were elevated in the CRSwNP group compared with the control group, but no significant difference in the serum total IgE and ECP levels were observed between the CRSwNP and control groups. In addition, the non-eosinophilic and eosinophilic CRSwNP groups showed significant elevations in supernatant total IgE, SEA and SEB levels compared with the control group. Thus, it may be concluded that allergy is a common pathogenesis of CRSwNP, and neutrophilic inflammation is present in most Chinese CRSwNP patients. Additionally, local indicators reflect the inflammatory status more accurately than do serum indicators. SEs may act as an infection factor rather than as a superantigen in Chinese non-eosinophilic CRSwNP patients. Thus, long-term antibiotic therapy may be an option for Chinese non-eosinophilic CRSwNP patients. PMID:28123509

  2. Staphylococcus aureus Isolates Encode Variant Staphylococcal Enterotoxin B Proteins That Are Diverse in Superantigenicity and Lethality

    PubMed Central

    Kohler, Petra L.; Greenwood, Seth D.; Nookala, Suba; Kotb, Malak; Kranz, David M.; Schlievert, Patrick M.

    2012-01-01

    Staphylococcus aureus produces superantigens (SAgs) that bind and cross-link T cells and APCs, leading to activation and proliferation of immune cells. SAgs bind to variable regions of the β-chains of T cell receptors (Vβ-TCRs), and each SAg binds a unique subset of Vβ-TCRs. This binding leads to massive cytokine production and can result in toxic shock syndrome (TSS). The most abundantly produced staphylococcal SAgs and the most common causes of staphylococcal TSS are TSS toxin-1 (TSST-1), and staphylococcal enterotoxins B and C (SEB and SEC, respectively). There are several characterized variants of humans SECs, designated SEC1-4, but only one variant of SEB has been described. Sequencing the seb genes from over 20 S. aureus isolates show there are at least five different alleles of seb, encoding forms of SEB with predicted amino acid substitutions outside of the predicted immune-cell binding regions of the SAgs. Examination of purified, variant SEBs indicates that these amino acid substitutions cause differences in proliferation of rabbit splenocytes in vitro. Additionally, the SEBs varied in lethality in a rabbit model of TSS. The SEBs were diverse in their abilities to cause proliferation of human peripheral blood mononuclear cells, and differed in their activation of subsets of T cells. A soluble, high-affinity Vβ-TCR, designed to neutralize the previously characterized variant of SEB (SEB1), was able to neutralize the variant SEBs, indicating that this high-affinity peptide may be useful in treating a variety of SEB-mediated illnesses. PMID:22815951

  3. Accessory cholera enterotoxin, Ace, from Vibrio cholerae: structure, unfolding, and virstatin binding.

    PubMed

    Chatterjee, Tanaya; Mukherjee, Debadrita; Dey, Sucharita; Pal, Aritrika; Hoque, Kazi Mirajul; Chakrabarti, Pinak

    2011-04-12

    Vibrio cholerae accessory cholera enterotoxin (Ace) is the third toxin, along with cholera toxin (CT) and zonula occludens toxin (Zot), that causes the endemic disease cholera. Structural characterization of Ace has been restricted because of the limited production of this toxic protein by V. cholerae. We have cloned, overexpressed, and purified Ace from V. cholerae strain O395 in Escherichia coli to homogeneity and determined its biological activity. The unfolding of the purified protein was investigated using circular dichroism and intrinsic tryptophan fluorescence. Because Ace is predominantly a hydrophobic protein, the degree of exposure of hydrophobic regions was identified from the spectral changes of the environment-sensitive fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) that quenches the fluorescence of tryptophan residues of Ace in a concentration-dependent manner. Results showed that bis-ANS binds one monomeric unit of Ace with a 1:1 stoichiometry and a K' of 0.72 μM. Ace exists as a dimer, with higher oligomeric forms appearing upon glutaraldehyde cross-linking. This study also reports the binding of virstatin, a small molecule that inhibits virulence regulation in V. cholerae, to Ace. The binding constant (K=9×10(4) M(-1)) and the standard free energy change (ΔG°=-12 kcal mol(-1)) of Ace-virstatin interaction have been evaluated by the fluorescence quenching method. The binding does not affect the oligomeric status of Ace. A cell viability assay of the antibacterial activity of Ace has been performed using various microbial strains. A homology model of Ace, consistent with the experimental results, has been constructed.

  4. Isolation and epitope mapping of staphylococcal enterotoxin B single-domain antibodies.

    PubMed

    Turner, Kendrick B; Zabetakis, Dan; Legler, Patricia; Goldman, Ellen R; Anderson, George P

    2014-06-19

    Single-domain antibodies (sdAbs), derived from the heavy chain only antibodies found in camelids such as llamas have the potential to provide rugged detection reagents with high affinities, and the ability to refold after denaturation. We have isolated and characterized sdAbs specific to staphylococcal enterotoxin B (SEB) which bind to two distinct epitopes and are able to function in a sandwich immunoassay for toxin detection. Characterization of these sdAbs revealed that each exhibited nanomolar binding affinities or better.  Melting temperatures for the sdAbs ranged from approximately 60 °C to over 70 °C, with each demonstrating at least partial refolding after denaturation and several were able to completely refold. A first set of sdAbs was isolated by panning the library using adsorbed antigen, all of which recognized the same epitope on SEB. Epitope mapping suggested that these sdAbs bind to a particular fragment of SEB (VKSIDQFLYFDLIYSI) containing position L45 (underlined), which is involved in binding to the major histocompatibility complex (MHC). Differences in the binding affinities of the sdAbs to SEB and a less-toxic vaccine immunogen, SEBv (L45R/Y89A/Y94A) were also consistent with binding to this epitope. A sandwich panning strategy was utilized to isolate sdAbs which bind a second epitope. This epitope differed from the initial one obtained or from that recognized by previously isolated anti-SEB sdAb A3. Using SEB-toxin spiked milk we demonstrated that these newly isolated sdAbs could be utilized in sandwich-assays with each other, A3, and with various monoclonal antibodies.

  5. Tagging staphylococcal enterotoxin B (SEB) with TGFaL3 for breast cancer therapy.

    PubMed

    Yousefi, Forough; Siadat, Seyed Davar; Saraji, Alireza Azizi; Hesaraki, Saeed; Aslani, Mohammad Mehdi; Mousavi, Seyed Fazlollah; Imani Fooladi, Abbas Ali

    2016-04-01

    Recent research has attempted to direct superantigens towards tumors by means of tumor-targeted superantigen (TTS) strategy. In this study, we explored the antitumor property of TTS by fusing the third loop of transforming growth factor α (TGFαL3) to staphylococcal enterotoxin type B (SEB) and investigated the possibility of the therapeutic application of TGFαL3-SEB as a novel antitumor candidate in mice bearing breast cancer. Treatment was performed through intratumoral and intravenous injection of TGFαL3-SEB. Tumor size/volume, long-term survival, and cytokine secretion were assessed. In addition, the toxicity of each treatment on liver and kidneys was examined. Our results indicated that the relative tumor volume significantly increased in the mice receiving intratumoral TGFaL3-SEB (p < 0.05). Surprisingly, 5 out of the 14 mice were cleared from the tumor thoroughly in 10-25 days after intratumoral administration of TGFaL3-SEB. Quantification of cytokines clearly showed that the mice receiving intratumoral SEB significantly secreted higher interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) compared with the other groups (p < 0.05). The antitumor effect was followed by inhibition of cell proliferation (Ki-67) and micro vascularization (CD31). The highest and lowest levels of tumor necrosis were observed in the intratumoral administration of TGFαL3-SEB (85 %) and PBS (14 %), respectively. Intratumoral injection of TGFαL3-SEB increased the lifespan of the mice so 37.5 % of them could survive for more than 6 months (p < 0.05). Overall, our findings indicated that intratumoral administration of TGFαL3-SEB effectively inhibited the growth of breast tumors through induction of necrosis and suppressing proliferation and angiogenesis without systemic toxicity.

  6. Staphylococcal enterotoxin B/texosomes as a candidate for breast cancer immunotherapy.

    PubMed

    Imani Fooladi, Abbas Ali; Halabian, Raheleh; Mahdavi, Mehdi; Amin, Mohsen; Mahmoodzadeh Hosseini, Hamideh

    2016-01-01

    A new recombinant construct made up of two components, texosomes (TEX) and staphylococcal enterotoxin B (SEB), showed cytostatic properties against several types of tumor cells in vitro. Here, we aimed to assess the construct's antitumor immunogenicity in a murine tumor model. SEB was anchored onto purified texosomes and was used for immunization of mice before challenge with 4T1 cells. Tumor size, survival time, necrosis, metastasis rate, and the levels of IL-2, IL-4, IL-17, IL-12, TNF-α, and IFN-γ were measured. Immunization of the mice with TEX-SEB increased the stimulation index of splenocytes significantly compared with the PBS-treated mice (p < 0.01). In addition, there was a significant increase of TNF-α, IL-2, and IFN-γ secreted from isolated splenocytes of the mice immunized by either TEX-SEB, TEX + SEB, TEX, or SEB in comparison with PBS (p < 0.001, p < 0.001, and p < 0.05, respectively), whereas no significant change of IL-4 secretion was observed in any treated groups. Finding from tumor tissue homogenate testing showed that the level of IL-17 and IFN-γ among mice immunized with TEX-SEB was significantly lower than PBS-treated group (p < 0.05). IL-12, IL-4, and TNF-α levels were not significantly different from PBS- and TEX-SEB-immunized groups except in the SEB-immunized mice. Although TEX-SEB immunization relatively prolonged the survival of the mice, it had no inhibitory impact on tumor size. Pathologic manifestations showed the significant rise of necrosis after immunization with TEX-SEB compared to PBS (p < 0.01). Overall, our findings suggest that the presence of SEB rescues tumorigenesis effects of TEX making the construct an appropriate candidate for tumor immunotherapy.

  7. Expression of Clostridium perfringens enterotoxin receptors claudin-3 and claudin-4 in prostate cancer epithelium.

    PubMed

    Long, H; Crean, C D; Lee, W H; Cummings, O W; Gabig, T G

    2001-11-01

    The mRNA for Rvp.1 (rat ventral prostate) increases in abundance before gland involution after androgen deprivation. Rvp.1 is homologous to CPE-R, the high-affinity intestinal epithelial receptor for Clostridium perfringens enterotoxin (CPE), and is sufficient to mediate CPE binding and trigger subsequent toxin-mediated cytolysis. Rvp.1 (claudin-3) and CPE-R (claudin-4) are members of a larger family of transmembrane tissue-specific claudin proteins that are essential components of intercellular tight junction structures regulating paracellular ion flux. However, claudin-3 and claudin-4 are the only family members capable of mediating CPE binding and cytolysis. The present study was designed to study the expression of claudin-3 and claudin-4 in human prostate tissue as potential targets for CPE toxin-mediated therapy for prostate cancer. On human multiple-tissue Northern blot analysis, mRNAs for both claudin-3 and claudin-4 were expressed at high levels in prostate tissue. In normal prostate tissue, expression of claudin-3 was localized exclusively within acinar epithelial cells by in situ mRNA hybridization. Compared with expression within prostate epithelial cells in surrounding normal glandular tissue, expression of claudin-3 mRNA remained high in the epithelium of prostate adenocarcinoma (10 of 10) and prostatic intraepithelial neoplasia (five of five). Prostate adenocarcinoma cells metastatic to bone were obtained from a patient with disease progression during antiandrogen therapy. These metastatic cells were prostate-specific antigen-positive by immunohistochemical staining and also expressed functional CPE receptors as measured by sensitivity to CPE-induced cell lysis. The persistent high level of claudin-3 expression in prostate adenocarcinoma and functional cytotoxicity of CPE in metastatic androgen-independent prostate adenocarcinoma suggests a new potential therapeutic strategy for prostate cancer.

  8. Rhinosinusitis derived Staphylococcal enterotoxin B possibly associates with pathogenesis of ulcerative colitis

    PubMed Central

    Yang, Ping-Chang; Liu, Tao; Wang, Bin-Quan; Zhang, Tao-Yuan; An, Zi-Yuan; Zheng, Peng-Yuan; Tian, Dao-Fa

    2005-01-01

    Background During clinical practice, we noticed that some patients with both ulcerative colitis (UC) and chronic rhinosinusitis (CRS) showed amelioration of UC after treatment of CRS. This study was designed to identify a possible association between CRS and UC. Methods Thirty-two patients with both CRS and UC received treatment with functional endoscopic sinus surgery (FESS) for CRS. Clinical symptom scores for CRS and UC, as well as serum levels of anti-Staphylococcal enterotoxin B (SEB) were evaluated at week 0 and week 12. Sinus wash fluid SEB content was measured with enzyme-linked immunosorbent assay (ELISA). The surgically removed tissues were cultured to identify growth of Staphylococcus. aureus (S. aureus). Immunohistochemistry was employed to identify anti-SEB positive cells in the colonic mucosa. Colonic biopsies were obtained and incubated with SEB. Mast cell activation in the colonic mucosa in response to incubation with SEB was observed with electron microscopy and immunoassay. Results The clinical symptom scores of CRS and UC severe scores (UCSS) were significantly reduced in the UC-CRS patients after FESS. The number of cultured S. aureus colonies from the surgically removed sinus mucosa significantly correlated with the decrease in UCSS. High levels of SEB were detected in the sinus wash fluids of the patients with UC-CRS. Histamine and tryptase release was significantly higher in the culture supernate in the patients with UC-CRS than the patients with UC-only and normal controls. Anti-SEB positive cells were located in the colonic mucosa. Conclusion The pathogenesis of UC in some patients may be associated with their pre-existing CRS by a mechanism of swallowing sinusitis-derived SEB. We speculate that SEB initiates inappropriate immune reactions and inflammation in the colonic mucosa that further progresses to UC. PMID:16144553

  9. Formation of cereulide and enterotoxins by Bacillus cereus in fermented African locust beans.

    PubMed

    Thorsen, Line; Azokpota, Paulin; Munk Hansen, Bjarne; Rønsbo, Mie Hvillum; Nielsen, Kristian Fog; Hounhouigan, D Joseph; Jakobsen, Mogens

    2011-12-01

    Afitin, iru and sonru are three spontaneously fermented African locust bean Benin condiments. The fermentation processes are exothermic, with temperatures mostly being above 40 °C. A total of 19 predominant Bacillus cereus isolates from afitin, iru and sonru, were investigated. The enterotoxin genes nhe (A, B, C) were present in all 19 isolates, the hbl (A, C, D) in one (afitin), and the cytK gene in three isolates (afitin). Levels of cytotoxicity to Vero cells and NheA production in BHI-broth was within the range of known diarrheal outbreak strains. Autoclaved cooked African locust beans inoculated with emetic (cereulide producing) B. cereus Ba18H2/RIF supported growth at 25, 30 and 40 °C with highly different maximum cereulide productions of 6 ± 5, 97 ± 3 and 0.04 ± 0.02 μg/g beans, respectively (48 h). For non-autoclaved cooked beans inoculated with 2, 4 and 6 log(10)B. cereus Ba18H2/RIF spores/g beans, cereulide production was 5 ± 4, 64 ± 8 and 69 ± 34 μg/g beans, respectively at 24 h, while it was 70 ± 43, 92 ± 53 and 99 ± 31 μg/g at 48 h of fermentation at 30 °C. Even though high toxin levels were observed, to date there are no known reports on diarrhea or vomiting due to the consumption or afitin, iru and sonru in Benin, which also according to the present study is likely to be expected from the low levels of cereulide produced at 40 °C.

  10. Prolonged expression and production of Staphylococcus aureus enterotoxin A in processed pork meat.

    PubMed

    Wallin-Carlquist, Nina; Márta, Dóra; Borch, Elisabeth; Rådström, Peter

    2010-07-31

    The bacteriophage-encoded staphylococcal enterotoxin A (SEA) is the toxin most frequently reported to be involved in staphylococcal food poisoning. In this study, sea expression and SEA formation were studied in four processed pork products: boiled ham, hot-smoked ham, Serrano ham (dry-cured Spanish ham) and black pepper salami. The products were selected because of their differences in intrinsic factors. As a reference, Staphylococcus aureus was cultivated under favorable planktonic growth conditions. Expression was mainly linked to bacterial growth for both meat products and broth cultures. In liquid broth, however, the relative level of sea mRNA peaked in the late exponential phase and then rapidly declined, while in the meat products allowing immediate growth, i.e. boiled and smoked ham, active sea expression occurred throughout the incubation period of seven days. Lower levels of sea mRNA and SEA were found in smoked ham compared to boiled ham, although viable counts of S. aureus on the two products were similar. Furthermore, the SEA concentration in the boiled ham reached a maximum after three days of incubation and then unpredictably decreased. In the Serrano ham, no increase in cell number was observed until day seven, and sea expression and extracellular SEA could only be detected on days five and seven. Finally, the black pepper salami with low pH and competing microbiota proved to be a difficult environment for the survival of S. aureus. The molecular mechanism behind the behaviour of S. aureus SEA expression is discussed in connection to the life-cycle of the SEA-encoding bacteriophage and the microbial communities in these pork products.

  11. Clonal spread of catalase-negative ST5/SCCmec II Staphylococcus aureus carrying the staphylococcal enterotoxin A (sea), staphylococcal enterotoxin b (seb), and toxic shock toxin (tst) virulence genes.

    PubMed

    Lee, Hae Kyung; Kim, Jung-Beom; Kim, Hyunjung; Jekarl, Dong Wook; Kim, Yang Ree; Yu, Jin Kyung; Park, Yeon-Joon

    2014-01-01

    17 catalase-negative methicillin-resistant Staphylococcus aureus (MRSA) isolates were recovered from respiratory specimens of patients at a 700-bed hospital in Korea. The goal of this study was to determine the molecular characteristics of catalase-negative MRSA strains in Korea for the first time. Characteristics that we explored included kat A gene mutation sequence, sequence type, staphylococcal cassette chromosome (SCC) mec subtype classification, and toxin gene profiles. All 17 isolates showed similar pulsed field gel electrophoresis (PFGE) pattern. Four mutations were identified in the kat A gene of a representative catalase-negative MRSA strain: A602G causing a histidine 201 to arginine change, A695T causing a glutamic acid 232 to valine change, T778A causing a tryptophan 260 to arginine change, and G1438A causing a glycine 480 to serine change. Previous studies suggest that the A695T and T778A mutations may have strong effects on the catalase activity of catalase-negative MRSA. The sequence type (ST) and SCCmec type of this isolate were ST 5 and SCCmec type II, respectively. All 17 isolates harbored toxic shock toxin (tst), staphylococcal enterotoxin A (sea), and staphylococcal enterotoxin B (seb) virulence genes. The mortality rate of the present study was 11.8%, suggesting that the clinical relevance of catalase-negative MRSA requires further study in the future.

  12. Development of a near-real-time procedure to detect Staphylococcus aureus enterotoxin A in military rations

    NASA Astrophysics Data System (ADS)

    Richardson, Michelle J.; Rand, Arthur G.; Senecal, Andre G.

    2002-02-01

    Using a chemiluminescent fiber optic biosensor and magnetic particles, a simple, sensitive and rapid method to determine Staphylococcus aureus enterotoxin A (SEA) in military ration components was developed. Anti-staphylococcal enterotoxin A (Anti-SEA) was immobilized on magnetic particles and incubated with SEA. The beads were then collected and rinsed on a membrane filter (0.45um). The captured toxin was then selectively labeled with a monoclonal-horseradish peroxidase (POD) conjugate. SEA concentration was detected with a luminometer and a chemiluminescent enhancing reagent. Total assay time was 1.25 hours. Chemiluminescent signal due to nonspecific binding was tested with various blocking agents. Phosphate buffered saline with casein had the lowest background signal. Primary antibody concentration, secondary labeled antibody concentration and chemiluminescent substrate type were also evaluated to optimize signal intensity. The chemiluminescent fiber optic biosensor assay was compared to the Analyte 2000, a commercial fluorescent fiber optic biosensor. This assay consisted of immobilizing Anti-SEA on polystyrene waveguides, and incubating the waveguides with the toxin. The waveguide was incubated with a selectively labeled monoclonal-CY5 Dye conjugate. The sensitivity of chemiluminescent and fluorescent immunoassays were 1 ng, significantly lower than the levels needed to cause illness.

  13. Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli.

    PubMed

    Chen, Weifeng; Hu, Li; Liu, Aiping; Li, Jinquan; Chen, Fusheng; Wang, Xiaohong

    2014-11-01

    The staphylococcal enterotoxins (SEs) are potent gastrointestinal exotoxins synthesized by Staphylococcus aureus, which is responsible for various diseases including septicemia, food poisoning, and toxic shock syndrome, as well as bovine mastitis. Among them, staphylococcal enterotoxin A (SEA) is one of the most commonly present serotypes in staphylococcal food poisoning cases. In this study, the stable hybridoma 3C12 producing anti-SEA monoclonal antibody was established with an equilibrium dissociation constant (KD) of 1.48 × 10(-8) mol·L(-1), its ScFv-coding genes were obtained and then the anti-SEA single chain variable fragment (ScFv) protein was expressed in Escherichia coli. Characterization of the expressed target ScFv protein was analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis, Western blot, and enzyme-linked immunosorbent assay. The results demonstrated that the recombinant anti-SEA ScFv protein retained a specific binding activity for SEA, and the KD value of the soluble ScFv was about 3.75 × 10(-7) mol·L(-1). The overall yield of bioactive anti-SEA ScFv in E. coli flask culture was more than 10 mg·L(-1).

  14. Quantitative analysis of Staphylococcus enterotoxin A by differential expression of IFN-gamma in splenocyte and CD4+ T-cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcus aureus is an important bacterial pathogen that produces a range of Staphylococcal Enterotoxins (SEs) which cause gastroenteritis and superantigen activation of T cells, the mechanism of which is not well understood. The ability to rapidly detect and quantify SEs is very important in ...

  15. Enterotoxigenic profiling of emetic toxin- and enterotoxin-producing Bacillus cereus, Isolated from food, environmental, and clinical samples by multiplex PCR.

    PubMed

    Forghani, Fereidoun; Kim, Jung-Beom; Oh, Deog-Hwan

    2014-11-01

    Bacillus cereus comprises the largest group of endospore-forming bacteria and can cause emetic and diarrheal food poisoning. A total of 496 B. cereus strains isolated from various sources (food, environmental, clinical) were assessed by a multiplex PCR for the presence of enterotoxin genes. The detection rate of nheA, entFM, hblC, and cytK enterotoxin genes among all B. cereus strains was 92.33%, 77.21%, 59.47%, and 47.58%, respectively. Enterotoxigenic profiles were determined in emetic toxin- (8 patterns) and enterotoxin-producing strains (12 patterns). The results provide important information on toxin prevalence and toxigenic profiles of B. cereus from various sources. Our findings revealed that B. cereus must be considered a serious health hazard and Bacillus thuringiensis should be considered of a greater potential concern to food safety among all B. cereus group members. Also, there is need for intensive and continuous monitoring of products embracing both emetic toxin and enterotoxin genes.

  16. Development of a Multiplex PCR Method for Detection of the Genes Encoding 16S rRNA, Coagulase, Methicillin Resistance and Enterotoxins in Staphylococcus aureus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex PCR method was developed for simultaneous detection of the genes encoding methicillin resistance (mecA), staphylococcal enterotoxins A, B and C (sea, seb and sec), coagulase (coa) and 16S rRNA. The primers for amplification of the 16S rRNA gene were specific for Staphylococcus spp., and ...

  17. Linkage of heat-stable enterotoxin activity and ampicillin resistance in a plasmid isolated from an Escherichia coli strain of human origin.

    PubMed Central

    Stieglitz, H; Fonseca, R; Olarte, J; Kupersztoch-Portnoy, Y M

    1980-01-01

    In an Escherichia coli strain of human origin, ampicillin resistance and heat-stable enterotoxin activity were shown by EcoRI restriction endonuclease and genetic analysis to be in an 80-megadalton plasmid. Images Fig. 1 Fig. 2 PMID:6254890

  18. Electrical percolation-based biosensor for real-time direct detection of staphylococcal enterotoxin B (SEB).

    PubMed

    Yang, Minghui; Sun, Steven; Bruck, Hugh Alan; Kostov, Yordan; Rasooly, Avraham

    2010-08-15

    Electrical percolation-based biosensing is a new technology. This is the first report of an electrical percolation-based biosensor for real-time detection. The label-free biosensor is based on electrical percolation through a single-walled carbon nanotubes (SWNTs)-antibody complex that forms a network functioning as a "Biological Semiconductor" (BSC). The conductivity of a BSC is directly related to the number of contacts facilitated by the antibody-antigen "connectors" within the SWNT network. BSCs are fabricated by immobilizing a pre-functionalized SWNTs-antibody complex directly on a poly(methyl methacrylate) (PMMA) and polycarbonate (PC) surface. Each BSC is connected via silver electrodes to a computerized ohmmeter, thereby enabling a continuous electronic measurement of molecular interactions (e.g. antibody-antigen binding) via the change in resistance. Using anti-staphylococcal enterotoxin B (SEB) IgG to functionalize the BSC, we demonstrate that the biosensor was able to detect SEB at concentrations as low as 5 ng/mL at a signal to baseline (S/B) ratio of 2. Such measurements were performed on the chip in wet conditions. The actuation of the chip by SEB is immediate, permitting real-time signal measurements. In addition to this "direct" label-free detection mode, a secondary antibody can be used to "label" the target molecule bound to the BSC in a manner analogous to an immunological sandwich "indirect" detection-type assay. Although a secondary antibody is not needed for direct detection, the indirect mode of detection may be useful as an additional measurement to verify or amplify signals from direct detection in clinical, food safety and other critical assays. The BSC was used to measure SEB both in buffer and in milk, a complex matrix, demonstrating the potential of electrical percolation-based biosensors for real-time label-free multi-analyte detection in clinical and complex samples. Assembly of BSCs is simple enough that multiple sensors can be

  19. Staphylococcal enterotoxin type A internal deletion mutants: serological activity and induction of T-cell proliferation.

    PubMed Central

    Harris, T O; Hufnagle, W O; Betley, M J

    1993-01-01

    Previous findings indicate that the N-terminal region of staphylococcal enterotoxin type A (SEA) is required for its ability to induce T-cell proliferation. To better localize internal peptides of SEA that are important for induction of murine T-cell proliferation, SEA mutants that had internal deletions in their N-terminal third were constructed. A series of unique restriction enzyme sites were first engineered into sea; only one of these changes resulted in an amino acid substitution (the aspartic acid residue at position 60 of mature SEA was changed to a glycine [D60G]). Because the D60G substitution had no discernible effect on serological or biological activity, the sea allele encoding this mutant SEA was used to construct a panel of mutant SEAs lacking residues 3 to 17, 19 to 23, 24 to 28, 29 to 49, 50 to 55, 56 to 59, 61 to 73, 68 to 74, or 74 to 85. Recombinant plasmids with the desired mutations were constructed in Escherichia coli and transferred to Staphylococcus aureus. Staphylococcal culture supernatants containing the mutant SEAs were examined. Western immunoblot analysis with polyclonal anti-SEA antiserum revealed that each of the recombinant S. aureus strains produced a mutant SEA of the predicted size. All the mutant SEAs exhibited increased sensitivity to monkey stomach lavage fluid in vitro, which is consistent with these mutants having conformations unlike that of wild-type SEA or the SEA D60G mutant. In general, deletion of internal peptides had a deleterious effect on the ability to induce T-cell proliferation; only SEA mutants lacking either residues 3 to 17 or 56 to 59 consistently produced a statistically significant increase in the incorporation of [3H]thymidine. In the course of this work, two monoclonal antibodies that had different requirements for binding to SEA in Western blots were identified. The epitope for one monoclonal antibody was contained within residues 108 to 230 of mature SEA. Binding of the other monoclonal antibody to

  20. Suppression of colorectal tumorigenesis by recombinant Bacteroides fragilis enterotoxin-2 in vivo

    PubMed Central

    Lv, You; Ye, Tao; Wang, Hui-Peng; Zhao, Jia-Ying; Chen, Wen-Jie; Wang, Xin; Shen, Chen-Xia; Wu, Yi-Bin; Cai, Yuan-Kun

    2017-01-01

    AIM To evaluate the impact of recombinant Bacteroides fragilis enterotoxin-2 (BFT-2, or Fragilysin) on colorectal tumorigenesis in mice induced by azoxymethane/dextran sulfate sodium (AOM/DSS). METHODS Recombinant proBFT-2 was expressed in Escherichia coli strain Rosetta (DE3) and BFT-2 was obtained and tested for its biological activity via colorectal adenocarcinoma cell strains SW-480. Seventy C57BL/6J mice were randomly divided into a blank (BC; n = 10), model (AD; n = 20), model + low-dose toxin (ADLT; n = 20, 10 μg), and a model + high-dose toxin (ADHT; n = 20, 20 μg) group. Mice weight, tumor formation and pathology were analyzed. Immunohistochemistry determined Ki-67 and Caspase-3 expression in normal and tumor tissues of colorectal mucosa. RESULTS Recombinant BFT-2 was successfully obtained, along with its biological activity. The most obvious weight loss occurred in the AD group compared with the ADLT group (21.82 ± 0.68 vs 23.23 ± 0.91, P < 0.05) and the ADHT group (21.82 ± 0.68 vs 23.57 ± 1.06, P < 0.05). More tumors were found in the AD group than in the ADLT and ADHT groups (19.75 ± 3.30 vs 6.50 ± 1.73, P < 0.05; 19.75 ± 3.30 vs 6.00 ± 2.16, P < 0.05). Pathology showed that 12 mice had adenocarcinoma and 6 cases had adenoma in the AD group. Five mice had adenocarcinoma and 15 had adenoma in the ADLT group. Four mice had adenocarcinoma and 16 had adenoma in the ADHT group. The incidence of colorectal adenocarcinoma in both the ADHT group and the ADHT group was reduced compared to that in the AD group (P < 0.05, P < 0.05). The positive rate of Ki-67 in the ADLT group and the ADHT group was 50% and 40%, respectively, both of which were lower than that found in the AD group (94.44%, P < 0.05, P < 0.05). Caspase-3 expression in the ADLT group and the ADHT group was 45% and 55%, both of which were higher than that found in the BC group (16.67%, P < 0.05, P < 0.05). CONCLUSION Oral administration with lower-dose biologically active recombinant BFT-2

  1. Clostridium perfringens enterotoxin as a potential drug for intravesical treatment of bladder cancer.

    PubMed

    Gabig, Theodore G; Waltzer, Wayne C; Whyard, Terry; Romanov, Victor

    2016-09-16

    The current intravesical treatment of bladder cancer (BC) is limited to a few chemotherapeutics that show imperfect effectiveness and are associated with some serious complications. Thus, there is an urgent need for alternative therapies, especially for patients with high-risk non-muscle invasive (NMIBC). Clostridium perfringens enterotoxin (CPE), cytolytic protein binds to its receptors: claudin 3 and 4 that are expressed in epithelial cells. This binding is followed by rapid cell death. Claudin 4 is present in several epithelial tissue including bladder urothelium and its expression is elevated in some forms of BC. In addition to directly targeting BC cells, binding of CPE to claudins increases urothelium permeability that creates conditions for better accession of the tumor. Therefore, we evaluated CPE as a candidate for intravesical treatment of BC using a cellular model. We examined cytotoxicity of CPE against BC cells lines and 3D cultures of cells derived from surgical samples. To better elucidate cellular mechanisms, activated by CPE and to consider the use of CPE non-toxic fragment (C-CPE) for combination treatment with other drugs we synthesized C-CPE, compared its cytotoxic activity with CPE and examined claudin 4 expression and intracellular localization after C-CPE treatment. CPE induced cell death after 1 h in low aggressive RT4 cells, in moderately aggressive 5637 cells and in the primary 3D cultures of BC cells derived from NMIBC. Conversely, non-transformed urothelial cells and cells derived from highly aggressive tumor (T24) survived this treatment. The reason for this resistance to CPE might be the lower expression of CLDNs or their inaccessibility for CPE in these cells. C-CPE treatment for 48 h did not affect cell viability in tested cells, but declined expression of CLDN4 in RT4 cells. C-CPE increased sensitivity of RT4 cells to Mitommycin C and Dasatinib. To better understand mechanisms of this effect we examined expression and

  2. Structure of Staphylococcal Enterotoxin E in Complex with TCR Defines the Role of TCR Loop Positioning in Superantigen Recognition.

    PubMed

    Rödström, Karin E J; Regenthal, Paulina; Lindkvist-Petersson, Karin

    2015-01-01

    T cells are crucial players in cell-mediated immunity. The specificity of their receptor, the T cell receptor (TCR), is central for the immune system to distinguish foreign from host antigens. Superantigens are bacterial toxins capable of inducing a toxic immune response by cross-linking the TCR and the major histocompatibility complex (MHC) class II and circumventing the antigen specificity. Here, we present the structure of staphylococcal enterotoxin E (SEE) in complex with a human T cell receptor, as well as the unligated T cell receptor structure. There are clear structural changes in the TCR loops upon superantigen binding. In particular, the HV4 loop moves to circumvent steric clashes upon complex formation. In addition, a predicted ternary model of SEE in complex with both TCR and MHC class II displays intermolecular contacts between the TCR α-chain and the MHC, suggesting that the TCR α-chain is of importance for complex formation.

  3. Trends in detection of warfare agents. Detection methods for ricin, staphylococcal enterotoxin B and T-2 toxin.

    PubMed

    Ler, Siok Ghee; Lee, Fook Kay; Gopalakrishnakone, P

    2006-11-10

    An overview of the different detection methods available for ricin, staphylococcal enterotoxin B (SEB) and T-2 toxin is presented here. These toxins are potential biological warfare agents (BWA). The aim of this review is not to cover all the papers that had been published but rather to give an overall picture of the trend in the detection methodologies for potential biological warfare agents as we do see the emerging threats from these three toxins. The advantages and disadvantages of each methodology as well as the detection limit will be reviewed. It seems that mass spectrometry has created a niche for analysis of proteinaceous toxins, ricin and SEB as well as molecular toxin, T-2 toxin given its high sensitivity, high selectivity, high specificity and capability to identify and quantify unknown agents simultaneously in a short time frame. But its main drawbacks are its sophisticated instrumentation and its high cost. Improvised immunoassay may be an alternative.

  4. Prevention of nonspecific reactions on reversed passive latex agglutination assay (RPLA) for detecting low amounts of staphylococcal enterotoxins.

    PubMed

    Pereira, M L; Heneine, L G; Santos, E J; Carmo, L S; Pereira, J L; Bergdoll, M S

    1997-01-01

    The SET-RPLA, from Denka Seiken Co. Ltd., Tokio, a commercial reversed passive latex agglutination test kit, has been recommended to establish the enterotoxicity capacity of some staphylococcal strains, implicated in food poisoning outbreaks that produce low levels of enterotoxins (SE). Despite the RPLA specificity, the occurrence of nonspecific reactions when testing low-SE-producing is common. In order to control these nonspecific reactions the addition of purified normal rabbit IgG purified was applied on approximately 350 staphylococcal isolates from human milk and anatomic sites of healthy dental student carriers. The results indicated that addition of 5% (v/v) of purified normal rabbit IgG (0.74 mg/mL) to the culture supernatant fluid is a simple and reliable tool for the controlling of nonspecific reactions in the RPLA assay.

  5. Estimation of human dose of staphylococcal enterotoxin A from a large outbreak of staphylococcal food poisoning involving chocolate milk.

    PubMed

    Evenson, M L; Hinds, M W; Bernstein, R S; Bergdoll, M S

    1988-12-31

    An outbreak of gastroenteritis in a school district in the United States was determined to be staphylococcal food poisoning due to 2% chocolate milk containing staphylococcal enterotoxin A (SEA). Twelve one-half pint (approx 0.28 l) cartons of the 2% chocolate milk from this outbreak were analyzed for the quantity of SEA present in the milk. The amount of SEA in the cartons varied from 94 to 184 ng with the average being 144 ng (mean = 139 +/- 45). The attack rate for vomiting among those who consumed more than one carton was greater (38.3%) than among those who consumed only one carton (31.5%) with the highest attack rate among those who consumed three or more cartons (44.4%).

  6. Staphylococcal enterotoxin B toxic shock syndrome induced by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA).

    PubMed

    Kashiwada, Takeru; Kikuchi, Ken; Abe, Shinji; Kato, Hidehito; Hayashi, Hiroki; Morimoto, Taisuke; Kamio, Koichiro; Usuki, Jiro; Takeda, Shinhiro; Tanaka, Keiji; Imanishi, Ken'ichi; Yagi, Junji; Azuma, Arata; Gemma, Akihiko

    2012-01-01

    We herein report a case of toxic shock syndrome (TSS) associated with the 2009 pandemic H1N1 (pH1N1) influenza virus and a community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infection in a 16-year-old Vietnamese girl. Staphylococcal enterotoxin B (SEB) was detected in the patient's serum, and the level of anti-SEB antibodies was found to be elevated. A flow cytometric analysis showed evidence of activated SEB-reactive Vβ3+ and Vβ12+ T cells. These data suggest that the CA-MRSA-induced activation of SEB-reactive T cells may cause TSS in patients with pH1N1 virus infection. Moreover, this is the first report describing immunological confirmation of SEB contributing directly to TSS in a patient fulfilling the diagnostic criteria of TSS.

  7. Metaphase yields from staphylococcal enterotoxin A stimulated peripheral blood lymphocytes of unirradiated and irradiated aged rhesus monkeys

    NASA Technical Reports Server (NTRS)

    Hill, F. S.; Cox, A. B.; Salmon, Y. L.; Cantu, A. O.; Lucas, J. N.

    1994-01-01

    The mitogen phytohemagglutinin (PHA) works well in both human and cynomolgus monkey (Macaca fascicularis) lymphocyte cultures to stimulate T cell proliferation. T cells from rhesus monkeys (Macaca mulatta) are less responsive than human cells, producing few metaphases when thousands are required, e.g. in biological dosimetry studies. We show that staphylococcal enterotoxin A (SEA), one of the most potent mitogens known, at a concentration of 0.5 microgram/ml stimulated peripheral lymphocytes to grow with a mitotic index (MI) averaging 0.13 metaphases/cell in old, irradiated rhesus macaques. This was significantly greater (p < 0.001) than that produced by PHA (MI < 0.01) in lymphocytes from the same animals. Whole blood was cultured for 96, 120 and 144 h for five irradiated individuals and for two controls. All cells cultured with SEA produced a high MI with a peak response at 120 h whereas the same cultures showed low MI for each PHA stimulated culture.

  8. Antidiarrheal activity of wood creosote: inhibition of muscle contraction and enterotoxin-induced fluid secretion in rabbit small intestine.

    PubMed

    Ogata, N; Shibata, T

    2001-01-01

    Wood creosote has long been used as an antidiarrheal agent, but its mechanism of action is not well understood. To elucidate the mechanism of its antidiarrheal activity, we have addressed questions whether it inhibits fluid secretion induced by Escherichia coli heat-stable enterotoxin (STa) in rabbit jejunum in vivo, and whether it inhibits muscle contraction of isolated rabbit ileum ex vivo. Wood creosote (10-100 mg/l) instilled in a ligated loop of jejunum inhibited STa-induced fluid secretion (p < 0.05). It also inhibited the spontaneous phasic, acetylcholine-induced tonic and Ba2+-induced tonic contractions of longitudinal and circular muscles of ileum dose-dependently with IC(50) values of 130-530 mg/l. These data provide further evidence that the antidiarrheal activity of wood creosote is attributable to its antisecretory and antimotility effects.

  9. Characterization of heat-stable enterotoxin from a hypertoxigenic Escherichia coli strain that is pathogenic for cattle.

    PubMed Central

    Saeed, A M; Magnuson, N S; Gay, C C; Greenberg, R N

    1986-01-01

    An enterotoxigenic Escherichia coli (ETEC) strain isolated from a calf with clinical scours was found to produce over 17- to 60-fold more heat-stable enterotoxin (STa) than four laboratory-adapted bovine ETEC strains. The purified STa of this strain was identical to those produced by other ETEC strains. A severe form of scours was induced in 5- to 15-day-old colostrum-fed calves and in 1- to 2-week-old piglets by oral administration of the purified STa. This study demonstrates that STa is a mediator of diarrhea in newborn calves and piglets and that under identical growth conditions diverse strains of bovine ETEC may produce variable amounts of homologous STa's. PMID:3525417

  10. Prevalence of staphylococcal enterotoxins, toxin genes and genetic-relatedness of foodborne Staphylococcus aureus strains isolated in the Marmara Region of Turkey.

    PubMed

    Aydin, Ali; Sudagidan, Mert; Muratoglu, Karlo

    2011-08-02

    Staphylococcus aureus is a major foodborne pathogen and it has the ability to produce a number of extracellular toxins. We analyzed 1070 food samples obtained from retail markets and dairy farms in the Marmara Region of Turkey for the presence of S. aureus. Out of 147 isolates, 92 (62.6%) were enterotoxigenic. PCR was used to investigate the presence of staphylococcal enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq and seu), exfoliative toxin genes (eta and etb) and the toxic-shock syndrome toxin gene (tst). The PCR results showed that 53.3% of the isolates contained staphylococcal enterotoxin-like (SEl) toxin genes (seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq and seu) which were more frequent than classical enterotoxin genes (sea to see). Furthermore, seo, sei, sem, seg, seu and sec were found in 37.0, 32.7, 30.4, 29.3, 29.3 and 27.2% of the isolates, respectively. The tst gene was detected and confirmed by DNA sequencing in 9 isolates. The presence of eta and etb were not found in the isolates. Enterotoxigenic capabilities of isolates with SEA-SEE were investigated by ELISA. Enterotoxigenic S. aureus isolates produced one to three enterotoxins, with the most frequently produced types being enterotoxin A and C. There was a correlation of 72.1% between production of a specific toxin and the presence of the respective genes. PFGE analysis was used to identify genetic-relatedness of enterotoxigenic S. aureus isolates and the results revealed that 13 groups of isolates from different or the same origin that contained the same genes showed 100% homology with indistinguishable band patterns. The other enterotoxigenic isolates showed related band patterns with 72-86% homology in sea-, 61-90% homology in sec-, 80-96% homology in seh-, and 69-96% homology in sep-positive isolates. To our knowledge, this is the first study to examine enterotoxins and related gene contents of S. aureus food isolates in the Marmara

  11. Crystal structure of staphylococcal enterotoxin I (SEI) in complex with a human major histocompatibility complex class II molecule.

    PubMed

    Fernández, Marisa M; Guan, Rongjin; Swaminathan, Chittoor P; Malchiodi, Emilio L; Mariuzza, Roy A

    2006-09-01

    Superantigens are bacterial or viral proteins that elicit massive T cell activation through simultaneous binding to major histocompatibility complex (MHC) class II and T cell receptors. This activation results in uncontrolled release of inflammatory cytokines, causing toxic shock. A remarkable property of superantigens, which distinguishes them from T cell receptors, is their ability to interact with multiple MHC class II alleles independently of MHC-bound peptide. Previous crystallographic studies have shown that staphylococcal and streptococcal superantigens belonging to the zinc family bind to a high affinity site on the class II beta-chain. However, the basis for promiscuous MHC recognition by zinc-dependent superantigens is not obvious, because the beta-chain is polymorphic and the MHC-bound peptide forms part of the binding interface. To understand how zinc-dependent superantigens recognize MHC, we determined the crystal structure, at 2.0 A resolution, of staphylococcal enterotoxin I bound to the human class II molecule HLA-DR1 bearing a peptide from influenza hemagglutinin. Interactions between the superantigen and DR1 beta-chain are mediated by a zinc ion, and 22% of the buried surface of peptide.MHC is contributed by the peptide. Comparison of the staphylococcal enterotoxin I.peptide.DR1 structure with ones determined previously revealed that zinc-dependent superantigens achieve promiscuous binding to MHC by targeting conservatively substituted residues of the polymorphic beta-chain. Additionally, these superantigens circumvent peptide specificity by engaging MHC-bound peptides at their conformationally conserved N-terminal regions while minimizing sequence-specific interactions with peptide residues to enhance cross-reactivity.

  12. Subinhibitory Concentrations of Thymol Reduce Enterotoxins A and B and α-Hemolysin Production in Staphylococcus aureus Isolates

    PubMed Central

    Xiang, Hua; Feng, Haihua; Jiang, Youshuai; Xia, Lijie; Dong, Jing; Lu, Jing; Yu, Lu; Deng, Xuming

    2010-01-01

    Background Targeting bacterial virulence factors is now gaining interest as an alternative strategy to develop new types of anti-infective agents. It has been shown that thymol, when used at low concentrations, can inhibit the TSST-1 secretion in Staphylococcus aureus. However, there are no data on the effect of thymol on the production of other exotoxins (e.g., α-hemolysin and enterotoxins) by S. aureus. Methodology/Principal Findings Secretion of α-hemolysin, SEA and SEB in both methicillin-sensitive and methicillin-resistant S. aureus isolates cultured with graded subinhibitory concentrations of thymol was detected by immunoblot analysis. Hemolysin and tumor necrosis factor (TNF) release assays were performed to elucidate the biological relevance of changes in α-hemolysin, SEA and SEB secretion induced by thymol. In addition, the influence of thymol on the transcription of hla, sea, and seb (the genes encoding α-hemolysin, SEA and SEB, respectively) was analyzed by quantitative RT-PCR. Thymol inhibited transcription of hla, sea and seb in S. aureus, resulting in a reduction of α-hemolysin, SEA and SEB secretion and, thus, a reduction in hemolytic and TNF-inducing activities. Conclusions/Significance Subinhibitory concentrations of thymol decreased the production of α-hemolysin, SEA and SEB in both MSSA and MRSA in a dose-dependent manner. These data suggest that thymol may be useful for the treatment of S. aureus infections when used in combination with β-lactams and glycopeptide antibiotics, which induce expression of α-hemolysin and enterotoxins at subinhibitory concentrations. Furthermore, the structure of thymol may potentially be used as a basic structure for development of drugs aimed against these bacterial virulence factors. PMID:20305813

  13. Production of staphylococcal enterotoxins in microbial broth and milk by Staphylococcus aureus strains harboring seh gene.

    PubMed

    Schubert, Justyna; Podkowik, Magdalena; Bystroń, Jarosław; Bania, Jacek

    2016-10-17

    Twenty Staphylococcus aureus strains harboring seh gene, including one carrying also sec gene and 11 sea gene, were grown in BHI+YE broth and milk and were tested for SEA, SEC and SEH production. All strains decreased pH of BHI+YE broth at 24h and increased them at 48h. Seventeen S. aureus strains grown in milk changed pH for no >0.3 unit until 48h. Three other S. aureus strains significantly decreased pH during growth in milk. All S. aureus produced SEH in BHI+YE broth in amounts ranging from 95 to 1292ng/ml, and from 170 to 4158ng/ml at 24 and 48h, respectively. SEH production in milk by 17 strains did not exceed 23ng/ml at 24h and 36ng/ml at 48h. Three S. aureus strains able to decrease milk pH produced 107-3029ng/ml and 320-4246ng/ml of SEH in milk at 24 and 48h, respectively. These strains were grown in milk and BHI+YE broth with pH stabilized at values near neutral leading to a significant decrease of SEH production. Representative weak SEH producers were grown in milk at reduced pH resulting in moderate increase in SEH production. SEA was produced in milk by 10S. aureus strains at 24-151ng/ml at 24h, and 31-303ng/ml at 48h. SEA production in milk was higher or comparable as in BHI+YE broth in 3 strains and lower for remaining strains. Production of SEC by sec-positive S. aureus strains was lower in milk than in BHI+YE broth, ranging from 131 to 2319ng/ml at 24 and 48h in milk and 296-30,087ng/ml in BHI+YE at 24 and 48h. Both lacE and lacG transcripts involved in lactose metabolism were significantly up-regulated in milk in strong SEH producers. In these strains hld, rot and sarA transcripts were up-regulated in milk as compared to weak SEH producers. Stabilization of milk pH at a value of raw milk significantly down-regulated hld, rot and sarA RNA in strong SEH producers. Milk was generally found unfavorable for enterotoxin production. However, certain S. aureus strains were not restricted in SEH and SEA expression in milk, unlike SEC which remained down

  14. Design of a Photoreactive Analogue of the Escherichia coli Heat-Stable Enterotoxin STIb: Use in Identifying Its Receptor on Rat Brush Border Membranes

    NASA Astrophysics Data System (ADS)

    Gariepy, Jean; Schoolnik, Gary K.

    1986-01-01

    The Escherichia coli heat-stable enterotoxin, STIb was prepared by solid-phase peptide synthesis and purified to homogeneity by high-pressure liquid chromatography. This analogue was iodinated and shown to bind specifically to rat intestinal membranes. The radiolabeled peptide was derivatized at the amino terminus with the photoreactive heterobifunctional crosslinking agent N-hydroxysuccinimidyl p-benzoylbenzoate. This photoreactive probe also exhibited binding specificity. It was mixed with rat intestinal brush border membranes and photolyzed in the presence or absence of excess unlabeled STIb. Polyacrylamide gel electrophoresis performed in the presence of sodium dodecyl sulfate and 2-mercaptoethanol indicated that the peptide probe was cross-linked specifically to two molecular species of 57 and 75 kDa. One or both of these molecules appear to constitute the enterotoxin receptor or to be in close proximity to it.

  15. Analysis of Heat-Labile Sites Generated by Reactions of Depleted Uranium and Ascorbate in Plasmid DNA

    PubMed Central

    Wilson, Janice; Young, Ashley; Civitello, Edgar R.

    2013-01-01

    The goal of this study was to characterize how depleted uranium (DU) causes DNA damage. Procedures were developed to assess the ability of organic and inorganic DNA adducts to convert to single strand breaks (SSB) in pBR322 plasmid DNA in the presence of heat or piperidine. DNA adducts formed by methyl methanesulfonate (MMS), cis-platin (cis-Pt), and chromic chloride were compared to those formed by reaction of uranyl acetate (UA) and ascorbate (Asc). Uranyl ion in the presence of Asc produced U-DNA adducts that converted to SSB upon heating. Piperidine, which acted on DNA methylated by MMS to convert methyl-DNA adducts to SSB, served in the opposite fashion with U-DNA adducts by decreasing SSB. The observation that piperidine also decreased the gel shift for metal-DNA adducts formed by monofunctional cis-Pt and chromic chloride was interpreted to suggest that piperidine served to remove U-DNA adducts. Radical scavengers did not affect formation of U-induced SSB, suggesting that SSB arose from the presence of U-DNA adducts and not from free radicals. A model is proposed to predict how U-DNA adducts may serve as initial lesions that convert to SSB or AP sites. Results suggest that DU can act as a chemical genotoxin that does not require radiation for its mode of action. Characterizing the DNA lesions formed by DU is necessary to assess the relative importance of different DNA lesions in the formation of DU-induced mutations. Understanding mechanisms of formation of DU-induced mutations may contribute to identification of biomarkers of DU exposures in humans. PMID:24218036

  16. Therapeutic Inhibition of Pro-Inflammatory Signaling and Toxicity to Staphylococcal Enterotoxin B by a Synthetic Dimeric BB-Loop Mimetic of MyD88

    DTIC Science & Technology

    2012-07-27

    C57BL/6 mice were obtained from Charles River (NCI-Frederick, Frederick, MD). Cell Isolation and Purification Peripheral blood mononuclear cells (MNC...7]. Consequently, both super- antigenic exotoxins (SEs) and bacterial LPS ( endotoxin ) have been implicated in the pathogenesis of TSS, supported by...enterotoxin B (SEB) and SEA was purchased from Porton Down, Inc. (Salisbury, UK) and stored at 250uC. SEB or SEA was endotoxin free and prepared under GMP

  17. Incidence of Salmonella, Listeria monocytogenes, Escherichia coli O157:H7, and Staphylococcal enterotoxin in two types of Mexican fresh cheeses.

    PubMed

    Torres-Vitela, M R; Mendoza-Bernardo, M; Castro-Rosas, J; Gomez-Aldapa, C A; Garay-Martinez, L E; Navarro-Hidalgo, V; Villarruel-López, A

    2012-01-01

    Handcrafted fresh cheeses are popular among consumers in Mexico. However, unsafe raw materials and inadequate food safety practices during cheese manufacture and preservation make them a potential public health risk. The incidence of Salmonella, Listeria, Escherichia coli O157:H7, and staphylococcal enterotoxin was analyzed in two types of fresh cheese (panela and adobera) commonly marketed in Mexico. A total of 200 samples, 100 panela and 100 adobera, were acquired from 100 wholesale milk product distributors who supply small retailers in the Guadalajara metropolitan area, Jalisco State, Mexico. Pathogens were identified using culture and immunoassay (miniVidas) methods. The presence of staphylococcal enterotoxin was determined by an immunoassay method. Of the 200 analyzed samples, 92 were positive for at least one of the pathogens. The incidence in the panela samples was 56%: 34% Salmonella, 16% E. coli O157:H7, and 6% L. monocytogenes. In the adobera samples, incidence was 36%: 20% Salmonella, 4% E. coli O157:H7, and 12% L. monocytogenes. Staphylococcal enterotoxin was not detected in any of the 200 samples. Choice of technique had no effect on detection of pathogen incidence, although the immunoassay method identified more Salmonella serotypes than the culture method. Handcrafted panela and adobera fresh cheeses in Mexico frequently contain pathogenic bacteria and therefore pose a public health risk.

  18. Effects of age and ambient temperature on the responses of infant mice to heat-stable enterotoxin of Escherichia coli: assay modifications.

    PubMed Central

    Moon, H W; Fung, P Y; Whipp, S C; Isaacson, R E

    1978-01-01

    The response of infant mice to heat-stable enterotoxin from Escherichia coli was affected by the age of the mice (2, 4, 6, and 8 days) and by the ambient temperature (25, 30, and 37 degrees C) after exposure to the enterotoxin. The younger mice and/or mice held at lower temperatures tended to accumulate intestinal fluid (high gut weight/body weight ratios), but older mice and/or mice held at higher temperatures tended to respond with diarrhea and low gut weight/body weight ratios. The standard infant mouse assay forheat-stable E. coli enterotoxin can be simplified, without loss of sensitivity or reliability, by holding the mice at 37 degrees C after exposure and using diarrhea as the index of response. Diarrhea can be detected easily by incorporating dye in the inocula and (at the end of the assay) checking for dye mixed with feces on the rear quarters of the mice or on a sheet of white paper placed under them during incubation. PMID:352935

  19. Development and application of a multiplex PCR assay for detection of the Clostridium perfringens enterotoxin-encoding genes cpe and becAB.

    PubMed

    Yonogi, Shinya; Kanki, Masashi; Ohnishi, Takahiro; Shiono, Masami; Iida, Tetsuya; Kumeda, Yuko

    2016-08-01

    Clostridium perfringens causes food-borne gastroenteritis following the consumption of contaminated food by producing C. perfringens enterotoxin (CPE) in the intestines. Recently, we reported a novel enterotoxin, binary enterotoxin of C. perfringens (BEC) in C. perfringens isolates, which caused two disease outbreaks in Japan. Consequently, in the event of food poisoning outbreaks caused by C. perfringens, it is now necessary to screen for both the cpe and becAB genes by diagnostic PCR. Here, we present a simple multiplex PCR method for simultaneous detection of cpe, becAB and a C. perfringens control locus, phospholipase C (plc). Applying this method, we investigated the prevalence of cpe- or becAB-carrying C. perfringens strains in human stool and bovine rectum swab samples. Using a total of 169 isolates, we found that the percentage of becAB-carrying strains was very small (0.59%), one-tenth that of cpe-carrying strains. The simple method presented in this study with high specificity and sensitivity to C. perfringens will be a useful tool to survey the global prevalence of becAB-carrying C. perfringens strains.

  20. Campomanesia adamantium Peel Extract in Antidiarrheal Activity: The Ability of Inhibition of Heat-Stable Enterotoxin by Polyphenols

    PubMed Central

    Lescano, Caroline Honaiser; de Oliveira, Ivan Pires; Zaminelli, Tiago; Baldivia, Débora da Silva; da Silva, Luan Ramos; Napolitano, Mauro; Silvério, Camila Bitencourt Mendes; Lincopan, Nilton; Sanjinez-Argandoña, Eliana Janet

    2016-01-01

    Campomanesia adamantium (Myrtaceae) is a medicinal plant distributed in Brazilian Cerrado. Different parts of this plant are used in popular medicine for treatment of several diseases like fever, diarrhea, hypercholesterolemia and rheumatism. The aim of this work was to evaluate the inhibition of heat-stable enterotoxin type A (STa) by gallic acid present in the peel of C. adamantium fruit and assays to assess the antidiarrheal activity, anti-inflammatory and cytotoxic properties of peel extract using the T84 cell line model. The possible inhibition exerted by the gallic acid of the peel extract on the STa peptide was inferred by molecular dynamics simulations. The antidiarrheal effects were investigated measuring cGMP accumulation in cells after stimulation by STa toxin and antibacterial activity was assessed. The anti-inflammatory activity was assessed by inhibition of COX-1 and COX-2. MTT and LDH assays were used to evaluate any possible cytotoxic action while the CyQUANT test was used to investigate the effect on cell proliferation. A representation showing how the possible interactions between STa and the gallic acid of the extract might reduce the action of the enterotoxin is presented. C. adamantium peel extract significantly decreased the levels of cGMP in T84 cells. However, no effect on the species of microorganisms was observed. The extract also inhibited COX-1 (IC50 255.70 ± 0.04 ng/mL) and COX-2 (IC50 569.50 ± 0.11 ng/mL) enzymes. Cytotoxicity assay have shown significant changes in cells treated with the extract, which inhibited the cell proliferation until 72 hours of treatment. Direct interactions of phenolic compounds present in the extract with the STa toxin may limit its activity. Curative effect in the diarrhea treatment and its anti-inflammatory action is based on the pharmacological properties, mechanism of action of the C. adamantium peel extract, and no toxic effects of the peel extract presented on this work. PMID:27764241

  1. Application of a SERS-based lateral flow immunoassay strip for the rapid and sensitive detection of staphylococcal enterotoxin B

    NASA Astrophysics Data System (ADS)

    Hwang, Joonki; Lee, Sangyeop; Choo, Jaebum

    2016-06-01

    A novel surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay (LFA) biosensor was developed to resolve problems associated with conventional LFA strips (e.g., limits in quantitative analysis and low sensitivity). In our SERS-based biosensor, Raman reporter-labeled hollow gold nanospheres (HGNs) were used as SERS detection probes instead of gold nanoparticles. With the proposed SERS-based LFA strip, the presence of a target antigen can be identified through a colour change in the test zone. Furthermore, highly sensitive quantitative evaluation is possible by measuring SERS signals from the test zone. To verify the feasibility of the SERS-based LFA strip platform, an immunoassay of staphylococcal enterotoxin B (SEB) was performed as a model reaction. The limit of detection (LOD) for SEB, as determined with the SERS-based LFA strip, was estimated to be 0.001 ng mL-1. This value is approximately three orders of magnitude more sensitive than that achieved with the corresponding ELISA-based method. The proposed SERS-based LFA strip sensor shows significant potential for the rapid and sensitive detection of target markers in a simplified manner.A novel surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay (LFA) biosensor was developed to resolve problems associated with conventional LFA strips (e.g., limits in quantitative analysis and low sensitivity). In our SERS-based biosensor, Raman reporter-labeled hollow gold nanospheres (HGNs) were used as SERS detection probes instead of gold nanoparticles. With the proposed SERS-based LFA strip, the presence of a target antigen can be identified through a colour change in the test zone. Furthermore, highly sensitive quantitative evaluation is possible by measuring SERS signals from the test zone. To verify the feasibility of the SERS-based LFA strip platform, an immunoassay of staphylococcal enterotoxin B (SEB) was performed as a model reaction. The limit of detection (LOD) for SEB, as

  2. Effect of Escherichia coli heat-stable enterotoxin, cholera toxin and theophylline on ion transport in porcine colon

    PubMed Central

    Argenzio, R. A.; Whipp, S. C.

    1981-01-01

    1. The effect of heat-stable enterotoxin (ST) of Escherichia coli, cholera toxin (CT), and theophylline (a phosphodiesterase inhibitor) on ion and water transport was studied with an in vivo isolated loop system of the pig colon. 2. All three agents abolished net Na absorption as a result of a decrease in the lumen to blood Na flux alone. With all three agents, net Cl absorption was reduced, but not abolished, and net HCO3 secretion was elicited. Luminal pCO2 was reduced with CT and theophylline from that observed in normal Ringer alone. 3. Theophylline resulted in a prompt and sustained increase in both cyclic adenosine monophosphate (cyclic AMP) and cyclic guanosine monophosphate (cyclic GMP) levels in colonic mucosa studied in vitro. ST selectively elevated cyclic GMP, whereas CT selectively elevated cyclic AMP. These responses paralleled the time course and magnitude of response of the transepithelial electrical potential difference (ψLB) measured in vivo. 4. Ion replacement studies in the presence or absence of theophylline showed that in the absence of Na, Cl absorption was slightly reduced and HCO3 secretion was elicited; no further additive effects of theophylline in the absence of luminal Na were observed. In the absence of luminal Cl, net Na absorption was abolished and HCO3 was absorbed; theophylline resulted in significant net Na and HCO3 secretion. Theophylline also increased ψLB in the absence of either luminal Na or Cl. 5. Results suggest that in the presence of theophylline or enterotoxin, the coupled Na—H and Cl—HCO3 exchange processes that are normally responsible for at least half of the net NaCl absorption by this tissue are interrupted. Active HCO3 secretion is observed and Cl absorption under these conditions can be entirely explained as a consequence of ψLB. Thus, these studies indicate that the colon may participate in the production of diarrhoea of enterotoxigenic origin. They also suggest an important functional role of cyclic

  3. Cloning Sequencing and Structural Manipulation of the Enterotoxin D and E Genes from Staphylococcus aureus

    DTIC Science & Technology

    1990-07-01

    the promoter regions of iron regulated genes in Gram- negative organisms (data not shown), we suspected that the sed gene might also be regulated by iron...HindIII digest of the plasmid pRWOO1 into the 5.5 kb HindIII fragment of the gram positive/gram negative shuttle plasrnid pDH5060. The ligation mixture was...genes in the gram negative genetic background. This suggests that at least two types of promoters exist in S. aureus. The 1.7 kb HindIII restriction

  4. Health Promotion

    DTIC Science & Technology

    1986-03-11

    Department of Defense DIRECTIVEAD-A269 638 , , AD-A29 638March 11, 1986 IIIIii!IN 111111111,11 Ii1111,111111[NUMBER 1010.10 SUBJECT: Health Promotion ...34 March 13, 1985 INC A. URPOSE SThis Directive establishes a health promotion policy within the Department of Defense to improve and maintain military...civilian employees. C. DEFINITIONS 1. Health Promotion . Any combination of health education and related organizational, social, economic or health care

  5. Structure of the food-poisoning Clostridium perfringens enterotoxin reveals similarity to the aerolysin-like pore-forming toxins.

    PubMed

    Briggs, David C; Naylor, Claire E; Smedley, James G; Lukoyanova, Natalya; Robertson, Susan; Moss, David S; McClane, Bruce A; Basak, Ajit K

    2011-10-14

    Clostridium perfringens enterotoxin (CPE) is a major cause of food poisoning and antibiotic-associated diarrhea. Upon its release from C. perfringens spores, CPE binds to its receptor, claudin, at the tight junctions between the epithelial cells of the gut wall and subsequently forms pores in the cell membranes. A number of different complexes between CPE and claudin have been observed, and the process of pore formation has not been fully elucidated. We have determined the three-dimensional structure of the soluble form of CPE in two crystal forms by X-ray crystallography, to a resolution of 2.7 and 4.0 Å, respectively, and found that the N-terminal domain shows structural homology with the aerolysin-like β-pore-forming family of proteins. We show that CPE forms a trimer in both crystal forms and that this trimer is likely to be biologically relevant but is not the active pore form. We use these data to discuss models of pore formation.

  6. Effect of Escherichia coli heat-stable enterotoxin on cell volume and intracellular inorganic ions of rat intestinal cells.

    PubMed

    Gilles-Baillien, M; Gerday, C; Kaeckenbeeck, A; Flamee, P A; Duchesne, P Y

    1987-11-01

    1.--Electron micrographs of rat jejunum mucosa incubated for 1 h in the presence of Escheria coli heat-stable enterotoxin (STa) in the lumen shows alterations of villous cells as well as of crypt cells. The brush border of mature enterocytes is partially desintegrated and covered with a thick mucus. Crypts are occupied on half of their height by cells very similar to Paneth cells, loaded with numerous large dark inclusions. 2.--Cell volume and intracellular inorganic ion concentrations have been estimated in mucosal scrapings of jejunum sacs, incubated in vitro for 1 or 3 h. The quick action (1 h of incubation) of STa is a swelling of the intestinal calls accompanied by an increase in Na+, Cl- and Ca2+ intracellular concentrations and a decrease in the K+ and Mg2+ ones. The delayed action (3 h of incubation) is an increase of extracellular space and a decrease in cell volume; and at the same time the intracellular concentration of Na+, Cl-, K+, Ca2+ and Mg2+ is augmented. 3.--After 3 h of incubation intestinal cells from the other levels of intestine (duodenum, ileum and colon) show the same variations in cell volume and intracellular inorganic ion concentrations under the influence of STa, as those recorded in the jejunum. 4.--The present work favours the hypothesis that all intestinal cells, villous or cryptic, are involved in the alteration of fluid ion transport ending in diarrhea.

  7. Crystal Structure of Staphylococcal Enterotoxin G (SEG) in Complex with a Mouse T-cell Receptor Beta Chain

    SciTech Connect

    Fernandez, M.M.; Robinson, H.; Cho, S.; De Marzi, M. C.; Kerzic, M. C.; Mariuzza, R. A.; Malchiodi, E. L.

    2011-01-14

    Superantigens (SAgs) are bacterial or viral toxins that bind MHC class II (MHC-II) molecules and T-cell receptor (TCR) in a nonconventional manner, inducing T-cell activation that leads to inflammatory cytokine production, which may result in acute toxic shock. In addition, the emerging threat of purpura fulminans and community-associated meticillin-resistant Staphylococcus aureus emphasizes the importance of a better characterization of SAg binding to their natural ligands that may allow the development of reagents to neutralize their action. The three-dimensional structure of the complex between a mouse TCR {beta} chain (mV{beta}8.2) and staphylococcal enterotoxin G (SEG) at 2.0 {angstrom} resolution revealed a binding site that does not conserve the 'hot spots' present in mV{beta}8.2-SEC2, mV{beta}8.2-SEC3, mV{beta}8.2-SEB, and mV{beta}8.2-SPEA complexes. Analysis of the mV{beta}8.2-SEG interface allowed us to explain the higher affinity of this complex compared with the others, which may account for the early activation of T-cells bearing mV{beta}8.2 by SEG. This mode of interaction between SEG and mV{beta}8.2 could be an adaptive advantage to bestow on the pathogen a faster rate of colonization of the host.

  8. Crystal Structure of Staphylococcal Enterotoxin G (SEG) in Complex with a Mouse T-cell Receptor β Chain*

    PubMed Central

    Fernández, Marisa M.; Cho, Sangwoo; De Marzi, Mauricio C.; Kerzic, Melissa C.; Robinson, Howard; Mariuzza, Roy A.; Malchiodi, Emilio L.

    2011-01-01

    Superantigens (SAgs) are bacterial or viral toxins that bind MHC class II (MHC-II) molecules and T-cell receptor (TCR) in a nonconventional manner, inducing T-cell activation that leads to inflammatory cytokine production, which may result in acute toxic shock. In addition, the emerging threat of purpura fulminans and community-associated meticillin-resistant Staphylococcus aureus emphasizes the importance of a better characterization of SAg binding to their natural ligands that may allow the development of reagents to neutralize their action. The three-dimensional structure of the complex between a mouse TCR β chain (mVβ8.2) and staphylococcal enterotoxin G (SEG) at 2.0 Å resolution revealed a binding site that does not conserve the “hot spots” present in mVβ8.2-SEC2, mVβ8.2-SEC3, mVβ8.2-SEB, and mVβ8.2-SPEA complexes. Analysis of the mVβ8.2-SEG interface allowed us to explain the higher affinity of this complex compared with the others, which may account for the early activation of T-cells bearing mVβ8.2 by SEG. This mode of interaction between SEG and mVβ8.2 could be an adaptive advantage to bestow on the pathogen a faster rate of colonization of the host. PMID:21059660

  9. Cytoplasmic calcium measurement in rotavirus enterotoxin-enhanced green fluorescent protein (NSP4-EGFP) expressing cells loaded with Fura-2.

    PubMed

    Berkova, Z; Morris, A P; Estes, M K

    2003-07-01

    The green fluorescent protein (GFP) and its analogs are standard markers of protein expression and intracellular localization of proteins. The fluorescent properties of GFP complicate accurate measurement of intracellular calcium using calcium sensitive fluorophores, which show a great degree of spectral overlap with GFP, or their K(d) values are too high for accurate measurement of subtle changes in cytoplasmic calcium concentrations. Here we describe a simple modification of the standard microscope-based Fura-2 calcium-imaging technique which permits the quantitative measurement of intracellular calcium levels in cells expressing enhanced green fluorescent protein (EGFP) fusion proteins. Longpass emission filtering of the Fura-2 signal in cells expressing an EGFP fusion protein is sufficient to eliminate the EGFP-Fura-2 emission spectra overlap and allows quantitative calibration of intracellular calcium. To validate this technique, we investigated the ability of rotavirus enterotoxin NSP4-EGFP to elevate intracellular calcium levels in mammalian HEK 293 cells. We show here that inducible intracellular expression of NSP4-EGFP fusion protein elevates basal intracellular calcium more than two-fold by a phospholipase C (PLC) independent mechanism.

  10. Production of staphylococcal enterotoxin F and pyrogenic exotoxin C by Staphylococcus aureus isolates from toxic shock syndrome-associated sources.

    PubMed Central

    Bonventre, P F; Weckbach, L; Staneck, J; Schlievert, P M; Thompson, M

    1983-01-01

    A total of 136 isolates of Staphylococcus aureus were tested for production of staphylococcal enterotoxin F (SEF) and pyrogenic exotoxin C (PEC), both of which have been identified as reliable indicators of toxic shock syndrome (TSS)-associated strains. SEF and PEC production by isolates from TSS-associated and other sources was tested independently in two laboratories, after which the two sets of data were compared. A 100% concordance between SEF and PEC production was obtained. The TSS toxin candidates were produced by 30 of 136 isolates, and in all instances SEF and PEC were made concurrently by the same strains; in no case was one toxin made and not the other. In the five groups of S. aureus tested, toxins were detected as follows: 23 of 25 (92%) acute TSS isolates, 2 of 48 (4.2%) genital non-TSS isolates, 2 of 16 (12.5%) recovered TSS isolates, 1 of 23 (4.3%) clinical nongenital isolates, and 2 of 24 (8.3%) enterotoxigenic food outbreak isolates. Comparison of purified SEF and purified PEC by immunological and biochemical criteria by immunodiffusion, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot analysis show that the toxins are immunologically identical and strongly suggest that the two nominal TSS toxins are in fact a single protein. Images PMID:6189784

  11. Cross-linking staphylococcal enterotoxin A bound to major histocompatibility complex class I is required for TNF-alpha secretion

    NASA Technical Reports Server (NTRS)

    Wright, A. D.; Chapes, S. K.

    1999-01-01

    The mechanism of how superantigens function to activate cells has been linked to their ability to bind and cross-link the major histocompatibility complex class II (MHCII) molecule. Cells that lack the MHCII molecule also respond to superantigens, however, with much less efficiency. Therefore, the purpose of this study was to confirm that staphylococcal enterotoxin A (SEA) could bind the MHCI molecule and to test the hypothesis that cross-linking SEA bound to MHCII-deficient macrophages would induce a more robust cytokine response than without cross-linking. We used a capture enzyme-linked immunosorbent assay and an immunprecipitation assay to directly demonstrate that MHCI molecules bind SEA. Directly cross-linking MHCI using monoclonal antibodies or cross-linking bound SEA with an anti-SEA antibody or biotinylated SEA with avidin increased TNF-alpha and IL-6 secretion by MHCII(-/-) macrophages. The induction of a vigorous macrophage cytokine response by SEA/anti-SEA cross-linking of MHCI offers a mechanism to explain how MHCI could play an important role in superantigen-mediated pathogenesis. Copyright 1999 Academic Press.

  12. Specific binding of Clostridium perfringens enterotoxin fragment to Claudin-b and modulation of zebrafish epidermal barrier.

    PubMed

    Zhang, Jingjing; Ni, Chen; Yang, Zhenguo; Piontek, Anna; Chen, Huapu; Wang, Sijie; Fan, Yiming; Qin, Zhihai; Piontek, Joerg

    2015-08-01

    Claudins (Cldn) are the major components of tight junctions (TJs) sealing the paracellular cleft in tissue barriers of various organs. Zebrafish Cldnb, the homolog of mammalian Cldn4, is expressed at epithelial cell-cell contacts and is important for regulating epidermal permeability. The bacterial toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to a subset of mammalian Cldns. In this study, we used the Cldn-binding C-terminal domain of CPE (194-319 amino acids, cCPE 194-319 ) to investigate its functional role in modulating zebrafish larval epidermal barriers. In vitro analyses show that cCPE 194-319 removed Cldn4 from epithelial cells and disrupted the monolayer tightness, which could be rescued by the removal of cCPE 194-319. Incubation of zebrafish larvae with cCPE 194-319 removed Cldnb specifically from the epidermal cell membrane. Dye diffusion analysis with 4-kDa fluorescent dextran indicated that the permeability of the epidermal barrier increased due to cCPE 194-319 incubation. Electron microscopic investigation revealed reversible loss of TJ integrity by Cldnb removal. Collectively, these results suggest that cCPE 194-319 could be used as a Cldnb modulator to transiently open the epidermal barrier in zebrafish. In addition, zebrafish might be used as an in vivo system to investigate the capability of cCPE to enhance drug delivery across tissue barriers.

  13. Structure of a C. perfringens enterotoxin mutant in complex with a modified Claudin-2 extracellular loop 2.

    PubMed

    Yelland, Tamas S; Naylor, Claire E; Bagoban, Tannya; Savva, Christos G; Moss, David S; McClane, Bruce A; Blasig, Ingolf E; Popoff, M; Basak, Ajit K

    2014-09-09

    CPE (Clostridium perfringens enterotoxin) is the major virulence determinant for C. perfringens type-A food poisoning, the second most common bacterial food-borne illness in the UK and USA. After binding to its receptors, which include particular human claudins, the toxin forms pores in the cell membrane. The mature pore apparently contains a hexamer of CPE, claudin and, possibly, occludin. The combination of high binding specificity with cytotoxicity has resulted in CPE being investigated, with some success, as a targeted cytotoxic agent for oncotherapy. In this paper, we present the X-ray crystallographic structure of CPE in complex with a peptide derived from extracellular loop 2 of a modified, CPE-binding Claudin-2, together with high-resolution native and pore-formation mutant structures. Our structure provides the first atomic-resolution data on any part of a claudin molecule and reveals that claudin's CPE-binding fingerprint (NPLVP) is in a tight turn conformation and binds, as expected, in CPE's C-terminal claudin-binding groove. The leucine and valine residues insert into the binding groove while the first residue, asparagine, tethers the peptide via an interaction with CPE's aspartate 225 and the two prolines are required to maintain the tight turn conformation. Understanding the structural basis of the contribution these residues make to binding will aid in engineering CPE to target tumor cells.

  14. Prophage-Encoded Staphylococcal Enterotoxin A: Regulation of Production in Staphylococcus aureus Strains Representing Different Sea Regions.

    PubMed

    Zeaki, Nikoleta; Susilo, Yusak Budi; Pregiel, Anna; Rådström, Peter; Schelin, Jenny

    2015-12-09

    The present study investigates the nature of the link between the staphylococcal enterotoxin A (SEA) gene and the lifecycle of Siphoviridae bacteriophages, including the origin of strain variation regarding SEA production after prophage induction. Five strains representing three different genetic lines of the sea region were studied under optimal and prophage-induced growth conditions and the Siphoviridae lifecycle was followed through the phage replicative form copies and transcripts of the lysogenic repressor, cro. The role of SOS response on prophage induction was addressed through recA transcription in a recA-disruption mutant. Prophage induction was found to increase the abundance of the phage replicative form, the sea gene copies and transcripts and enhance SEA production. Sequence analysis of the sea regions revealed that observed strain variances were related to strain capacity for prophage induction, rather than sequence differences in the sea region. The impact of SOS response activation on the phage lifecycle was demonstrated by the absence of phage replicative form copies in the recA-disruption mutant after prophage induction. From this study it emerges that all aspects of SEA-producing strain, the Siphoviridae phage and the food environment must be considered when evaluating SEA-related hazards.

  15. Reduction of the secretory response to Escherichia coli heat-stable enterotoxin by thiol and disulfide compounds. [Mice

    SciTech Connect

    Greenberg, R.N.; Dunn, J.A.; Guerrant, R.L.

    1983-07-01

    We examined the effects of disulfide and thiol compounds on Escherichia coli heat-stable enterotoxin (ST) and cyclic GMP-induced secretion. Both cystamine and cystine (disulfide compounds) reduced the secretory responses to submaximal doses of ST in suckling mice (at 0.5 mumol per mouse) and reduced ST activation of guanylate cyclase (by 33 to 73% at 1 mM). In higher doses, cystamine completely eradicated a maximally effective ST dose as well. In addition, the sulfhydryl (thiol) compounds cysteamine, cysteine, and acetylcysteine strikingly reduced the secretory response and the guanylate cyclase response to ST. Neither the disulfide nor the thiol compounds tested reduced cyclic GMP-induced secretion. These studies suggest that disulfide and thiol compounds both block ST-induced secretion before its activation of guanylate cyclase. Taken with the work of others, these findings suggest that disulfide compounds may alter the oxidation reduction state of a cell or act directly on the guanylate cyclase enzyme, whereas thiol compounds may inactivate ST itself by breaking its disulfide bridges, or it may alter guanylate cyclase activation by ST. Both families of compounds deserve further consideration among potential antisecretory agents for application in the control of ST-induced diarrhea.

  16. Localized surface plasmon resonance-based hybrid Au-Ag nanoparticles for detection of Staphylococcus aureus enterotoxin B

    NASA Astrophysics Data System (ADS)

    Zhu, Shaoli; Du, ChunLei; Fu, Yongqi

    2009-09-01

    A triangular hybrid Au-Ag nanoparticles array was proposed for the purpose of biosensing in this paper. Constructing the hybrid nanoparticles, an Au thin film is capped on the Ag nanoparticles which are attached on glass substrate. The hybrid nanoparticles array was designed by means of finite-difference and time-domain (FDTD) algorithm-based computational numerical calculation and optimization. Sensitivity of refractive index of the hybrid nanoparticles array was obtained by the computational calculation and experimental detection. Moreover, the hybrid nanoparticles array can prevent oxidation of the pure Ag nanoparticles from atmosphere environment because the Au protective layer was deposited on top of the Ag nanoparticles so as to isolate the Ag particles from the atmosphere. We presented a novel surface covalent link method between the localized surface plasmon resonance (LSPR) effect-based biosensors with hybrid nanoparticles array and the detected target molecules. The generated surface plasmon wave from the array carries the biological interaction message into the corresponding spectra. Staphylococcus aureus enterotoxin B (SEB), a small protein toxin was directly detected at nanogramme per milliliter level using the triangular hybrid Au-Ag nanoparticles. Hence one more option for the SEB detection is provided by this way.

  17. Cell-to-cell transformation in Escherichia coli: a novel type of natural transformation involving cell-derived DNA and a putative promoting pheromone.

    PubMed

    Etchuuya, Rika; Ito, Miki; Kitano, Seiko; Shigi, Fukiko; Sobue, Rina; Maeda, Sumio

    2011-01-20

    Escherichia coli is not assumed to be naturally transformable. However, several recent reports have shown that E. coli can express modest genetic competence in certain conditions that may arise in its environment. We have shown previously that spontaneous lateral transfer of non-conjugative plasmids occurs in a colony biofilm of mixed E. coli strains (a set of a donor strain harbouring a plasmid and a plasmid-free recipient strain). In this study, with high-frequency combinations of strains and a plasmid, we constructed the same lateral plasmid transfer system in liquid culture. Using this system, we demonstrated that this lateral plasmid transfer was DNase-sensitive, indicating that it is a kind of transformation in which DNase-accessible extracellular naked DNA is essential. However, this transformation did not occur with purified plasmid DNA and required a direct supply of plasmid from co-existing donor cells. Based on this feature, we have termed this transformation type as 'cell-to-cell transformation'. Analyses using medium conditioned with the high-frequency strain revealed that this strain released a certain factor(s) that promoted cell-to-cell transformation and arrested growth of the other strains. This factor is heat-labile and protease-sensitive, and its roughly estimated molecular mass was between ∼9 kDa and ∼30 kDa, indicating that it is a polypeptide factor. Interestingly, this factor was effective even when the conditioned medium was diluted 10(-5)-10(-6), suggesting that it acts like a pheromone with high bioactivity. Based on these results, we propose that cell-to-cell transformation is a novel natural transformation mechanism in E. coli that requires cell-derived DNA and is promoted by a peptide pheromone. This is the first evidence that suggests the existence of a peptide pheromone-regulated transformation mechanism in E. coli and in Gram-negative bacteria.

  18. Multiple-Strain Approach and Probabilistic Modeling of Consumer Habits in Quantitative Microbial Risk Assessment: A Quantitative Assessment of Exposure to Staphylococcal Enterotoxin A in Raw Milk.

    PubMed

    Crotta, Matteo; Rizzi, Rita; Varisco, Giorgio; Daminelli, Paolo; Cunico, Elena Cosciani; Luini, Mario; Graber, Hans Ulrich; Paterlini, Franco; Guitian, Javier

    2016-03-01

    Quantitative microbial risk assessment (QMRA) models are extensively applied to inform management of a broad range of food safety risks. Inevitably, QMRA modeling involves an element of simplification of the biological process of interest. Two features that are frequently simplified or disregarded are the pathogenicity of multiple strains of a single pathogen and consumer behavior at the household level. In this study, we developed a QMRA model with a multiple-strain approach and a consumer phase module (CPM) based on uncertainty distributions fitted from field data. We modeled exposure to staphylococcal enterotoxin A in raw milk in Lombardy; a specific enterotoxin production module was thus included. The model is adaptable and could be used to assess the risk related to other pathogens in raw milk as well as other staphylococcal enterotoxins. The multiplestrain approach, implemented as a multinomial process, allowed the inclusion of variability and uncertainty with regard to pathogenicity at the bacterial level. Data from 301 questionnaires submitted to raw milk consumers were used to obtain uncertainty distributions for the CPM. The distributions were modeled to be easily updatable with further data or evidence. The sources of uncertainty due to the multiple-strain approach and the CPM were identified, and their impact on the output was assessed by comparing specific scenarios to the baseline. When the distributions reflecting the uncertainty in consumer behavior were fixed to the 95th percentile, the risk of exposure increased up to 160 times. This reflects the importance of taking into consideration the diversity of consumers' habits at the household level and the impact that the lack of knowledge about variables in the CPM can have on the final QMRA estimates. The multiple-strain approach lends itself to use in other food matrices besides raw milk and allows the model to better capture the complexity of the real world and to be capable of geographical

  19. Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin

    PubMed Central

    Toniti, Waraphan; Yoshida, Toru; Tsurumura, Toshiharu; Irikura, Daisuke; Monma, Chie; Kamata, Yoichi

    2017-01-01

    Unusual outbreaks of food poisoning in Japan were reported in which Clostridium perfringens was strongly suspected to be the cause based on epidemiological information and fingerprinting of isolates. The isolated strains lack the typical C. perfringens enterotoxin (CPE) but secrete a new enterotoxin consisting of two components: C. perfringens iota-like enterotoxin-a (CPILE-a), which acts as an enzymatic ADP-ribosyltransferase, and CPILE-b, a membrane binding component. Here we present the crystal structures of apo-CPILE-a, NAD+-CPILE-a and NADH-CPILE-a. Though CPILE-a structure has high similarity with known iota toxin-a (Ia) with NAD+, it possesses two extra-long protruding loops from G262-S269 and E402-K408 that are distinct from Ia. Based on the Ia–actin complex structure, we focused on actin-binding interface regions (I-V) including two protruding loops (PT) and examined how mutations in these regions affect the ADP-ribosylation activity of CPILE-a. Though some site-directed mutagenesis studies have already been conducted on the actin binding site of Ia, in the present study, mutagenesis studies were conducted against both α- and β/γ-actin in CPILE-a and Ia. Interestingly, CPILE-a ADP-ribosylates both α- and β/γ-actin, but its sensitivity towards β/γ-actin is 36% compared with α-actin. Our results contrast to that only C2-I ADP-ribosylates β/γ-actin. We also showed that PT-I and two convex-concave interactions in CPILE-a are important for actin binding. The current study is the first detailed analysis of site-directed mutagenesis in the actin binding region of Ia and CPILE-a against both α- and β/γ-actin. PMID:28199340

  20. A study on the prevalence of Aeromonas spp. and its enterotoxin genes in samples of well water, tap water, and bottled water

    PubMed Central

    Didugu, Hareesh; Thirtham, Madhavarao; Nelapati, Krishnaiah; Reddy, K Kondal; Kumbhar, Baba Saheb; Poluru, Anusha; Pothanaboyina, Guruvishnu

    2015-01-01

    Aim: The aim of this work was to study the prevalence of Aeromonas spp. and its enterotoxin genes in various water sources. Materials and Methods: 125 samples (50 from well water, 50 from tap water, and 25 from bottled water) were collected from various sources in and around Greater Hyderabad Municipal Corporation and examined for the presence of aeromonads by both cultural and polymerase chain reaction (PCR) assay. Alkaline peptone water with ampicillin was used as enrichment. Aeromonas isolation medium and ampicillin dextrin agar were used as selective media. The boiling and snap chilling method was used for DNA extraction. Primers targeted against 16S rRNA, aer, and ast were used to identify aeromonads and its enterotoxins. Results: 48%, 18%, and 12% of well water, tap water, and bottled water samples were found positive by cultural assay with an overall prevalence of 28.8%. Aeromonads were detected in 32 % (52% in well water, 20% in tap water, and 16% in bottled water) of samples by PCR assay. Aerolysin (aer) gene was noticed in 34.6%, 20%, and 0% of well water, tap water, and bottled water samples, respectively, with an overall prevalence of 27.5%. Thermostable cytotonic enterotoxin (ast) was observed in 37.5% (42.3% in well water, 30% in tap water, and 25% in bottled mineral water) of samples. Conclusions: Presence of aeromonads and its toxin genes in various sources of water is of public health concern and emphasizes the need for necessary preventive measures to tackle the problem. PMID:27047024

  1. Health Promotion

    PubMed Central

    Wilson, Ron

    1992-01-01

    How physicians address issues of disease prevention and health promotion is discussed and current standards of screening for disease and counseling practices are reviewed. Collaboration among all health professionals is necessary if preventive medicine is to be effective. PMID:21221259

  2. Antibody Binding Studies Reveal Conformational Flexibility of the Bacillus cereus Non-Hemolytic Enterotoxin (Nhe) A-Component

    PubMed Central

    Märtlbauer, E.

    2016-01-01

    The non-hemolytic enterotoxin complex (Nhe) is supposed to be the main virulence factor of B. cereus causing a diarrheal outcome of food poisoning. This tripartite toxin consists of the single components NheA, -B and -C all of them being necessary for maximum toxicity. In the past, research activities aiming to elucidate the mode-of-action of Nhe were mostly focused on the B- and C-component. In this study the generation of novel monoclonal antibodies (mAb) and their thorough characterization enabled the determination of key features for NheA. By the means of immunoaffinity chromatography it could be shown that NheA does not interact with -B and -C in solution. Additionally, the establishment of a highly sensitive sandwich-EIA now enables the detection of NheA in B. cereus supernatants down to 20 pg ml-1.Peptide-based epitope mapping in combination with partially deleted recombinant NheA fragments allowed the allocation of the binding regions for the three mAbs under study. Furthermore, by different EIA set-ups the conformational flexibility of NheA could be shown. For two of the antibodies under study different mechanisms of NheA neutralization were proven. Due to prevention of complete pore formation by one of the antibodies, NheA could be detected in an intermediate stage of the tripartite complex on the cell surface. Taken together, the results obtained in the present study allow a refinement of the mode-of-action for the Nhe toxin-complex. PMID:27768742

  3. Review Over a 3-Year Period of European Union Proficiency Tests for Detection of Staphylococcal Enterotoxins in Food Matrices.

    PubMed

    Nia, Yacine; Mutel, Isabelle; Assere, Adrien; Lombard, Bertrand; Auvray, Frederic; Hennekinne, Jacques-Antoine

    2016-04-13

    Staphylococcal food poisoning outbreaks are a major cause of foodborne illnesses in Europe and their notifications have been mandatory since 2005. Even though the European regulation on microbiological criteria for food defines a criterion on staphylococcal enterotoxin (SE) only in cheese and dairy products, European Food Safety Authority (EFSA) data reported that various types of food matrices are involved in staphylococcal food poisoning outbreaks. The European Screening Method (ESM) of European Union Reference Laboratory for Coagulase Positive Staphylococci (EURL CPS) was validated in 2011 for SE detection in food matrices and is currently the official method used for screening purposes in Europe. In this context, EURLCPS is annually organizing Inter-Laboratory Proficiency Testing Trials (ILPT) to evaluate the competency of the European countries' National Reference Laboratories (NRLs) to analyse SE content in food matrices. A total of 31 NRLs representing 93% of European countries participated in these ILPTs. Eight food matrices were used for ILPT over the period 2013-2015, including cheese, freeze-dried cheese, tuna, mackerel, roasted chicken, ready-to-eat food, milk, and pastry. Food samples were spiked with four SE types (i.e., SEA, SEC, SED, and SEE) at various concentrations. Homogeneity and stability studies showed that ILPT samples were both homogeneous and stable. The analysis of results obtained by participants for a total of 155 blank and 620 contaminated samples allowed for evaluation of trueness (>98%) and specificity (100%) of ESM. Further to the validation study of ESM carried out in 2011, these three ILPTs allowed for the assessment of the proficiency of the NRL network and the performance of ESM on a large variety of food matrices and samples. The ILPT design presented here will be helpful for the organization of ILPT on SE detection by NRLs or other expert laboratories.

  4. MyD88-dependent pro-inflammatory cytokine response contributes to lethal toxicity of staphylococcal enterotoxin B in mice.

    PubMed

    Kissner, Teri L; Ruthel, Gordon; Cisney, Emily D; Ulrich, Robert G; Fernandez, Stefan; Saikh, Kamal U

    2011-10-01

    An elevated pro-inflammatory cytokine response is the primary cause of death by toxic shock after exposure to staphylococcal enterotoxin B (SEB). Identifying an intracellular signal mediator that predominantly controls the pro-inflammatory response is important for developing a therapeutic strategy. We examined the role of the signaling adaptor MyD88 in cell culture and in a mouse model of toxic shock. Our results indicated that elevated tumor necrosis factor-α, interferon-γ, interleukin (IL)-1α/β and IL-6 production from mouse spleen cells treated with SEB alone or in combination with lipopolysaccharide (LPS) was regulated by MyD88. Elevated levels of MyD88 protein in spleen cells, as well as in CD11c(+) or Mac3(+) cells, and activation of nuclear factor-κB in spleen cells were observed in mice treated with SEB. An SEB-dose dependent lethality was observed in LPS-potentiated and in D-galactosamine-sensitized mice. D-Galactosamine treatment of spleen cells had no effect in cytokine induction but rather increased the sensitivity to toxic shock in mice. Our results demonstrated an impaired pro-inflammatory cytokine production by spleen cells of MyD88(-/-) mice in response to SEB or SEB plus LPS. Most importantly, MyD88(-/-) mice were resistant to SEB-induced death. These results demonstrate that MyD88-dependent pro-inflammatory signaling is responsible for SEB intoxication. In addition, our studies also demonstrated that LPS potentiation, in comparison to D-galactosamine sensitization, contributes to a stronger SEB-induced lethality. This is due to the pro-inflammatory cytokine response elicited by MyD88 after exposure to SEB and LPS. These findings offer an important insight upon SEB intoxication and subsequent therapy targeting MyD88.

  5. Interlaboratory Profiency Testing trial on the Detection of Staphylococcal Enterotoxins types SEA to SEE in food in Germany 2013.

    PubMed

    Fetsch, Alexandra; Steege, Katja; Leeser, Daniel; Krause, Gladys

    2016-01-01

    In the autumn 2013, the National Reference Laboratory for coagulase positive staphylococci (CPS) including Staphylococcus (S.) aureus (NRL-Staph) at the Federal Institute for Risk Assessment has organized its first interlaboratory profiency testing (ILPT) trial for the detection of staphylococcal enterotoxins (SE) types SEA to SEE in food. The purpose of the ILPT was to assess the analytical competence of the official laboratories of the Federal German "Länder"authorities. Moreover, it was the intention to gain an overview of the standard methods implemented in the participating laboratories for the purpose of SE detection in food. Five cream cheese samples at three different contamination levels (blank, low, and high) were sent to each participant. In total, 15 laboratories participated to the ILPT: 14 laboratories from 11 Federal German "Länder", and the European Reference Laboratory for CPS including S. aureus (EU-RL for CPS). Data sets from 14 participating laboratories were included in the analysis. Overall, a specificity of 100% (14/14 true negative results), a sensitivity of 55% (31/56 true positive results), and an accuracy of 64% (45/60 true results) was achieved. The majority of participants (9/15) used other analytical methods for the detection of SE in food than the suggested European Screening Method (ESM) v5. To conclude on the ILPT in general it is to state that the majority of participating laboratories failed to correctly identify SE-low-contaminated samples. Further efforts are necessary to improve the analytical capacity and sensitivity as regards the detection of SE in food in Germany.

  6. Shigella enterotoxin-2 is a type III effector that participates in Shigella-induced interleukin 8 secretion by epithelial cells

    PubMed Central

    Farfán, Mauricio J.; Toro, Cecilia S.; Barry, Eileen M.; Nataro, James P.

    2011-01-01

    We have previously described a protein termed Shigella enterotoxin 2 (ShET-2), which induces rises in short circuit current in rabbit ileum mounted in the Ussing chamber. Published reports have postulated that ShET-2 may be secreted by the Shigella type III secretion system (T3SS). In this study we show that ShET-2 secretion into the extracellular space requires the T3SS in S. flexneri 2a strain 2457T and a ShET-2-TEM fusion was translocated into epithelial cells in a T3SS-dependent manner. The ShET-2 gene, sen, is encoded downstream of the ospC1 gene of S. flexneri, and we show that sen is co-transcribed with this T3SS-secreted product. Considering that T3SS effectors have diverse roles in Shigella infection and that vaccine constructs lacking ShET-2 are attenuated in volunteers, we asked whether ShET-2 has a function other than its enterotoxic activity. We constructed a ShET-2 mutant in 2457T and tested its effect on epithelial cell invasion, plaque formation, guinea pig keratoconjunctivitis and interleukin 8 (IL-8) secretion from infected monolayers. Though other phenotypes were not different compared to the wild-type parent, we found that HEp-2 and T84 cells infected with the ShET-2 mutant exhibited significantly reduced IL-8 secretion into the basolateral compartment, suggesting that ShET-2 might participate in the Shigella-induced inflammation of epithelial cells. PMID:21219446

  7. Spray-dried animal plasma prevents the effects of Staphylococcus aureus enterotoxin B on intestinal barrier function in weaned rats.

    PubMed

    Pérez-Bosque, Anna; Amat, Concepció; Polo, Javier; Campbell, Joy M; Crenshaw, Joe; Russell, Louis; Moretó, Miquel

    2006-11-01

    In this study, we investigated intestinal barrier function during inflammation as well as the effects of dietary supplementation with porcine spray-dried animal plasma (SDAP) proteins and porcine immunoglobulin concentrate (IC). Wistar Lewis rats were fed from d 21 (weaning) until d 34 or 35 either a control diet or a diet containing SDAP or IC. On d 30 and d 33, rats received an intraperitoneal dose of Staphylococcus aureus enterotoxin B (SEB; 0.5 mg/kg body wt; groups SEB, SEB-SDAP, and SEB-IC). SEB reduced the potential difference across the jejunum by 60%, the short-circuit current by 70%, and Na-K-ATPase activity in intestinal mucosa (all P < 0.05). The fluxes of dextran flux (4 kDa) and horseradish peroxidase (HRP, 40 kDa) across the intestinal wall also increased in SEB-treated rats (P < 0.01, P = 0.068, respectively). SEB also increased HRP flux across the paracellular space (P < 0.05). Moreover, SEB-treated rats had a reduced expression of tight junction proteins, such as ZO-1 (10% reduction; P < 0.05) and beta-catenin (20% reduction; P < 0.05). Dietary supplementation with SDAP or IC prevented dextran (P < 0.05) and HRP (P < 0.05) paracellular flux across the intestinal epithelium. SDAP supplementation also prevented SEB effects on Na-K-ATPase activity (P < 0.05). In our model of SEB-induced intestinal inflammation, the increased permeability across the intestinal mucosa was due to the lower expression of tight junction proteins, an effect that can be prevented by both SDAP and IC supplementation.

  8. Enteropathogenic bacteria and enterotoxin-producing Staphylococcus aureus isolated from ready-to-eat foods in Khon Kaen, Thailand.

    PubMed

    Chomvarin, Chariya; Chantarasuk, Yingrit; Srigulbutr, Sugunya; Chareonsudjai, Sorujsiri; Chaicumpar, Kunyaluk

    2006-09-01

    The objective of this study was to investigate the microbiological quality of ready-to-eat food in the Municipality of Khon Kaen, Thailand. Four categories of 186 food samples were collected: (1) high heat food; (2) low heat food; (3) no heat food; and, 4) on-site prepared fruit juices and beverages. Of the food samples, 145 (78%) failed to meet acceptable microbiological standards, including fruit juice and beverages (100%), no heat food (91.7%), low heat food (81.7%) and high heat food (57.9%). The most frequent bacterial indexes indicating unacceptability were the most probable number (MPN) of coliforms (78%), the bacterial colony count (58%), and the MPN of E. coli (46%). Pathogenic bacteria were found in 6.5% of food samples. Salmonella, Vibrio cholerae non O1 and Aeromonas hydrophila were found in 4.3, 1.6 and 0.5% of the total food samples, respectively. The serovars of Salmonella found in food were S. Derby, S. Give, S. Krefield, S. Paratyphi B, S. Verchow, S. Lexington and S. Senftenberg. Staphylococcus aureus concentrations of >10(2) CFU/g and >10(5) CFU/g were found in 10.8% and 1.1% of the food samples. Enterotoxin types AB and A of S. aureus were found in 2.7% of the food samples. These results indicate that more than half of the ready-to-eat foods tested in Khon Kaen municipality did not meet microbiological national standards and many kinds of enteropathogenic bacteria were found, suggesting food stalls may be a source of foodborne disease.

  9. Establishment of a new animal model of allergic rhinitis with biphasic sneezing by intranasal sensitization with Staphylococcal enterotoxin B.

    PubMed

    Sun, Rong; Tang, Xinye; Yao, Hongbing; Hong, Suling; Yang, Yang; Kou, Wei; Wei, Ping

    2015-08-01

    Allergic rhinitis (AR) is a global health problem. The effectiveness of currently available medications is limited and therefore investigation for more effective drugs is essential. The aim of the present study was to establish a model of AR in guinea pigs that can be utilized for the further investigation of new drugs. Guinea pigs were intranasally sensitized with 1 µg Staphylococcal enterotoxin B (SEB) dissolved in 40 µl saline once daily for 14 days. One week after the last sensitization, the same treatment was applied intranasally once every four days for a total of 30 times. In the treatment group, terfenadine was administered orally 70 min before the 4th, 14th and 24th challenge. Sneezing and nasal scratching were evaluated following each of the 30 challenges. The quantity of antigen-specific antibodies in the serum was measured. Between the 19th and 30th challenges, the guinea pigs in the model group produced significant biphasic elevations in sneezing number, with peaks 10 min-2 h and 4-8 h after the SEB challenges. In addition, the guinea pigs produced significantly more sneezing in the first peak during the 19th to 30th challenges than during the first to 18th challenges (P<0.01). Terfenadine significantly inhibited the early- and late-phase sneezing at all challenge times. The serum levels of SEB-specific immunoglobulin (Ig) E and IgG1 were higher in the model group in comparison with those in the control group (P<0.01). This experiment demonstrated that SEB can induce typical AR with biphasic sneezing in guinea pigs. Histamine may play an important role in the early- and the late-phase sneezing in the model of AR. This model can be potentially used for the investigation of new drugs.

  10. Regulation of intestinal guanylate cyclase by the heat-stable enterotoxin of Escherichia coli (STa) and protein kinase C.

    PubMed Central

    Crane, J K; Wehner, M S; Bolen, E J; Sando, J J; Linden, J; Guerrant, R L; Sears, C L

    1992-01-01

    The heat-stable enterotoxin of Escherichia coli (STa) stimulates membrane-bound guanylate cyclase in intestinal epithelium and induces fluid and ion secretion. Using the T84 human colon carcinoma cell line as a model, we observed that phorbol esters markedly enhanced STa-stimulated cyclic GMP accumulation in T84 cells (C. S. Weikel, C. L. Spann, C. P. Chambers, J. K. Crane, J. Linden, and E. L. Hewlett, Infect. Immun. 58:1402-1407, 1990). In this study we document that the phorbol ester treatment increases 125I-STa-binding sites as well as membrane-bound guanylate cyclase activity in T84 cells and provide evidence that both effects are mediated by phosphorylation. Guanylate cyclase activity was increased approximately 50% in membranes prepared from intact T84 cells treated with phorbol-12,13-dibutyrate (beta-PDB) and after treatment of homogenates with beta-PDB in a manner dependent on ATP, MgCl2, and cytosol. Similarly, treatment of membranes with purified bovine brain protein kinase C in the presence of appropriate cofactors and beta-PDB resulted in an increase in STa-stimulated guanylate cyclase activity of about 70%. Likewise, the number of 125I-STa-binding sites was increased by about 25 to 40% in membranes prepared from intact cells or homogenates treated with beta-PDB; no effect on binding affinity (Kd = 0.15 nM) was noted. These experiments suggest that protein kinase C may phosphorylate the STa receptor-guanylate cyclase or a closely related protein and increase guanylate cyclase activity. The stimulatory effects of protein kinase C on STa-sensitive guanylate cyclase are opposite in direction to the profound inhibitory effects of the kinase on atrial natriuretic peptide-stimulated guanylate cyclase, demonstrating differential regulation by protein kinases within the guanylate cyclase-receptor family. PMID:1360449

  11. Comparison of enterotoxic activities of heat-stable enterotoxins from class 1 and class 2 Escherichia coli of swine origin.

    PubMed Central

    Whipp, S C; Moon, H W; Argenzio, R A

    1981-01-01

    Pig small intestine develops age-dependent resistance to some (class 2 strains) enterotoxigenic Escherichia coli while remaining susceptible to others (class 1 strains). This study tested the hypothesis that class 1 and class 2 strains produce different subtypes of heat-stable enterotoxin (ST). The dose-response curves of small intestine to crude ST preparations from a class 1 and a class 2 strain were compared in several species. In infant mice, the class 1 ST preparation was less active than the class 2 ST preparation, whereas in rabbits the preparations were equally potent. However, in 1-, 7-, and 14-week-old pigs, the class 1 ST preparation was more active than the class 2 preparation. At low doses, both preparations caused reduced absorption in pigs of all three age groups, and at high doses the class 1 preparation caused secretion in all three age groups. In contrast, at high doses the class 2 preparation caused secretion in 1-week-old pigs but only reduced absorption in older pigs. when class 1 and class 2 ST preparations were fractionated by methanol extraction, in both cases the mouse-negative, pig-positive activity was associated with the methanol-insoluble fraction and mouse-positive, pig-positive activity was associated with the methanol-soluble fraction. The results are consistent with a hypothesis that class 1 and class 2 strains of enterotoxigenic E. coli produce different subtypes of ST and that the response of pig intestine to ST varies with both age and toxin subtype. PMID:7011991

  12. Tissue distribution and safety evaluation of a claudin-targeting molecule, the C-terminal fragment of Clostridium perfringens enterotoxin.

    PubMed

    Li, Xiangru; Saeki, Rie; Watari, Akihiro; Yagi, Kiyohito; Kondoh, Masuo

    2014-02-14

    We previously found that claudin (CL) is a potent target for cancer therapy using a CL-3 and -4-targeting molecule, namely the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE). Although CL-3 and -4 are expressed in various normal tissues, the safety of this CL-targeting strategy has never been investigated. Here, we evaluated the tissue distribution of C-CPE in mice. Ten minutes after intravenous injection into mice, C-CPE was distributed to the liver and kidney (24.0% and 9.5% of the injected dose, respectively). The hepatic level gradually fell to 3.2% of the injected dose by 3 h post-injection, whereas the renal C-CPE level gradually rose to 46.5% of the injected dose by 6 h post-injection and then decreased. A C-CPE mutant protein lacking the ability to bind CL accumulated in the liver to a much lesser extent (2.0% of the dose at 10 min post-injection) than did C-CPE, but its renal profile was similar to that of C-CPE. To investigate the acute toxicity of CL-targeted toxin, we intravenously administered C-CPE-fused protein synthesis inhibitory factor to mice. The CL-targeted toxin dose-dependently increased the levels of serum biomarkers of liver injury, but not of kidney injury. Histological examination confirmed that injection of CL-targeted toxin injured the liver but not the kidney. These results indicate that potential adverse hepatic effects should be considered in C-CPE-based cancer therapy.

  13. Effects of age, ambient temperature, and heat-stable Escherichia coli enterotoxin on intestinal transit in infant mice.

    PubMed

    Moon, H W; Fung, P Y; Isaacson, R E; Booth, G D

    1979-07-01

    Some interrelationships among age, ambient temperature, intestinal transit, and enterotoxigenic Escherichia coli infection were studied in an infant mouse model. The transit of dye in the small intestine was accelerated during the response to heat-stable E. coli enterotoxin. Transit in the small intestine of normal mice accelerated with increased age (from less than 17 h to 8 days old) and accelerated with increased ambient temperature (from 25 to 37 degrees C). Transit was more rapid in the jejunum than in the ileum throughout the range of experimental conditions studied. E. coli strains that do not produce any of the pili known facilitate intestinal colonization were cleared from the small intestine more rapidly at 37 degrees C than at 25 degrees C. This clearance was thought to be due to accelerated transit at the higher temperature. In contrast, a strain of E. coli that produces K99 (pili previously shown to facilitate intestinal colonization in other species) was not cleared from the small intestine and colonized more intensively at 37 degrees C than at 25 degrees C. Intensified colonization by this strain was thought to be due to increased production of K99 at the higher temperature. It was suggested that sluggish intestinal transit may also be characteristic of the neonates of other species and be one of the factors predisposing them to intestinal colonization by enteropathogens. It was speculated that this predisposition may be enhanced if the neonates are chilled. However, the effect of ambient temperature on intestinal transit in homeothermic neonates such as pigs, calves, and humans may be different from that in mice because neonatal mice are poikilothermic.

  14. The Cytotoxic Enterotoxin of Aeromonas hydrophila Induces Proinflammatory Cytokine Production and Activates Arachidonic Acid Metabolism in Macrophages

    PubMed Central

    Chopra, A. K.; Xu, X.-J.; Ribardo, D.; Gonzalez, M.; Kuhl, K.; Peterson, J. W.; Houston, C. W.

    2000-01-01

    An aerolysin-related cytotoxic enterotoxin (Act) of Aeromonas hydrophila possesses multiple biological activities, which include its ability to lyse red blood cells, destroy tissue culture cell lines, evoke a fluid secretory response in ligated intestinal loop models, and induce lethality in mice. The role of Act in the virulence of the organism has been demonstrated. In this study, we evaluated the potential of Act to induce production of proinflammatory cytokines associated with Act-induced tissue injury and Act's capacity to activate in macrophages arachidonic acid (AA) metabolism that leads to production of eicosanoids (e.g., prostaglandin E2 [PGE2]). Our data indicated that Act stimulated the production of tumor necrosis factor alpha and upregulated the expression of genes encoding interleukin-1β (IL-1β) and IL-6 in the murine macrophage cell line RAW264.7. Act also activated transcription of the gene encoding inducible nitric oxide synthase. Act evoked the production of PGE2 coupled to the cyclooxygenase-2 (COX-2) pathway. AA is a substrate for PGE2, and Act produced AA from phospholipids by inducing group V secretory phospholipase A2. We also demonstrated that Act increased cyclic AMP (cAMP) production in macrophages. cAMP, along with PGE2, could potentiate fluid secretion in animal models because of infiltration and activation of macrophages resulting from Act-induced tissue injury. After Act treatment of RAW cells, we detected an increased translocation of NF-κB and cAMP-responsive element binding protein (CREB) to the nucleus using gel shift assays. Act also upregulated production of antiapoptotic protein Bcl-2 in macrophages, suggesting a protective role for Bcl-2 against cell death induced by proinflammatory cytokines. The increased expression of genes encoding the proinflammatory cytokines, COX-2, and Bcl-2 appeared correlated with the activation of NF-κB and CREB. This is the first report of the detailed mechanisms of action of Act from A

  15. Sublethal staphylococcal enterotoxin B challenge model in pigs to evaluate protection following immunization with a soybean-derived vaccine.

    PubMed

    Hudson, Laura C; Seabolt, Brynn S; Odle, Jack; Bost, Kenneth L; Stahl, Chad H; Piller, Kenneth J

    2013-01-01

    In an effort to develop a sustainable platform for manufacturing protein-based vaccine candidates, we expressed a triple mutant of staphylococcal enterotoxin B carrying the L45R, Y89A, and Y94A modifications in transgenic soybean seeds (soy-mSEB). Soy-mSEB possessed no detectable superantigen activity in vitro. We found that this soybean-derived, nontoxic mutant of SEB could be stably expressed, stored in seeds for extended periods at room temperature without degradation, and easily purified from contaminating soy proteins. Vaccination of pigs with purified soy-mSEB, or the identical triple mutant expressed in Escherichia coli (E. coli-mSEB), resulted in high antibody titers against the native toxin in immunized animals. In fact, titers were indistinguishable regardless of the immunogen used, demonstrating the equivalence of soy-mSEB and E. coli-mSEB vaccinations. Antisera from either immunized group were able to block native SEB superantigen activity in an in vitro neutralization assay. Similar results were obtained when immunized animals were challenged with a sublethal dose of native toxin. Significant reductions in toxin-induced serum cytokine levels were observed in soy-mSEB- and E. coli-mSEB-immunized pigs compared to control animals. The reductions in SEB-induced cytokine responses were similar regardless of the immunogen used for vaccination. Surprisingly, however, some clinical symptoms, such as prostration, lethargy, emesis, and/or diarrhea, were still observed in all immunized animals. These studies demonstrate the potential for soybean-derived proteins as a platform technology for sustainable vaccine manufacturing and the usefulness of a sublethal challenge model in pigs for evaluating the efficacy of potential SEB vaccine candidates.

  16. Health Promotion

    PubMed Central

    Karmali-Rawji, Shameela; Kassim-Lakha, Shaheen; Taylor, Karmel

    1992-01-01

    Perceived lack or loss of control, stress, a rapidly again population and rising costs of health care necessitate effective health promotion and disease prevention in the elderly. In a collaborative health promotion effort, the private sector, public sector, and community partners have joined to increase the South Asian elders' sense of control over the decisions and circumstances that affect their everyday lives. The project was designed to help elders come to terms with the fragmentation of their extended families, cultural alienation, decreased autonomy, need for information, and greater risk of cardiovascular disease. Imagesp622-a

  17. Clostridium perfringens enterotoxin and Clostridium difficile toxin A/B do not play a role in acute haemorrhagic diarrhoea syndrome in dogs.

    PubMed

    Busch, K; Suchodolski, J S; Kühner, K A; Minamoto, Y; Steiner, J M; Mueller, R S; Hartmann, K; Unterer, S

    2015-03-07

    Although an association between clostridial pathogens and canine idiopathic acute haemorrhagic diarrhoea syndrome (AHDS) has been described, the relevance of those bacteria and their toxins remains unclear. The aim of this study was to evaluate the association between severity of clinical signs and presence of Clostridium perfringens enterotoxin (CPE) and Clostridium difficile toxin A/B (CDT A/B) in faeces of dogs with AHDS. Faecal samples of 54 dogs with idiopathic AHDS were tested by qualitative CPE and CDT A/B ELISA, and PCR was performed to detect enterotoxin genes of C. perfringens (cpe) and toxin B genes of C. difficile (cdt b). Prevalence of cdt b and CDT A/B in dogs with AHDS was 10/54 and 2/54 versus 3/23 and 0/23 in control dogs. Prevalence of cpe was 35/54 in affected versus 9/23 in control dogs. Prevalence of CPE in dogs with AHDS (13/54) was higher compared with control dogs (0/23). No significant difference was detected between CPE-positive and -negative and between cpe-positive and -negative dogs in severity of clinical signs, duration of hospitalisation, mortality rate and selected laboratory parameters. This study suggests that CPE and CDT A/B do not play a role in idiopathic AHDS, are not associated with clinical parameters in affected dogs and cannot be used to predict disease outcome.

  18. Staphylococcus aureus enterotoxins A- and B: binding to the enterocyte brush border and uptake by perturbation of the apical endocytic membrane traffic.

    PubMed

    Danielsen, E Michael; Hansen, Gert H; Karlsdóttir, Edda

    2013-04-01

    Enterotoxins of Staphylococcus aureus are among the most common causes of food poisoning. Acting as superantigens they intoxicate the organism by causing a massive uncontrolled T cell activation that ultimately may lead to toxic shock and death. In contrast to our detailed knowledge regarding their interaction with the immune system, little is known about how they penetrate the epithelial barrier to gain access to their targets. We therefore studied the uptake of two staphylococcal enterotoxins (SEs), SEA and SEB, using organ cultured porcine jejunal explants as model system. Attachment of both toxins to the villus surface was scarce and patchy compared with that of cholera toxin B (CTB). SEA and SEB also bound to microvillus membrane vesicles in vitro, but less efficiently than CTB, and the binding was sensitive to treatment with endoglycoceramidase II, indicating that a glycolipid, possibly digalactosylceramide, acts as cell surface receptor at the brush border. Both SEs partitioned poorly with detergent resistant membranes (DRMs) of the microvillus, suggesting a weak association with lipid raft microdomains. Where attachment occurred, cellular uptake of SEA and SEB was also observed. In enterocytes, constitutive apical endocytosis normally proceeds only to subapical early endosomes present in the actomyosin-rich "terminal web" region. But, like CTB, both SEA and SEB penetrated deep into the cytoplasm. In conclusion, the data show that after binding to the enterocyte brush border SEA and SEB perturb the apical membrane trafficking, enabling them to engage in transcytosis to reach their target cells in the subepithelial lamina propria.

  19. Influence of carvacrol and thymol on the physiological attributes, enterotoxin production and surface characteristics of Staphylococcus aureus strains isolated from foods

    PubMed Central

    Souza, E.L.; Oliveira, C.E.V.; Stamford, T.L.M.; Conceição, M.L.; Neto, N.J. Gomes

    2013-01-01

    This study evaluated the influence of the phenolic compounds carvacrol (CAR) and thymol (THY) on some physiological characteristics and on the modulation of the secretion of some staphylococcal virulence factors, that is, coagulase and enterotoxin. This study also investigated possible mechanisms for the establishment of the anti-staphylococcal activity of these compounds. Sublethal concentrations (0.3 and 0.15 μL/mL) of CAR and THY inhibited the activity of the enzymes coagulase and lipase and led to a decrease in salt tolerance. At the tested sublethal concentrations, both CAR and THY led to a total suppression of enterotoxin production. The loss of a 260-nm-absorbing material and an efflux of potassium ions occurred immediately after the addition of CAR and THY at 0.6 and 1.2 μL/mL and increased up to 120 min of exposure. Electron microscopy of cells exposed to CAR and THY (0.6 μL/mL) revealed that individual cells appeared to be deformed, with projections of cellular material. The observations of leakage of cellular material and an altered cell surface suggest that gross damage to a cell’s cytoplasmic membrane, which results in a disruption in protein secretion, could be responsible for the anti-staphylococcal properties of CAR and THY. PMID:24159280

  20. Immunogenicity and Efficacy of a Heat-Killed Whole Cell/B Subunit Cholera Vaccine

    DTIC Science & Technology

    1993-05-15

    occurring enterotoxigenic E . coli infection in U.S. adults in Mexico with a comparison between the two study groups of the following endpoints: rates of...enterotoxigenic E . coli due to heat-labile (LT) and heat-stable (ST) enterotoxin producing strains during a 5-week period, timing of protection in...relationship to vaccine administration, and relationship between enterotoxigenic E . coli diarrhea attack rates and serum IgG as well as fecal IgA

  1. Characterization of a Shiga-Like Toxin Converting Phage Isolated from an Escherichia coli Strain Responsible for Hemorrhagic Colitis in Humans

    DTIC Science & Technology

    1985-01-01

    Specific Airs 12 METHODS AND MATERIALS 14 Bacterial Strains 14 Culture Media and Diluents 15 Ultraviolet Light Bacteriophage Induction 15 Plaque...coli 026 strain H-19 were carried on a bacteriophage . Escherichia coli 026 strain H-19 does not produce heat-labile or heat-stable enterotoxin and is...strain that had become toxinogenic after coculti- vation with Escherichia coli 026 strain H-19. Attempts to isolate the bacteriophage from Escherichia

  2. In vivo emergence of enterotoxigenic Escherichia coli variants lacking genes for K99 fimbriae and heat-stable enterotoxin.

    PubMed Central

    Mainil, J G; Sadowski, P L; Tarsio, M; Moon, H W

    1987-01-01

    Neonatal pigs were inoculated with porcine enterotoxigenic Escherichia coli 431, which carries genes for K99 fimbriae and STaP enterotoxin. Colonies of strain 431 were recovered from feces of pigs for up to 17 days after inoculation and tested for hybridization with gene probes for K99 and STaP. Variants of strain 431 that did not hybridize with the probes were considered to have lost the genes. Variants were recovered from 10 of 13 suckling pigs that survived the infection. Only 0.4% of the isolates recovered during the first 2 days after inoculation were variants. Of the isolates recovered 3 to 5 days after inoculation, 20 to 36% were variants. Variant colonies were detected more frequently among pigs in some litters than in others. The litter with the highest number of variant-shedding pigs had the dam with the highest titer of K99 antibody in her colostrum. Variants also occurred in colostrum-deprived, artificially reared pigs. However, the number of variants detected was lower and they occurred later in the course of the infection in colostrum-deprived pigs than in suckling pigs. More variants were detected and they were detected earlier in colostrum-deprived pigs fed anti-K99 monoclonal antibody than in controls fed anti-K88 monoclonal antibody. Loss of STaP appeared to be secondary to loss of K99 in that some variants lacked only K99 (K99- STaP+) and some lacked both genes (K99- STaP-), but none was of the K99+ STaP- type. Our results confirmed reports of gene loss from enterotoxigenic E. coli during infection. They are consistent with the hypothesis that variants emerge under in vivo selection pressure of K99 antibody and with the speculation that gene loss may be an important component of protection in vaccinated populations. However, the emergence of variants did not appear to play a major role in the recovery of individual pigs from clinical disease. PMID:2890584

  3. Prevalence and diversity of enterotoxin genes with genetic background of Staphylococcus aureus isolates from different origins in China.

    PubMed

    Chao, Guoxiang; Bao, Guangyu; Cao, Yongzhong; Yan, Wenguang; Wang, Yan; Zhang, Xiaorong; Zhou, Liping; Wu, Yantao

    2015-10-15

    Staphylococcal enterotoxins (SE) induce toxin-mediated diseases, such as food poisoning. In the present study, 568 isolates from different sources were tested for the prevalence of 18 SE genes and performed spa typing. In addition, we characterized the relationships between the distribution of SE genes and molecular clones based on multilocus sequence typing (MLST), spa and staphylococcal cassette chromosome mec (SCCmec) typing in selected 250 isolates. Approximately 54.40% of the isolates from different sources harbored one or more SE genes forming 120 distinct gene profiles. Seven genes, sea, seb, seg, seo, sem, seq, and sel were more frequently detected. The distributions of the SE genes among the isolates from human, animals, and foodborne origins were highly different with isolates from environments (P<0.01). The classic SE genes in both foodborne and human origin isolates were significantly higher than that in animal origin isolates (P<0.01), whereas the prevalence of genes of egc cluster and the other genes was similar in human, animal, and foodborne origin isolates (P>0.05). We identified two important gene clusters, sea-sek-seq, which is closely related to hospital-acquired (HA) methicillin-resistant Staphylococcus aureus (MRSA)-III, and the egc cluster, which accounts for nearly half of all genes. Approximately 71% isolates could be typed by spa, yielding 103 spa types, of which 18 spa types were primary types. In clonal complex (CC) 239, an important Asian HA-MRSA-III clone from humans, nearly all isolates harbored complete or partial sea-sek-seq cluster; the main spa types were t030 and t037. In CC630, an important new community-associated (CA) MRSA-V CC in China, only sporadic SE genes, three main spa types, t4549, t2196, and t377 were observed. The egc cluster coexisting with other genes was present in isolates of CC5, CC9, CC1281, CC1301, CC30 and sequence type (ST) 25, but completely absent in isolates of CC239, CC59, CC7, and CC88. The results

  4. The three-dimensional crystal structure of cholera toxin

    SciTech Connect

    Zhang, Rong-Guang; Westbrook, M.L.; Nance, S.; Spangler, B.D.; Scott, D.L.; Westbrook, E.M.

    1996-02-01

    The clinical manifestations of cholera are largely attributable to the actions of a secreted hexameric AB{sub 5} enterotoxin (choleragen). We have solved the three-dimensional structure of choleragen at 2.5 {Angstrom} resolution and compared the refined coordinates with those of choleragenoid (isolated B pentamer) and the heat-labile enterotoxin from Escherichia coli (LT). The crystalline coordinates provide a detailed view of the stereochemistry implicated in binding to GM1 gangliosides and in carrying out ADP-ribosylation. The A2 chain of choleragen, in contrast to that of LT, is a nearly continuous {alpha}-helix with an interpretable carboxyl tail.

  5. Genetic probes for enterotoxigenic Escherichia coli isolated from childhood diarrhea in the Central African Republic.

    PubMed Central

    Georges, M C; Wachsmuth, I K; Birkness, K A; Moseley, S L; Georges, A J

    1983-01-01

    Escherichia coli strains were isolated from 778 children with diarrhea and 151 well children in the Central African Republic over a period of 1 year. These 929 strains were assayed for heat-labile and heat-stable enterotoxin production and were hybridized (probed) with structural genes for these enterotoxins. Twenty-four isolates from diarrheal patients and one isolate from a well child were found to be toxigenic by assay and probe. Minor discrepancies were encountered with both assays and probes during initial screening procedures, but the two methodologies were ultimately comparable. Images PMID:6350346

  6. Structure of the superantigen staphylococcal enterotoxin B in complex with TCR and peptide-MHC demonstrates absence of TCR-peptide contacts.

    PubMed

    Rödström, Karin E J; Elbing, Karin; Lindkvist-Petersson, Karin

    2014-08-15

    Superantigens are immune-stimulatory toxins produced by Staphylococcus aureus, which are able to interact with host immune receptors to induce a massive release of cytokines, causing toxic shock syndrome and possibly death. In this article, we present the x-ray structure of staphylococcal enterotoxin B (SEB) in complex with its receptors, the TCR and MHC class II, forming a ternary complex. The structure, in combination with functional analyses, clearly shows how SEB adopts a wedge-like position when binding to the β-chain of TCR, allowing for an interaction between the α-chain of TCR and MHC. Furthermore, the binding mode also circumvents contact between TCR and the peptide presented by MHC, which enables SEB to initiate a peptide-independent activation of T cells.

  7. A synthetic peptide corresponding to the extracellular loop 2 region of claudin-4 protects against Clostridium perfringens enterotoxin in vitro and in vivo.

    PubMed

    Shrestha, Archana; Robertson, Susan L; Garcia, Jorge; Beingasser, Juliann; McClane, Bruce A; Uzal, Francisco A

    2014-11-01

    Clostridium perfringens enterotoxin (CPE) action starts when the toxin binds to claudin receptors. Claudins contain two extracellular loop domains, with the second loop (ECL-2) being slightly smaller than the first. CPE has been shown to bind to ECL-2 in receptor claudins. We recently demonstrated that Caco-2 cells (a naturally CPE-sensitive enterocyte-like cell line) can be protected from CPE-induced cytotoxicity by preincubating the enterotoxin with soluble full-length recombinant claudin-4 (rclaudin-4), which is a CPE receptor, but not with recombinant nonreceptor claudins, such as rclaudin-1. The current study evaluated whether a synthetic peptide corresponding to the claudin-4 ECL-2 sequence can similarly inhibit CPE action in vitro and in vivo. Significant protection of Caco-2 cells was also observed using either rclaudin-4 or the claudin-4 ECL-2 peptide in both a preincubation assay and a coincubation assay. This inhibitory effect was specific, since rclaudin-1 and a synthetic peptide based on the claudin-1 ECL-2 offered no protection to Caco-2 cells. However, the claudin-4 ECL-2 peptide was unable to neutralize cytotoxicity if CPE had already bound to Caco-2 cells. When the study was repeated in vivo using a rabbit small intestinal loop assay, preincubation or coincubation of CPE with the claudin-4 ECL-2 peptide significantly and specifically inhibited the development of CPE-induced luminal fluid accumulation and histologic lesions in rabbit small intestinal loops. No similar in vivo protection from CPE was afforded by the claudin-1 ECL-2 peptide. These results suggest that claudin-4 ECL-2 peptides should be further investigated for their potential therapeutic application against CPE-associated disease.

  8. Quantitative Analysis of Staphylococcal Enterotoxins A and B in Food Matrices Using Ultra High-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS)

    PubMed Central

    Zuberovic Muratovic, Aida; Hagström, Thomas; Rosén, Johan; Granelli, Kristina; Hellenäs, Karl-Erik

    2015-01-01

    A method that uses mass spectrometry (MS) for identification and quantification of protein toxins, staphylococcal enterotoxins A and B (SEA and SEB), in milk and shrimp is described. The analysis was performed using a tryptic peptide, from each of the toxins, as the target analyte together with the corresponding 13C-labeled synthetic internal standard peptide. The performance of the method was evaluated by analyzing spiked samples in the quantification range 2.5–30 ng/g (R2 = 0.92–0.99). The limit of quantification (LOQ) in milk and the limit of detection (LOD) in shrimp was 2.5 ng/g, for both SEA and SEB toxins. The in-house reproducibility (RSD) was 8%–30% and 5%–41% at different concentrations for milk and shrimp, respectively. The method was compared to the ELISA method, used at the EU-RL (France), for milk samples spiked with SEA at low levels, in the quantification range of 2.5 to 5 ng/g. The comparison showed good coherence for the two methods: 2.9 (MS)/1.8 (ELISA) and 3.6 (MS)/3.8 (ELISA) ng/g. The major advantage of the developed method is that it allows direct confirmation of the molecular identity and quantitative analysis of SEA and SEB at low nanogram levels using a label and antibody free approach. Therefore, this method is an important step in the development of alternatives to the immune-assay tests currently used for staphylococcal enterotoxin analysis. PMID:26378579

  9. Antimicrobial resistance and detection of the mecA gene besides enterotoxin-encoding genes among coagulase-negative Staphylococci isolated from clam meat of Anomalocardia brasiliana.

    PubMed

    Batista, Jacqueline Ellen Camelo; Ferreira, Ewerton Lucena; Nascimento, Danielle Cristina de Oliveira; Ventura, Roberta Ferreira; de Oliveira, Wagner Luis Mendes; Leal, Nilma Cintra; Lima-Filho, José Vitor

    2013-12-01

    The marine clam Anomalocardia brasiliana is a candidate as a sentinel animal to monitor the contamination levels of coliforms in shellfish-harvesting areas of Brazil's northeastern region. The aim of the present study was to search enterotoxin-encoding genes plus the mecA gene among coagulase-negative staphylococci (CNS) isolates from shellfish meats of A. brasiliana. The specimen clam (n=48; 40 clams per sample) was collected during low tide in the bay area of Mangue Seco from April through June 2009, and random samples of chilled and frozen shelled clam meat (n=33; 250 g per sample) were obtained from retail shops from January through March 2012. Seventy-nine CNS isolates were identified, including Staphylococcus xylosus, S. cohnii spp. urealyticus, S. sciuri, and S. lentus. A high percentage of isolates resistant to erythromycin (58.5%), penicillin (51.2%), and tetracycline (43.9%), and the fluoroquinolones levofloxacin (39%) and ciprofloxacin (34.1%) were recorded from those environmental samples. Isolates from retail shops were particularly resistant to oxacillin (55.3%) and penicillin (36.8%). All CNS resistant to oxacillin and/or cefoxitin were positive for the presence of the mecA gene, but phenotypically susceptible to vancomycin. Also, the enterotoxin-encoding genes seg and seh were detected through multiplex-polymerase chain reaction in 77.7% and 88.8% of the isolates from environmental samples, versus 90.5% and 100% of the isolates from retail shops, respectively. The data reveal the risk to public health due to consuming raw or undercooked shellfish containing enterotoxigenic plus methicillin-resistant CNS.

  10. Immunization with recombinant bivalent chimera r-Cpae confers protection against alpha toxin and enterotoxin of Clostridium perfringens type A in murine model.

    PubMed

    Shreya, Das; Uppalapati, Siva R; Kingston, Joseph J; Sripathy, Murali H; Batra, Harsh V

    2015-05-01

    Clostridium perfringens type A, an anaerobic pathogen is the most potent cause of soft tissue infections like gas gangrene and enteric diseases like food poisoning and enteritis. The disease manifestations are mediated via two important exotoxins, viz. myonecrotic alpha toxin (αC) and enterotoxin (CPE). In the present study, we synthesized a bivalent chimeric protein r-Cpae comprising C-terminal binding regions of αC and CPE using structural vaccinology rationale and assessed its protective efficacy against both alpha toxin (αC) and enterotoxin (CPE) respectively, in murine model. Active immunization of mice with r-Cpae generated high circulating serum IgG (systemic), significantly increased intestinal mucosal s-IgA antibody titres and resulted in substantial protection to the immunized animals (100% and 75% survival) with reduced tissue morbidity when administered with 5×LD(100) doses of αC (intramuscular) and CPE (intra-gastric gavage) respectively. Mouse RBCs and Caco-2 cells incubated with a mixture of anti-r-Cpae antibodies and αC and CPE respectively, illustrated significantly higher protection against the respective toxins. Passive immunization of mice with a similar mixture resulted in 91-100% survival at the end of the 15 days observation period while mice immunized with a concoction of sham sera and respective toxins died within 2-3 days. This work demonstrates the efficacy of the rationally designed r-Cpae chimeric protein as a potential sub unit vaccine candidate against αC and CPE of C. perfringens type A toxemia.

  11. Development and evaluation of IgY ImmunoCapture PCR ELISA for detection of Staphylococcus aureus enterotoxin A devoid of protein A interference.

    PubMed

    Reddy, Prakash; Ramlal, Shylaja; Sripathy, Murali Harishchandra; Batra, Harsh Vardhan

    2014-06-01

    In the present study, a sensitive and specific IgY mediated ImmunoCapture-PCR-ELISA (IC-PCR-ELISA) was developed for the detection of staphylococcal enterotoxin A (SEA) from culture supernatants and suspected contaminated samples. Due to the virtue of avian immunoglobulins (IgY) to have the least affinity towards staphylococcal protein A (SpA) responsible for false positives, we employed anti-SEA IgY for capture of SEA toxin and revealed with SEA specific rabbit antibodies conjugated to a 524bp DNA marker. Biotin-11-dUTP was incorporated during PCR amplification and post PCR analysis was performed by PCR-ELISA. Unlike IgG immunocapture, IgY mediated immunocapture of SEA was free from false positives due to protein A. The developed assay was specific to SEA except for minor cross reactivity with staphylococcal enterotoxin E (SEE). Several raw milk samples were evaluated for the presence of SEA with and without enrichment. Three samples were found to be positive for SEA after enrichment for 8h. Though IC-PCR-ELISA for SEA showed 100% correlation with PCR analysis for sea gene, the assay was unique in terms of sensitivity of detecting ~10pg/ml of SEA toxin from spiked milk samples. Result of IC-PCR-ELISA was further confirmed by conventional methods of isolation and characterization. The presented method can be very useful for rapid analysis of milk samples for SEA contamination and can be further extended for detection of multiple SE's in different wells of same PCR plate using common DNA substrate.

  12. Quantitative Analysis of Staphylococcal Enterotoxins A and B in Food Matrices Using Ultra High-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS).

    PubMed

    Muratovic, Aida Zuberovic; Hagström, Thomas; Rosén, Johan; Granelli, Kristina; Hellenäs, Karl-Erik

    2015-09-11

    A method that uses mass spectrometry (MS) for identification and quantification of protein toxins, staphylococcal enterotoxins A and B (SEA and SEB), in milk and shrimp is described. The analysis was performed using a tryptic peptide, from each of the toxins, as the target analyte together with the corresponding (13)C-labeled synthetic internal standard peptide. The performance of the method was evaluated by analyzing spiked samples in the quantification range 2.5-30 ng/g (R² = 0.92-0.99). The limit of quantification (LOQ) in milk and the limit of detection (LOD) in shrimp was 2.5 ng/g, for both SEA and SEB toxins. The in-house reproducibility (RSD) was 8%-30% and 5%-41% at different concentrations for milk and shrimp, respectively. The method was compared to the ELISA method, used at the EU-RL (France), for milk samples spiked with SEA at low levels, in the quantification range of 2.5 to 5 ng/g. The comparison showed good coherence for the two methods: 2.9 (MS)/1.8 (ELISA) and 3.6 (MS)/3.8 (ELISA) ng/g. The major advantage of the developed method is that it allows direct confirmation of the molecular identity and quantitative analysis of SEA and SEB at low nanogram levels using a label and antibody free approach. Therefore, this method is an important step in the development of alternatives to the immune-assay tests currently used for staphylococcal enterotoxin analysis.

  13. Patterns of loss of enterotoxigenicity by Escherichia coli isolated from adults with diarrhea: suggestive evidence for an interrelationship with serotype.

    PubMed Central

    Evans, D J; Evans, D G; DuPont, H L; Orskov, F; Orskov, I

    1977-01-01

    Enterotoxigenic Escherichia coli isolates obtained in Mexico from adult subjects with diarrhea and from healthy controls were examined for the production of heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT) after serial passage in the laboratory. Isolates were found to be either stable for the production of ST and LT or unstable with respect to ST, LT, or both. Unilateral loss of either ST or LT production allowed classification of E. coli isolates into four groups according to stability/instability of enterotoxin production. Fewer serotypes, with more representative isolates, were in group I (stable) than in group IV (completely unstable). Isolates from Dacca, Bangladesh, could be similarly classified into stability groups. There is an apparent relationship between serotype, stability of enterotoxin production, particularly LT, and isolation from diarrhea cases as opposed to isolation from healthy controls. PMID:328392

  14. Staphylococcal Enterotoxin A (SEA)

    DTIC Science & Technology

    1981-01-01

    751 and [77]. Assay Method C Serologic Activity. Many immunodiffusion procedures have been suc- cessfully employed, but we find the radial diffusion...method of Mancini et Ao E. P. Casman, MI. S. Beftdoll, and J. Robinson, J. Bacterial. 8S, 715 (1963). NTIS MU& ’M. S. Bergdoll. C. R. Burla R. N...A. Oeorgiades, G. J. Stanton. F. Dianzani. and H. M. Johnson. Infect. Inman. 26, 36 (1979). "G,. Mancini , A. 0. Carbonara. and 3. F. Heremans

  15. Enterotoxins of Staphylococci

    DTIC Science & Technology

    1988-01-01

    is observed by analy- tical ultracentrifugation, ion-exchange chromatography, gel filtration, and polyacrylamide gel electrophoresis in sodium dodecyl...significant heterogeneity is apparent in purified SEB in starch gel electrophoresis (Baird-Parker and Joseph, 1964). This was confirmed by Schantz et al...al., 1972). A better separation is obtained by iso- electric focusing in polyacrylamide gels as shown in Fig. 1. A similar heterogeneity is also seen

  16. Promoting Retention

    PubMed Central

    Hall, LaToya N.; Ficker, Lisa J.; Chadiha, Letha A.; Green, Carmen R.; Jackson, James S.; Lichtenberg, Peter A.

    2016-01-01

    Objectives: The objectives of this study were to evaluate the capability of a research volunteer registry to retain community-dwelling African American older adults, and to explore demographic and health factors associated with retention. Method: A logistic regression model was used to determine the influence of demographics, health factors, and registry logic model activities on retention in a sample of 1,730 older African American adults. Results: Almost 80% of participants active in the volunteer research registry between January 2012 and June 2015 were retained. Employment, being referred to research studies, a higher number of medical conditions, and more follow-up contacts were associated with an increased likelihood of retention. Older age, more months in the registry, and more mobility problems decreased the likelihood of retention. Discussion: These results suggest the Michigan Center for Urban African American Aging Research logic model promotes retention through involving older African American adults in research through study referrals and intensive follow-up. The loss of participants due to age- and mobility-related issues indicate the registry may be losing its most vulnerable participants. PMID:28138501

  17. Assessment of Enterotoxin Production and Cross-Contamination of Staphylococcus aureus between Food Processing Materials and Ready-To-Eat Cooked Fish Paste.

    PubMed

    Tango, Charles Nkufi; Hong, Sung-Sam; Wang, Jun; Oh, Deog-Hwan

    2015-12-01

    This study evaluated Staphylococcus aureus growth and subsequent staphylococcal enterotoxin A production in tryptone soy broth and on ready-to-eat cooked fish paste at 12 to 37 °C, as well as cross-contamination between stainless steel, polyethylene, and latex glove at room temperature. A model was developed using Barany and Roberts's growth model, which satisfactorily described the suitable growth of S. aureus with R(2)-adj from 0.94 to 0.99. Except at 12 °C, S. aureus cells in TSB presented a lag time lower (14.64 to 1.65 h), grew faster (0.08 to 0.31 log CFU/h) and produced SEA at lower cell density levels (5.65 to 6.44 log CFU/mL) compare to those inoculated on cooked fish paste with data of 16.920 to 1.985 h, 0.02 to 0.23 log CFU/h, and 6.19 to 7.11 log CFU/g, respectively. Staphylococcal enterotoxin type A (SEA) visual immunoassay test showed that primary SEA detection varied considerably among different storage temperature degrees and media. For example, it occurred only during exponential phase at 30 and 37 °C in TSB, but in cooked fish paste it took place at late exponential phase of S. aureus growth at 20 and 25 °C. The SEA detection test was negative on presence of S. aureus on cooked fish paste stored at 12 and 15 °C, although cell density reached level of 6.12 log CFU/g at 15 °C. Cross-contamination expressed as transfer rate of S. aureus from polyethylene surface to cooked fish paste surface was slower than that observed with steel surface to cooked fish paste under same conditions. These results provide helpful information for controlling S. aureus growth, SEA production and cross-contamination during processing of cooked fish paste.

  18. Characteristics of enterotoxin distribution, hemolysis, lecithinase, and starch hydrolysis of Bacillus cereus isolated from infant formulas and ready-to-eat foods.

    PubMed

    Hwang, Ji-Yeon; Park, Jong-Hyun

    2015-03-01

    Bacillus cereus is a ubiquitous environmental microbe implicated as a main cause of food poisoning with various symptoms, depending on the strain type and the isolation source. In this study, the potential virulence factors and biochemical properties of B. cereus isolated from infant formulas and ready-to-eat (RTE) foods were analyzed and compared. A total of 347 B. cereus strains were isolated and identified from 687 infant food formulas and RTE food samples. All the isolates had one or more enterotoxin genes, and one-half of the strains had all 3 enterotoxin genes (hbl, nhe, and cytK) that are involved in food poisoning in humans. Here, all the 3 genes were detected in 50% of the B. cereus isolates from RTE foods and only 14% of the isolates were identified from infant formulas. The latter harbored low cytK and bceT, and very low hbl genes. Most B. cereus isolates possessed the hemolysis gene, but not the ces gene. The infant formula isolates showed stronger hemolysis activity than the other isolates. In addition, 26% of the total isolates showed low lecithinase activities and 10% showed high lecithinase activities. A greater number of isolates from the infant formula showed high lecithinase activity than those from the RTE foods. Approximately 83% of the isolates were positive and 17% were negative for starch hydrolysis. Over 90% of the RTE food isolates and only 35% of the infant formula isolates were positive for starch hydrolysis. However, all the strains possessed nhe, but their harboring patterns of hbl and cytK were significantly different. Most starch-hydrolyzing strains possessed hbl, but only 23% nonstarch-hydrolyzing isolates possessed this gene. Moreover, very low nonstarch hydrolyzing strains harbored cytK. Most nonstarch-hydrolyzing isolates showed high lecithinase and strong hemolysis activities, and very low hbl and cytK harboring. In summary, most infant formula isolates showed stronger hemolysis and higher lecithinase activities with lower

  19. Wet-milling transgenic maize seed for fraction enrichment of recombinant subunit vaccine.

    PubMed

    Moeller, Lorena; Taylor-Vokes, Raye; Fox, Steve; Gan, Qinglei; Johnson, Lawrence; Wang, Kan

    2010-01-01

    The production of recombinant proteins in plants continues to be of great interest for prospective large-scale manufacturing of industrial enzymes, nutrition products, and vaccines. This work describes fractionation by wet-milling of transgenic maize expressing the B subunit of the heat-labile enterotoxin of Escherichia coli (LT-B), a potent immunogen and candidate for oral vaccine and vaccine components. The LT-B gene was directed to express in seed by an endosperm specific promoter. Two steeping treatments, traditional steeping (TS, 0.2% SO(2) + 0.5% lactic acid) and water steeping (WS, water only), were evaluated to determine effects on recovery of functional LT-B in wet-milled fractions. The overall recovery of the LT-B protein from WS treatment was 1.5-fold greater than that from TS treatment. In both steeping types, LT-B was distributed similarly among the fractions, resulting in enrichment of functional LT-B in fine fiber, coarse fiber and pericarp fractions by concentration factors of 1.5 to 8 relative to the whole kernels on a per-mass basis. Combined with endosperm-specific expression and secretory pathway targeting, wet-milling enables enrichment of high-value recombinant proteins in low-value fractions, such as the fine fiber, and co-utilization of remaining fractions in alternative industrial applications.

  20. Reduction of Non-Specific Protein Adsorption Using Poly(ethylene) Glycol (PEG) Modified Polyacrylate Hydrogels In Immunoassays for Staphylococcal Enterotoxin B Detection

    PubMed Central

    Charles, Paul T.; Stubbs, Veronte R.; Soto, Carissa M.; Martin, Brett D.; White, Brandy J.; Taitt, Chris R.

    2009-01-01

    Three PEG molecules (PEG-methacrylate, -diacrylate and -dimethacrylate) were incorporated into galactose-based polyacrylate hydrogels and their relative abilities to reduce non-specific protein adsorption in immunoassays were determined. Highly crosslinked hydrogels containing amine-terminated functionalities were formed and used to covalently attach antibodies specific for staphylococcal enterotoxin B (SEB). Patterned arrays of immobilized antibodies in the PEG-modified hydrogels were created with a PDMS template containing micro-channels for use in sandwich immunoassays to detect SEB. Different concentrations of the toxin were applied to the hydrogel arrays, followed with a Cy3-labeled tracer antibody specific for the two toxins. Fluorescence laser scanning confocal microscopy of the tracer molecules provided both qualitative and quantitative measurements on the detection sensitivity and the reduction in non-specific binding as a result of PEG incorporation. Results showed the PEG-modified hydrogel significantly reduced non-specific protein binding with a detection limit for SEB of 1 ng/mL. Fluorescence signals showed a 10-fold decrease in the non-specific binding and a 6-fold increase in specific binding of SEB. PMID:22389622

  1. Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier.

    PubMed

    Sonoda, N; Furuse, M; Sasaki, H; Yonemura, S; Katahira, J; Horiguchi, Y; Tsukita, S

    1999-10-04

    Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.

  2. Establishment of highly specific and quantitative immunoassay systems for staphylococcal enterotoxin A, B, and C using newly-developed monoclonal antibodies.

    PubMed

    Sasaki, Takanori; Terano, Yoshitake; Shibata, Tadayoshi; Kawamoto, Hiroyoshi; Kuzuguchi, Tsuyoshi; Kohyama, Erina; Watanabe, Toshihiro; Ohyama, Tohru; Gemba, Munekazu

    2005-01-01

    Staphylococcal enterotoxin (SE) activities remain after boiling or treating with proteases. The main symptoms such as vomiting and diarrhea, are caused by the ingestion of SEs. Among SEs, SEA has been reported to be the major and most toxic protein. A highly specific and simple assay system is required to diagnose staphylococcal food poisoning. Therefore, the development of a suitable assay system is strongly anticipated. In this study, we have established a highly specific and sensitive avidin-biotin sandwich ELISA (ABS-ELISA) system for SEA, SEB, and SEC1 using newly-developed monoclonal antibodies. The linearity of these systems obtained was in the range of 0.78-25 ng/ml for each SE, and furthermore, the lower concentrations of SEs could also be detected. The recoveries of SEs from murine serum, skim milk solution, and raw milk were found to be over 90%, suggesting that our systems could detect SEs without any interventions, such as these from milk or serum proteins. We were also able to quantify SEs in 22 specimens of culture supernatants of S. aureus isolated in past occurrences. Our established system should be very useful not only in the clinical field but also in various fields of investigation because of its quantifi-cation and simplicity in detecting SEs.

  3. Virulence gene profiling of enteroaggregative Escherichia coli heat-stable enterotoxin 1-harboring E. coli (EAST1EC) derived from sporadic diarrheal patients.

    PubMed

    Konno, Takayuki; Yatsuyanagi, Jun; Saito, Shioko

    2012-04-01

    Between 2007 and 2009, a total of 2168 Escherichia coli strains derived from diarrheal patients, defined as putative diarrheagenic E. coli (DEC), were collected from medical institutions in Akita prefecture, Japan. Thirty five of the strains lacked typical pathogenic determinants of DEC other than astA, which encodes enteroaggregative E. coli (EAggEC) heat-stable enterotoxin 1 (EAST1). These E. coli strains are referred to as EAST1EC. Several studies have suggested a role of EAST1 in diarrhea; however, the correlation between diarrhea and the presence of astA remains inconclusive. To investigate whether EAST1EC strains derived from diarrheal patients shared pathogenic factors other than EAST1, virulence gene profiling of 12 virulence genes - iha, lpfA, ldaG, pilS, pic, pet, irp2, daa, aah, aid, cdtB and hlyA - was carried out. PCR analysis revealed that four of the 35 EAST1EC strains harbored only astA, 24 harbored genes associated with adhesins and intestinal colonization, three strains harbored the gene for α-hemolysin, and 24 strains harbored the gene for a siderophore. These results indicated that some EAST1EC strains harbor various virulence genes associated with distinct E. coli pathotypes, primarily enterohemorrhagic E. coli and EAggEC, which may represent additional pathogenic determinants of EAST1EC.

  4. Quantitative defect in staphylococcal enterotoxin A binding and presentation by HLA-DM-deficient T2.Ak cells corrected by transfection of HLA-DM genes.

    PubMed

    Albert, L J; Denzin, L K; Ghumman, B; Bangia, N; Cresswell, P; Watts, T H

    1998-01-10

    HLA-DM facilitates peptide acquisition by MHC class II proteins within the endosomes of APC by facilitating release of invariant chain peptide intermediates (CLIP) from the class II molecules. T2 cells have a deletion in the MHC II region which deletes HLA-DM and MHC II genes. T2 cells transfected with MHC class II proteins are defective in protein presentation, a defect that is corrected by HLA-DM transfection. Here we show that T2 cells transfected with Ak are also impaired in binding and presentation of the superantistaphylococcal enterotoxin A and that HLA-DM transfection corrects this defect. The poor ability of SEA to bind to Ak on DM-deficient cells is somewhat surprising since Ak has a low affinity for CLIP and is not predominantly occupied with CLIP on T2 cells compared to wide-type APC. These data suggest an influence of HLA-DM on the structure or composition of the Ak/peptide complex beyond its role in the release of invariant chain peptides.

  5. Skin reactivity of unsensitized monkeys upon challenge with staphylococcal enterotoxin B: a new approach for investigating the site of toxin action.

    PubMed Central

    Scheuber, P H; Golecki, J R; Kickhöfen, B; Scheel, D; Beck, G; Hammer, D K

    1985-01-01

    The correlation between skin tests and emetic responses in unsensitized monkeys was used to elucidate the cellular site of action of staphylococcal enterotoxin B (SEB). Evidence is presented that SEB administered intradermally provoked immediate-type skin reactions associated with mild degranulation of cutaneous mast cells. The cytoplasma showed signs of synthetic and metabolic activity, with formation of vesicles and increased prominence of mitochondria. Carboxymethylation of histidine residues of SEB altered the molecule (cSEB) from more alkaline components to more acidic species with increased microheterogeneity. This modification caused a loss in toxicity and completely abrogated the skin-sensitizing activity without changing the immunological specificity. cSEB, however, could compete with SEB for binding sites on the target cell surface. Previously, compound 48/80-treated skin sites behaved refractively to challenge with SEB, indicating that mediators from cutaneous mast cells are required for SEB-induced skin reactions. Skin reactions as well as emetic responses challenged with SEB were completely inhibited by H2 receptor antagonists and calcium channel blockers but not by H1 antihistamine or competitive antagonists of serotonin. This new approach provides a model for investigating the mechanisms of SEB action. Images PMID:2866161

  6. Oligonucleotide probes for detection and differentiation of Staphylococcus aureus strains containing genes for enterotoxins A, B, and C and toxic shock syndrome toxin 1.

    PubMed Central

    Neill, R J; Fanning, G R; Delahoz, F; Wolff, R; Gemski, P

    1990-01-01

    Different synthetic DNA nucleotide sequences were evaluated as gene probes for the specific detection and differentiation of Staphylococcus aureus strains encoding enterotoxins A (SEA), B (SEB), and C (SEC) and toxic shock syndrome toxin 1 (TSST-1). Identification of sequences unique to each toxin, based on knowledge of their nucleotide sequences, led to preparation of the specific 18-base oligonucleotide probes EA1 (encoding amino acids 177 to 182 of SEA), EB2 (encoding amino acids 105 to 110 of SEB), EC5 (encoding amino acids 125 to 131 of SEC1), and TS1 (encoding amino acids 160 to 166 of TSST-1). In colony blot hybridization analyses, these probes hybridized specifically with DNA from strains that produced the respective toxin serotypes. An excellent (greater than or equal to 93%) correlation between hybridization results (genotype) and toxin protein detection by an enzyme-linked immunosorbent assay (phenotype) was observed in the characterization of both reference and clinical strains of S. aureus for SEA, SEB, and TSST-1. A lower correlation (64%) for SEC reflected a lack of sensitivity in detecting toxin production. Our findings demonstrate that molecular DNA hybridization with synthetic oligonucleotide probes provides another approach for establishing the toxigenicity of S. aureus. Images PMID:2380378

  7. Detection of Staphylococcus aureus enterotoxin B at femtomolar levels with a miniature integrated two-channel surface plasmon resonance (SPR) sensor.

    PubMed

    Naimushin, Alexei N; Soelberg, Scott D; Nguyen, Di K; Dunlap, Lucinda; Bartholomew, Dwight; Elkind, Jerry; Melendez, Jose; Furlong, Clement E

    2002-06-01

    Surface plasmon resonance (SPR) biosensors offer the capability for continuous real-time monitoring. The commercial instruments available have been large in size, expensive, and not amenable to field applications. We report here an SPR sensor system based on a prototype two-channel system similar to the single channel Spreeta devices. This system is an ideal candidate for field use. The two-channel design provides a reference channel to compensate for bulk refractive index (RI), non-specific binding and temperature variations. The SPR software includes a calibration function that normalizes the response from both channels, thus enabling accurate referencing. In addition, a temperature-controlled enclosure utilizing a thermo-electric module based on the Peltier effect provides the temperature stability necessary for accurate measurements of RI. The complete SPR sensor system can be powered by a 12V battery. Pre-functionalized, disposable, gold-coated thin glass slides provide easily renewable sensor elements for the system. Staphylococcus aureus enterotoxin B (SEB), a small protein toxin was directly detectable at sub-nanomolar levels and with amplification at femtomolar levels. A regeneration procedure for the sensor surface allowed for over 60 direct detection cycles in a 1-month period.

  8. In vitro cytotoxicity induced by Clostridium perfringens isolate carrying a chromosomal cpe gene is exclusively dependent on sporulation and enterotoxin production.

    PubMed

    Yasugi, Mayo; Sugahara, Yuki; Hoshi, Hidenobu; Kondo, Kaori; Talukdar, Prabhat K; Sarker, Mahfuzur R; Yamamoto, Shigeki; Kamata, Yoichi; Miyake, Masami

    2015-08-01

    Clostridium perfringens type A is a common source of food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases in humans. In the intestinal tract, the vegetative cells sporulate and produce a major pathogenic factor, C. perfringens enterotoxin (CPE). Most type A FP isolates carry a chromosomal cpe gene, whereas NFB type A isolates typically carry a plasmid-encoded cpe. In vitro, the purified CPE protein binds to a receptor and forms pores, exerting a cytotoxic activity in epithelial cells. However, it remains unclear if CPE is indispensable for C. perfringens cytotoxicity. In this study, we examined the cytotoxicity of cpe-harboring C. perfringens isolates co-cultured with human intestinal epithelial Caco-2 cells. The FP strains showed severe cytotoxicity during sporulation and CPE production, but not during vegetative cell growth. While Caco-2 cells were intact during co-culturing with cpe-null mutant derivative of strain SM101 (a FP strain carrying a chromosomal cpe gene), the wild-type level cytotoxicity was observed with cpe-complemented strain. In contrast, both wild-type and cpe-null mutant derivative of the NFB strain F4969 induced Caco-2 cell death during both vegetative and sporulation growth. Collectively, the Caco-2 cell cytotoxicity caused by C. perfringens strain SM101 is considered to be exclusively dependent on CPE production, whereas some additional toxins should be involved in F4969-mediated in vitro cytotoxicity.

  9. Preliminary investigation of human serum albumin-Vβ inhibition on toxic shock syndrome induced by staphylococcus enterotoxin B in vitro and in vivo.

    PubMed

    Yuan, Qifeng; Li, Lin; Pian, Yaya; Hao, Huaijie; Zheng, Yuling; Zang, Yating; Jiang, Hua; Jiang, Yongqiang

    2016-04-01

    Staphylococcus enterotoxin B (SEB) is a superantigen that can induce massive activation of T cells with specific Vβ and inflammatory cytokine cascades, which mediate shock. To date, no SEB vaccine has been developed for preventing toxic shock syndrome (TSS). Here, we evaluated the therapeutic effect of a fusion protein human serum albumin-Vβ (HSA-Vβ) on TSS induced by SEB. Compared with Vβ, the preparation of HSA-Vβ was much easier to handle owing to its solubility. Affinity testing showed that HSA-Vβ had high affinity for SEB. In vitro results showed that HSA-Vβ could effectively inhibit interferon (IFN)-γ and tumor necrosis factor (TNF)-α secretion by human peripheral blood mononuclear cells. Moreover, in vivo, HSA-Vβ reduced IFN-γ and TNF-α levels in the serum and protected mice from SEB lethal challenge when administered simultaneously with SEB or 30 min after SEB. In summary, we simplified the preparation of Vβ by fusion with HSA, creating the HSA-Vβ protein, which effectively inhibited cytokine production and protected mice from lethal challenge with SEB. These data indicated that HSA-Vβ may represent a novel therapeutic strategy for the treatment of SEB-induced TSS.

  10. Clostridium perfringens enterotoxin elicits rapid and specific cytolysis of breast carcinoma cells mediated through tight junction proteins claudin 3 and 4.

    PubMed

    Kominsky, Scott L; Vali, Mustafa; Korz, Dorian; Gabig, Theodore G; Weitzman, Sigmund A; Argani, Pedram; Sukumar, Saraswati

    2004-05-01

    Clostridium perfringens enterotoxin (CPE) induces cytolysis very rapidly through binding to its receptors, the tight junction proteins CLDN 3 and 4. In this study, we investigated CLDN 3 and 4 expression in breast cancer and tested the potential of CPE-mediated therapy. CLDN 3 and 4 proteins were detected in all primary breast carcinomas tested (n = 21) and, compared to normal mammary epithelium, were overexpressed in approximately 62% and 26%, respectively. Treatment of breast cancer cell lines in culture with CPE resulted in rapid and dose-dependent cytolysis exclusively in cells that expressed CLDN 3 and 4. Intratumoral CPE treatment of xenografts of T47D breast cancer cells in immunodeficient mice resulted in a significant reduction in tumor volume (P = 0.007), with accompanying necrosis. Necrotic reactions were also seen in three freshly resected primary breast carcinoma samples treated with CPE for 12 hours, while isolated primary breast carcinoma cells underwent rapid and complete cytolysis within 1 hour. Thus, expression of CLDN 3 and 4 sensitizes primary breast carcinomas to CPE-mediated cytolysis and emphasizes the potential of CPE in breast cancer therapy.

  11. Clostridium perfringens Enterotoxin Elicits Rapid and Specific Cytolysis of Breast Carcinoma Cells Mediated through Tight Junction Proteins Claudin 3 and 4

    PubMed Central

    Kominsky, Scott L.; Vali, Mustafa; Korz, Dorian; Gabig, Theodore G.; Weitzman, Sigmund A.; Argani, Pedram; Sukumar, Saraswati

    2004-01-01

    Clostridium perfringens enterotoxin (CPE) induces cytolysis very rapidly through binding to its receptors, the tight junction proteins CLDN 3 and 4. In this study, we investigated CLDN 3 and 4 expression in breast cancer and tested the potential of CPE-mediated therapy. CLDN 3 and 4 proteins were detected in all primary breast carcinomas tested (n = 21) and, compared to normal mammary epithelium, were overexpressed in approximately 62% and 26%, respectively. Treatment of breast cancer cell lines in culture with CPE resulted in rapid and dose-dependent cytolysis exclusively in cells that expressed CLDN 3 and 4. Intratumoral CPE treatment of xenografts of T47D breast cancer cells in immunodeficient mice resulted in a significant reduction in tumor volume (P = 0.007), with accompanying necrosis. Necrotic reactions were also seen in three freshly resected primary breast carcinoma samples treated with CPE for 12 hours, while isolated primary breast carcinoma cells underwent rapid and complete cytolysis within 1 hour. Thus, expression of CLDN 3 and 4 sensitizes primary breast carcinomas to CPE-mediated cytolysis and emphasizes the potential of CPE in breast cancer therapy. PMID:15111309

  12. Involvement of protein kinase C in the mechanism of action of Escherichia coli heat-stable enterotoxin (STa) in a human colonic carcinoma cell line, COLO-205

    SciTech Connect

    Gupta, Dyuti Datta; Saha, Subhrajit; Chakrabarti, Manoj K. . E-mail: mkc_niced@yahoo.co.in

    2005-08-01

    The present study was undertaken to determine the involvement of calcium-protein kinase C pathway in the mechanism of action of Escherichia coli heat stable enterotoxin (STa) apart from STa-induced activation of guanylate cyclase in human colonic carcinoma cell line COLO-205, which was used as a model cultured cell line to study the mechanism of action of E. coli STa. In response to E. coli STa, protein kinase C (PKC) activity was increased in a time-dependent manner with its physical translocation from cytosol to membrane. Inhibition of the PKC activity in membrane fraction and inhibition of its physical translocation in response to IP{sub 3}-mediated calcium release inhibitor dantrolene suggested the involvement of intracellular store depletion in the regulation of PKC activity. Among different PKC isoforms, predominant involvement of calcium-dependent protein kinase C (PKC{alpha}) was specified using isotype-specific pseudosubstrate, which showed pronounce enzyme activity. Inhibition of enzyme activity by PKC{alpha}-specific inhibitor Goe6976 and immunoblott study employing isotype-specific antibody further demonstrated the involvement of calcium-dependent isoform of PKC in the mechanism of action of E. coli STa. Moreover, inhibition of guanylate cyclase activity by PKC{alpha}-specific inhibitor Goe6976 suggested the involvement of PKC{alpha} in the regulation of guanylate cyclase activity.

  13. Enterotoxin Gene Cluster-Encoded SEI and SElN from Staphylococcus aureus Isolates are Crucial for the Induction of Human Blood Cell Proliferation and Pathogenicity in Rabbits

    PubMed Central

    Roetzer, Andreas; Gruener, Corina S.; Haller, Guenter; Beyerly, John; Model, Nina; Eibl, Martha M.

    2016-01-01

    Among the toxin family of bacterial superantigens, the six members of the enterotoxin gene cluster (egc) seem to have unusual characteristics. They are present in the majority of Staphylococcus aureus strains, but their role in disease remains uncertain. We assessed secretion levels, immunogenicity, and toxicity of native and recombinant egc proteins. After having developed enzyme-linked immunosorbent assays, we found different quantities of egc proteins secreted by bacterial isolates. Supernatants induced proliferation of human peripheral blood mononuclear cells. However, purified recombinant egc proteins were shown to have differing superantigenicity potentials. Immunization with identical amounts of all members of egc, and the prominent toxic agent SEB, resulted in neutralizing antisera. Two egc proteins, SEI and SElN, were found to play a predominant role within the cluster. Both displayed the highest potential to activate blood cells, and were essential to be neutralized in supernatants. The application of a supernatant of a strain bearing only egc was sufficient for a lethal outcome in a rabbit model. Again, neutralization of SEI and SElN led to the survival of all tested animals. Finally, nanogram amounts of purified rSEI and rSElN led to lethality in vivo, pointing out the importance of both as virulence determinants among egc superantigens. PMID:27801832

  14. The mutation Glu151Asp in the B-component of the Bacillus cereus non-hemolytic enterotoxin (Nhe) leads to a diverging reactivity in antibody-based detection systems.

    PubMed

    Didier, Andrea; Jeßberger, Nadja; Krey, Victoria; Dietrich, Richard; Scherer, Siegfried; Märtlbauer, Erwin

    2015-11-09

    The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection. For the diarrhea-associated enterotoxins, the assessment of the non-hemolytic enterotoxin B (NheB) titer determined by a sandwich enzyme immunoassay (EIA) correlates best with in vitro cytotoxicity. In general, the regulation of enterotoxin expression of B. cereus is a coordinately-regulated process influenced by environmental, and probably also by host factors. As long as these factors are not completely understood, the currently-applied diagnostic procedures are based on indirect approaches to assess the potential virulence of an isolate. To date, sandwich EIA results serve as a surrogate marker to categorize isolates as either potentially low or highly toxic. Here, we report on a single amino acid exchange in the NheB sequence leading to an underestimation of the cytotoxic potential in a limited number of strains. During the screening of a large panel of B. cereus isolates, six showed uncommon features with low sandwich EIA titers despite high cytotoxicity. Sequence analysis revealed the point-mutation (Glu)151(Asp) in the potential binding region of the capture antibody. Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots. A commercially-available assay based on a lateral flow device detects all strains correctly as NheB producers in a qualitative manner. In conclusion, isolates showing low NheB titers should additionally be assayed in an indirect EIA or for their in vitro cytotoxicity to ensure a correct classification as either low or highly toxic.

  15. Anoctamin 6 Contributes to Cl- Secretion in Accessory Cholera Enterotoxin (Ace)-stimulated Diarrhea: AN ESSENTIAL ROLE FOR PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE (PIP2) SIGNALING IN CHOLERA.

    PubMed

    Aoun, Joydeep; Hayashi, Mikio; Sheikh, Irshad Ali; Sarkar, Paramita; Saha, Tultul; Ghosh, Priyanka; Bhowmick, Rajsekhar; Ghosh, Dipanjan; Chatterjee, Tanaya; Chakrabarti, Pinak; Chakrabarti, Manoj K; Hoque, Kazi Mirajul

    2016-12-23

    Accessory cholera enterotoxin (Ace) of Vibrio cholerae has been shown to contribute to diarrhea. However, the signaling mechanism and specific type of Cl(-) channel activated by Ace are still unknown. We have shown here that the recombinant Ace protein induced ICl of apical plasma membrane, which was inhibited by classical CaCC blockers. Surprisingly, an Ace-elicited rise of current was neither affected by ANO1 (TMEM16A)-specific inhibitor T16A(inh)-AO1(TAO1) nor by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker, CFTR inh-172. Ace stimulated whole-cell current in Caco-2 cells. However, the apical ICl was attenuated by knockdown of ANO6 (TMEM16F). This impaired phenotype was restored by re-expression of ANO6 in Caco-2 cells. Whole-cell patch clamp recordings of ANO currents in HEK293 cells transiently expressing mouse ANO1-mCherry or ANO6-GFP confirmed that Ace induced Cl(-) secretion. Application of Ace produced ANO6 but not the ANO1 currents. Ace was not able to induce a [Ca(2+)]i rise in Caco-2 cells, but cellular abundance of phosphatidylinositol 4,5-bisphosphate (PIP2) increased. Identification of the PIP2-binding motif at the N-terminal sequence among human and mouse ANO6 variants along with binding of PIP2 directly to ANO6 in HEK293 cells indicate likely PIP2 regulation of ANO6. The biophysical and pharmacological properties of Ace stimulated Cl(-) current along with intestinal fluid accumulation, and binding of PIP2 to the proximal KR motif of channel proteins, whose mutagenesis correlates with altered binding of PIP2, is comparable with ANO6 stimulation. We conclude that ANO6 is predominantly expressed in intestinal epithelia, where it contributes secretory diarrhea by Ace stimulation in a calcium-independent mechanism of RhoA-ROCK-PIP2 signaling.

  16. How T-Cell-Dependent and -Independent Challenges Access the Brain: Vascular and Neural Responses to Bacterial Lipopolysaccharide and Staphylococcal Enterotoxin B

    PubMed Central

    Serrats, Jordi; Sawchenko, Paul E.

    2009-01-01

    Bacterial lipopolysaccharide (LPS) is widely used to study immune influences on the CNS, and cerebrovascular prostaglandin (PG) synthesis is implicated in mediating LPS influences on some acute phase responses. Other bacterial products, such as staphylococcal enterotoxin B (SEB), impact target tissues differently in that their effects are T-lymphocyte-dependent, yet both LPS and SEB recruit a partially overlapping set of subcortical central autonomic cell groups. We sought to compare neurovascular responses to the two pathogens, and the mechanisms by which they may access the brain. Rats received iv injections of LPS (2 μg/kg), SEB (1 mg/kg) or vehicle and were sacrificed 0.5–3 hr later. Both challenges engaged vascular cells as early 0.5 hr, as evidenced by induced expression of the vascular early response gene (Verge), and the immediate-early gene, NGFI-B. Cyclooxygenase-2 (COX-2) expression was detected in both endothelial and perivascular cells (PVCs) in response to LPS, but only in PVCs of SEB-challenged animals. The non-selective COX inhibitor, indomethacin (1 mg/kg, iv), blocked LPS-induced activation in a subset of central autonomic structures, but failed to alter SEB-driven responses. Liposome mediated ablation of PVCs modulated the CNS response to LPS, did not affect the SEB-induced activational profile. By contrast, disruptions of interoceptive signaling by area postrema lesions or vagotomy (complete or hepatic) markedly attenuated SEB-, but not LPS-, stimulated central activational responses. Despite partial overlap in their neuronal and vascular response profiles, LPS and SEB appear to use distinct mechanisms to access the brain. PMID:19524662

  17. Effects of Small Molecule Calcium-Activated Chloride Channel Inhibitors on Structure and Function of Accessory Cholera Enterotoxin (Ace) of Vibrio cholerae

    PubMed Central

    Chatterjee, Tanaya; Sheikh, Irshad Ali; Chakravarty, Devlina; Chakrabarti, Pinak; Sarkar, Paramita; Saha, Tultul; Chakrabarti, Manoj K.; Hoque, Kazi Mirajul

    2015-01-01

    Cholera pathogenesis occurs due to synergistic pro-secretory effects of several toxins, such as cholera toxin (CTX) and Accessory cholera enterotoxin (Ace) secreted by Vibrio cholerae strains. Ace activates chloride channels stimulating chloride/bicarbonate transport that augments fluid secretion resulting in diarrhea. These channels have been targeted for drug development. However, lesser attention has been paid to the interaction of chloride channel modulators with bacterial toxins. Here we report the modulation of the structure/function of recombinant Ace by small molecule calcium-activated chloride channel (CaCC) inhibitors, namely CaCCinh-A01, digallic acid (DGA) and tannic acid. Biophysical studies indicate that the unfolding (induced by urea) free energy increases upon binding CaCCinh-A01 and DGA, compared to native Ace, whereas binding of tannic acid destabilizes the protein. Far-UV CD experiments revealed that the α-helical content of Ace-CaCCinh-A01 and Ace-DGA complexes increased relative to Ace. In contrast, binding to tannic acid had the opposite effect, indicating the loss of protein secondary structure. The modulation of Ace structure induced by CaCC inhibitors was also analyzed using docking and molecular dynamics (MD) simulation. Functional studies, performed using mouse ileal loops and Ussing chamber experiments, corroborate biophysical data, all pointing to the fact that tannic acid destabilizes Ace, inhibiting its function, whereas DGA stabilizes the toxin with enhanced fluid accumulation in mouse ileal loop. The efficacy of tannic acid in mouse model suggests that the targeted modulation of Ace structure may be of therapeutic benefit for gastrointestinal disorders. PMID:26540279

  18. Effects of Small Molecule Calcium-Activated Chloride Channel Inhibitors on Structure and Function of Accessory Cholera Enterotoxin (Ace) of Vibrio cholerae.

    PubMed

    Chatterjee, Tanaya; Sheikh, Irshad Ali; Chakravarty, Devlina; Chakrabarti, Pinak; Sarkar, Paramita; Saha, Tultul; Chakrabarti, Manoj K; Hoque, Kazi Mirajul

    2015-01-01

    Cholera pathogenesis occurs due to synergistic pro-secretory effects of several toxins, such as cholera toxin (CTX) and Accessory cholera enterotoxin (Ace) secreted by Vibrio cholerae strains. Ace activates chloride channels stimulating chloride/bicarbonate transport that augments fluid secretion resulting in diarrhea. These channels have been targeted for drug development. However, lesser attention has been paid to the interaction of chloride channel modulators with bacterial toxins. Here we report the modulation of the structure/function of recombinant Ace by small molecule calcium-activated chloride channel (CaCC) inhibitors, namely CaCCinh-A01, digallic acid (DGA) and tannic acid. Biophysical studies indicate that the unfolding (induced by urea) free energy increases upon binding CaCCinh-A01 and DGA, compared to native Ace, whereas binding of tannic acid destabilizes the protein. Far-UV CD experiments revealed that the α-helical content of Ace-CaCCinh-A01 and Ace-DGA complexes increased relative to Ace. In contrast, binding to tannic acid had the opposite effect, indicating the loss of protein secondary structure. The modulation of Ace structure induced by CaCC inhibitors was also analyzed using docking and molecular dynamics (MD) simulation. Functional studies, performed using mouse ileal loops and Ussing chamber experiments, corroborate biophysical data, all pointing to the fact that tannic acid destabilizes Ace, inhibiting its function, whereas DGA stabilizes the toxin with enhanced fluid accumulation in mouse ileal loop. The efficacy of tannic acid in mouse model suggests that the targeted modulation of Ace structure may be of therapeutic benefit for gastrointestinal disorders.

  19. T cell-mediated lethal shock triggered in mice by the superantigen staphylococcal enterotoxin B: critical role of tumor necrosis factor

    PubMed Central

    1992-01-01

    Because mice are more resistant than humans to the pathogenic effects of bacterial toxins, we used D-Galactosamine- (D-Gal) sensitized mice as a model system to evaluate potential toxic shock symptoms triggered by the superantigen staphylococcal enterotoxin B (SEB). We show that similar to endotoxin (lipopolysaccharide) [LPS], the exotoxin SEB causes lethal shock within 8 h in D-Gal-sensitized mice, inducing 100% and about 50% lethality with 20 and 2 micrograms SEB, respectively. The lethal shock triggered by the superantigen SEB is mediated by T cells, a conclusion based on the observation that T cell repopulation of SCID mice conferred sensitivity to SEB. Since CSA also conferred protection, the role of T cell-derived lymphokines in mediating lethal shock was evaluated. Within 30-60 min after SEB injection, serum tumor necrosis factor (TNF) levels peaked, followed immediately by interleukin-2 (IL- 2). Serum-borne lymphokines were detected well in advance of signs of T cell activation, as assessed by IL-2 receptor expression of SEB- reactive V beta 8+ T cells. Passive immunization with anti-TNF- alpha/beta-neutralizing monoclonal antibody also conferred protection, indicating that it is TNF which is critical for initiating toxic shock symptoms. Taken together, this study defines basic differences between endotoxin (LPS)- and exotoxin (SEB)-mediated lethal shock, in that the former is mediated by macrophages and the latter by T cells. Yet the pathogenesis distal to the lymphokine/cytokine-producing cells appears surprisingly similar in that TNF represents a key mediator in inducing shock. PMID:1730929

  20. Detection and genetic analysis of the enteroaggregative Escherichia coli heat-stable enterotoxin (EAST1) gene in clinical isolates of enteropathogenic Escherichia coli (EPEC) strains

    PubMed Central

    2014-01-01

    Background The enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) encoded by astA gene has been found in enteropathogenic E. coli (EPEC) strains. However, it is not sufficient to simply probe strains with an astA gene probe due to the existence of astA mutants (type 1 and type 2 SHEAST) and EAST1 variants (EAST1 v1-4). In this study, 222 EPEC (70 typical and 152 atypical) isolates were tested for the presence of the astA gene sequence by PCR and sequencing. Results The astA gene was amplified from 54 strains, 11 typical and 43 atypical. Sequence analysis of the PCR products showed that 25 strains, 7 typical and 18 atypical, had an intact astA gene. A subgroup of 7 atypical strains had a variant type of the astA gene sequence, with four non-synonymous nucleotide substitutions. The remaining 22 strains had mutated astA gene with nucleotide deletions or substitutions in the first 8 codons. The RT-PCR results showed that the astA gene was transcribed only by the strains carrying either the intact or the variant type of the astA gene sequence. Southern blot analysis indicated that astA is located in EAF plasmid in typical strains, and in plasmids of similar size in atypical strains. Strains carrying intact astA genes were more frequently found in diarrheic children than in non-diarrheic children (p < 0.05). Conclusion In conclusion, our data suggest that the presence of an intact astA gene may represent an additional virulence determinant in both EPEC groups. PMID:24884767

  1. Formation of small transmembrane pores: An intermediate stage on the way to Bacillus cereus non-hemolytic enterotoxin (Nhe) full pores in the absence of NheA.

    PubMed

    Zhu, Kui; Didier, Andrea; Dietrich, Richard; Heilkenbrinker, Uta; Waltenberger, Eva; Jessberger, Nadja; Märtlbauer, Erwin; Benz, Roland

    2016-01-15

    The non-hemolytic enterotoxin (Nhe) of Bacillus cereus is a three-partite toxin formed of the components NheA, -B and -C. Pore formation and subsequent lysis of target cells caused by Nhe is an orchestrated process comprising three steps: (i) formation of NheB/C oligomers in solution, (ii) attachment of the oligomers to the cell membrane, (iii) binding of NheA to the oligomers. The present study aimed to characterize the properties of the NheB/C complex and the fate of the target cell upon binding. An enzyme immunoassay allowing kinetic measurements and surface plasmon resonance revealed the fast and high affinity formation of the NheB/C oligomers. The benefit of these complexes is a more stable cell binding as well as stronger and earlier cytotoxic effect. High molecular mass hetero-oligomers (620 kDa) probably consisting of one NheC and up to 15 NheB were detected by size-exclusion chromatography and on native PAGE immunoblots. Due to the NheBC application the morphology and membrane permeability of Vero cells is partly disturbed. Formation of stable transmembrane channels with a conductance of about 870 pS and a diameter of about 2 nm due to the application of NheBC could be demonstrated in lipid bilayer experiments. Thus, the NheBC complex itself has a tendency to increase the membrane permeability prior to the emergence of full pores containing also NheA.

  2. Immunogenicity and efficacy against lethal aerosol staphylococcal enterotoxin B challenge in monkeys by intramuscular and respiratory delivery of proteosome-toxoid vaccines.

    PubMed Central

    Lowell, G H; Colleton, C; Frost, D; Kaminski, R W; Hughes, M; Hatch, J; Hooper, C; Estep, J; Pitt, L; Topper, M; Hunt, R E; Baker, W; Baze, W B

    1996-01-01

    Staphylococcal enterotoxin B (SEB), a primary cause of food poisoning, is also a superantigen that can cause toxic shock after traumatic or surgical staphylococcal wound [correction of would] infections or viral influenza-associated staphylococcal superinfections or when aerosolized for use as a potential biologic warfare threat agent. Intranasal or intramuscular (i.m.) immunization with formalinized SEB toxoid formulated with meningococcal outer membrane protein proteosomes has previously been shown to be immunogenic and protective against lethal respiratory or parenteral SEB challenge in murine models of SEB intoxication. Here, it is demonstrated that immunization of nonhuman primates with the proteosome-SEB toxoid vaccine is safe, immunogenic, and protective against lethal aerosol challenge with 15 50% lethal doses of SEB. Monkeys (10 per group) were primed i.m. and given booster injections by either the i.m. or intratracheal route without adverse side effects. Anamnestic anti-SEB serum immunoglobulin G (IgG) responses were elicited in all monkeys, but strong IgA responses in sera and bronchial secretions were elicited both pre- and post-SEB challenge only in monkeys given booster injections intratracheally. The proteosome-SEB toxoid vaccine was efficacious by both routes in protecting 100% of monkeys against severe symptomatology and death from aerosolized-SEB intoxication. These data confirm the safety, immunogenicity, and efficacy in monkeys of parenteral and respiratory vaccination with the proteosome-SEB toxoid, thereby supporting clinical trials of this vaccine in humans. The safety and enhancement of both bronchial and systemic IgA and IgG responses by the proteosome vaccine delivered by a respiratory route are also encouraging for the development of mucosally delivered proteosome vaccines to protect against SEB and other toxic or infectious respiratory pathogens. PMID:8890226

  3. The Bacillus cereus Hbl and Nhe tripartite enterotoxin components assemble sequentially on the surface of target cells and are not interchangeable.

    PubMed

    Sastalla, Inka; Fattah, Rasem; Coppage, Nicole; Nandy, Poulomi; Crown, Devorah; Pomerantsev, Andrei P; Leppla, Stephen H

    2013-01-01

    Bacillus cereus is a spore-forming, Gram-positive bacterium commonly associated with outbreaks of food poisoning. It is also known as an opportunistic pathogen causing clinical infections such as bacteremia, meningitis, pneumonia, and gas gangrene-like cutaneous infections, mostly in immunocompromised patients. B. cereus secretes a plethora of toxins of which four are associated with the symptoms of food poisoning. Two of these, the non-hemolytic enterotoxin Nhe and the hemolysin BL (Hbl) toxin, are predicted to be structurally similar and are unique in that they require the combined action of three toxin proteins to induce cell lysis. Despite their dominant role in disease, the molecular mechanism of their toxic function is still poorly understood. We report here that B. cereus strain ATCC 10876 harbors not only genes encoding Nhe, but also two copies of the hbl genes. We identified Hbl as the major secreted toxin responsible for inducing rapid cell lysis both in cultured cells and in an intraperitoneal mouse toxicity model. Antibody neutralization and deletion of Hbl-encoding genes resulted in significant reductions of cytotoxic activity. Microscopy studies with Chinese Hamster Ovary cells furthermore showed that pore formation by both Hbl and Nhe occurs through a stepwise, sequential binding of toxin components to the cell surface and to each other. This begins with binding of Hbl-B or NheC to the eukaryotic membrane, and is followed by the recruitment of Hbl-L1 or NheB, respectively, followed by the corresponding third protein. Lastly, toxin component complementation studies indicate that although Hbl and Nhe can be expressed simultaneously and are predicted to be structurally similar, they are incompatible and cannot complement each other.

  4. Developing a Promotional Video

    ERIC Educational Resources Information Center

    Epley, Hannah K.

    2014-01-01

    There is a need for Extension professionals to show clientele the benefits of their program. This article shares how promotional videos are one way of reaching audiences online. An example is given on how a promotional video has been used and developed using iMovie software. Tips are offered for how professionals can create a promotional video and…

  5. Cell surface binding site for Clostridium difficile enterotoxin: evidence for a glycoconjugate containing the sequence Gal alpha 1-3Gal beta 1-4GlcNAc.

    PubMed Central

    Krivan, H C; Clark, G F; Smith, D F; Wilkins, T D

    1986-01-01

    This study was undertaken to determine whether a binding site for Clostridium difficile enterotoxin (toxin A) exists in the brush border membranes (BBMs) of the hamster, an animal known to be extremely sensitive to the action of the toxin. Toxin A was the only antigen adsorbed by the BBMs from the culture filtrate of C. difficile. The finding that binding activity could not be destroyed by heat indicated that a carbohydrate moiety might be involved. We therefore examined erythrocytes from various animal species for binding activity since erythrocytes provide a variety of carbohydrate sequences on their cell surfaces. Only rabbit erythrocytes bound the toxin, and the cells agglutinated. A binding assay based on an enzyme-linked immunosorbent assay method for quantifying C. difficile toxin A was used to compare binding of the toxin to hamster BBMs, rabbit erythrocytes, and BBMs from rats, which are less susceptible to the action of C. difficile toxin A than hamsters. Results of this comparison indicated the following order of toxin-binding frequency: rabbit erythrocytes greater than hamster BBMs greater than rat BBMs. Binding of toxin A to hamster BBMs at 37 degrees C was comparable to what has been observed with cholera toxin, but binding was enhanced at 4 degrees C. A similar binding phenomenon was observed with rabbit erythrocytes. Examination of the cell surfaces of hamster BBMs and rabbit erythrocytes with lectins and specific glycosidases revealed a high concentration of terminal alpha-linked galactose. Treatment of both membrane types with alpha-galactosidase destroyed the binding activity. The glycoprotein, calf thyroglobulin, also bound the toxin and inhibited toxin binding to cells. Toxin A did not bind to human erythrocytes from blood group A, B, or O donors. However, after fucosidase treatment of human erythrocytes, only blood group B erythrocytes, which possess the blood group B structure Gal alpha 1-3[Fuc alpha 1-2]Gal beta 1-4GlcNAc-R, bound the toxin

  6. Natural indoles, indole-3-carbinol and 3,3′-diindolymethane, inhibit T cell activation by staphylococcal enterotoxin B through epigenetic regulation involving HDAC expression

    SciTech Connect

    Busbee, Philip B.; Nagarkatti, Mitzi; Nagarkatti, Prakash S.

    2014-01-01

    Staphylococcal enterotoxin B (SEB) is a potent exotoxin produced by the Staphylococcus aureus. This toxin is classified as a superantigen because of its ability to directly bind with MHC-II class molecules followed by activation of a large proportion of T cells bearing specific Vβ-T cell receptors. Commonly associated with classic food poisoning, SEB has also been shown to induce toxic shock syndrome, and is also considered to be a potential biological warfare agent because it is easily aerosolized. In the present study, we assessed the ability of indole-3-carbinol (I3C) and one of its byproducts, 3,3′-diindolylmethane (DIM), found in cruciferous vegetables, to counteract the effects of SEB-induced activation of T cells in mice. Both I3C and DIM were found to decrease the activation, proliferation, and cytokine production by SEB-activated Vβ8{sup +} T cells in vitro and in vivo. Interestingly, inhibitors of histone deacetylase class I (HDAC-I), but not class II (HDAC-II), showed significant decrease in SEB-induced T cell activation and cytokine production, thereby suggesting that epigenetic modulation plays a critical role in the regulation of SEB-induced inflammation. In addition, I3C and DIM caused a decrease in HDAC-I but not HDAC-II in SEB-activated T cells, thereby suggesting that I3C and DIM may inhibit SEB-mediated T cell activation by acting as HDAC-I inhibitors. These studies not only suggest for the first time that plant-derived indoles are potent suppressors of SEB-induced T cell activation and cytokine storm but also that they may mediate these effects by acting as HDAC inhibitors. - Highlights: • I3C and DIM reduce SEB-induced T cell activation and inflammatory cytokines. • Inhibiting class I HDACs reduces T cell activation and inflammatory cytokines. • Inhibiting class II HDACs increases T cell activation and inflammatory cytokines. • I3C and DIM selectively reduce mRNA expression of class I HDACs. • Novel use and mechanism to counteract

  7. Preparation and activity of conjugate of monoclonal antibody HAb18 against hepatoma F(ab’)2 fragment and staphylococcal enterotoxin A Lian

    PubMed Central

    Yang, Jun; Sui, Yan Fang; Chen, Zhi Nan

    2001-01-01

    AIM: To prepare the conjugate of staphy lococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab’)2 fragment of mAb HAb18 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC. METHODS: MAb HAb18 was extracted from the abdominal dropsy of Balb/c mice, and was purified through chromatography column SP 40HR with Fast protein liquid chromatography (FPLC) system. The F(ab’)2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab’)2 fragment and SEA was prepared with chemical conjugating reagent N succinimidyl 3 (2pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12 with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry’s method. The antibody activity of HAb18 F(ab’)2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay. RESULTS: The IgG mAb HAb18 was extracted, and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab’)2 SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F(ab’)2 SEA conjugate and HAb18 F(ab’)2, i.e.the cytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F(ab’)2 SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033 ± 0.009, and there was significant difference between them (P

  8. Health promotion in Brazil.

    PubMed

    Buss, Paulo Marchiori; de Carvalho, Antonio Ivo

    2007-01-01

    The evolution of health promotion within the Brazilian health system is examined, including an assessment of the intersectoral and development policies that have influenced the process. Particular attention is paid to the legal characteristics of the Unified Health System. Human resources formation and research initiatives in health promotion are outlined, with a summary of the obstacles that need to be overcome in order to ensure the effective implementation of health promotion in the future. Up to the end of the 20th Century health promotion was not used as a term in the Brazilian public heath context. Health promoting activities were concentrated in the area of health education, although targeting the social determinants of health and the principle of intersectoral action were part of the rhetoric. The situation has changed during the last decade, with the publication of a national policy of health promotion, issued by the Ministry of Health and jointly implemented with the States and Municipals Health Secretaries. More recently there has been a re-emergence of the discourse on the social determinants of health and the formation of intersectoral public policies as the basis of a comprehensive health promotion. Health promotion infrastructure, particularly around human resources and financing, requires strengthening in order to ensure capacity and sustainability in health promotion practice.

  9. Skin exposure promotes a Th2-dependent sensitization to peanut allergens.

    PubMed

    Tordesillas, Leticia; Goswami, Ritobrata; Benedé, Sara; Grishina, Galina; Dunkin, David; Järvinen, Kirsi M; Maleki, Soheila J; Sampson, Hugh A; Berin, M Cecilia

    2014-11-01

    Sensitization to foods often occurs in infancy, without a known prior oral exposure, suggesting that alternative exposure routes contribute to food allergy. Here, we tested the hypothesis that peanut proteins activate innate immune pathways in the skin that promote sensitization. We exposed mice to peanut protein extract on undamaged areas of skin and observed that repeated topical exposure to peanut allergens led to sensitization and anaphylaxis upon rechallenge. In mice, this epicutaneous peanut exposure induced sensitization to the peanut components Ara h 1 and Ara h 2, which is also observed in human peanut allergy. Both crude peanut extract and Ara h 2 alone served as adjuvants, as both induced a bystander sensitization that was similar to that induced by the atopic dermatitis-associated staphylococcal enterotoxin B. In cultured human keratinocytes and in murine skin, peanut extract directly induced cytokine expression. Moreover, topical peanut extract application induced an alteration dependent on the IL-33 receptor ST2 in skin-draining DCs, resulting in Th2 cytokine production from T cells. Together, our data support the hypothesis that peanuts are allergenic due to inherent adjuvant activity and suggest that skin exposure to food allergens contributes to sensitization to foods in early life.

  10. High expression Zymomonas promoters

    DOEpatents

    Viitanen, Paul V.; Tao, Luan; Zhang, Yuying; Caimi, Perry G.; McCole, Laura : Zhang, Min; Chou, Yat-Chen; McCutchen, Carol M.; Franden, Mary Ann

    2011-08-02

    Identified are mutants of the promoter of the Z. mobilis glyceraldehyde-3-phosphate dehydrogenase gene, which direct improved expression levels of operably linked heterologous nucleic acids. These are high expression promoters useful for expression of chimeric genes in Zymomonas, Zymobacter, and other related bacteria.

  11. Health Promotion Program.

    ERIC Educational Resources Information Center

    McClary, Cheryl

    The Health Promotion Program began with establishment of a one-credit course in health promotion and wellness and the training of family practice residents at the Mountain Area Health Education Center to serve as lab leaders in the course. The course later became part of the university's general education requirements. In addition, a health…

  12. Promoting Resilience in Children.

    ERIC Educational Resources Information Center

    Rolfe, Sharne A.

    2002-01-01

    This booklet invites reflection on ways in which childhood resilience can be promoted, thereby helping children to adapt effectively in the face of adversity. The attributes of resilient children are described, as is the importance of protective factors in building or promoting resilience. The booklet discusses the complex interplay between risk…

  13. Immunostimulant patches containing Escherichia coli LT enhance immune responses to DNA- and recombinant protein-based Alzheimer's disease vaccines.

    PubMed

    Davtyan, Hayk; Ghochikyan, Anahit; Hovakimyan, Armine; Petrushina, Irina; Yu, Jianmei; Flyer, David; Madsen, Peter Juul; Pedersen, Lars Ostergaard; Cribbs, David H; Agadjanyan, Michael G

    2014-03-15

    Immunotherapeutic approaches to treating Alzheimer's disease (AD) using vaccination strategies must overcome the obstacle of achieving adequate responses to vaccination in the elderly. Here we demonstrate for the first time that application of the Escherichia coli heat-labile enterotoxin adjuvant-laden immunostimulatory patches (LT-IS) dramatically enhances the onset and magnitude of immune responses to DNA- and protein-based vaccines for Alzheimer's disease following intradermal immunization via gene gun and conventional needles, respectively. Our studies suggest that the immune activation mediated by LT-IS offers improved potency for generating AD-specific vaccination responses that should be investigated as an adjuvant in the clinical arena.

  14. Investigating potential exogenous tumor initiating and promoting factors for Cutaneous T-Cell Lymphomas (CTCL), a rare skin malignancy.

    PubMed

    Litvinov, Ivan V; Shtreis, Anna; Kobayashi, Kenneth; Glassman, Steven; Tsang, Matthew; Woetmann, Anders; Sasseville, Denis; Ødum, Niels; Duvic, Madeleine

    2016-07-01

    Most skin malignancies are caused by external and often preventable environmental agents. Multiple reports demonstrated that cutaneous T-cell lymphomas (CTCL) can occur in married couples and cluster in families. Furthermore, recent studies document geographic clustering of this malignancy in Texas as well as in other areas of the United States. Multiple infectious, occupational, and medication causes have been proposed as triggers or promoters of this malignancy including hydrochlorothiazide diuretics, Staphylococcus aureus, dermatophytes, Mycobacterium leprae, Chlamydia pneumoniae, human T-Cell lymphotropic virus type 1 (HTLV1), Epstein-Barr virus (EBV), and herpes simplex virus (HSV). In this report, we review recent evidence evaluating the involvement of these agents in cancer initiation/progression. Most importantly, recent molecular experimental evidence documented for the first time that S. aureus can activate oncogenic STAT3 signaling in malignant T cells. Specifically, S. aureus Enterotoxin type A (SEA) was recently shown to trigger non-malignant infiltrating T cells to release IL-2 and other cytokines. These signals upon binging to their cognate receptors on malignant T cells are then able to activate STAT3 and STAT5 oncogenic signaling and promote cancer progression and IL-17 secretion. In light of these findings, it might be important for patients with exacerbation of their CTCL symptoms to maintain high index of suspicion and treat these individuals for S. aureus colonization and/or sepsis with topical and systemic antibiotics.

  15. The influence of headspace and dissolved oxygen level on growth and haemolytic BL enterotoxin production of a psychrotolerant Bacillus weihenstephanensis isolate on potato based ready-to-eat food products.

    PubMed

    Samapundo, S; Everaert, H; Wandutu, J N; Rajkovic, A; Uyttendaele, M; Devlieghere, F

    2011-04-01

    The major objective of this study was to determine the influence of the initial headspace and dissolved O(2) level and vacuum packaging on growth and diarrhoeal enterotoxin production by Bacillus weihenstephanensis on potato based ready-to-eat food products. In general, the lower the initial headspace or dissolved O(2) level the slower the maximum growth rate (μ(max), log(10) CFU g(-1) d(-1)), the longer the lag phase duration (λ, d) and the smaller the maximum population density (N(max), log(10) CFU g(-1)) became. The slowest μ(max), the longest λ and the smallest N(max) were generally found for growth under vacuum packaging. This implies shorter shelf-lives will occur at higher initial headspace or dissolved O(2) levels as the growth of B. weihenstephanensis to the infective dose of 10(5) CFU g(-1) in such atmospheres takes a shorter time. Significant consumption of dissolved O(2) only occurred when growth shifted from the lag to the exponential phase and growth generally transitioned from the exponential to the stationary phase when the dissolved O(2) levels fell below ca. 75 ppb. Diarrhoeal enterotoxin production (determined via detection of the L2 component of haemolytic BL) was similar for growth under initial headspace O(2) levels of 1-20.9%, and was only reduced when growth took place under vacuum packaging. The reduction in L2 production when growth took place under vacuum was most probably related to the low final cell densities observed under this condition. Both growth and L2 production were inhibited over a 32-day incubation period at 7 °C by 40% CO(2) irrespective of the headspace or dissolved O(2) levels. The results illustrate the importance of residual O(2) and CO(2) on the shelf-stability and safety of modified atmosphere packaged potato based ready-to-eat food products with regards to B. weihenstephanensis.

  16. Staphylococcal Enterotoxin: Role of Intestinal Immunity in Enterotoxin B Intoxication

    DTIC Science & Technology

    1994-02-01

    radial immunodiffusion ( Mancini , Carbonara and Heremans 1965) was done using commercially available plates for mouse IgG total, IgG subclasses, IgA and IgM...present in sera of peroral immunized animals were found to be too low to be accurately measured by the radial immunodiffusion method. The quantitative...animal) and individual serum was quantified for immunoglobulin classes and subclasses by the radial immunodiffusion technique. Compared to control animals

  17. 77 FR 47820 - Invention Promoters/Promotion Firms Complaints

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-10

    ... United States Patent and Trademark Office Invention Promoters/Promotion Firms Complaints ACTION: Proposed... participate in any legal proceedings against invention promoters or promotion firms. Complaints submitted to... promotion firm, explain the basis for the complaint, and include the signature of the complainant....

  18. Novelty and Promotional Items

    EPA Pesticide Factsheets

    Small novelty or promotional products, primarily used for outreach and educational purposes, must effectively convey a message, and their purchase will only be allowed if the item will contribute to the accomplishment of the Agency's mission.

  19. Promoting "Global" Attitudes.

    ERIC Educational Resources Information Center

    Case, Roland

    1996-01-01

    Discusses and illustrates three ways to promote prosocial attitudes towards global issues among students. Includes classroom environments that reinforce desired attitudes; facilitating direct "emotional" experiences that influence attitudes; and engaging students in thoughtful deliberation about global issues. Offers illustrative…

  20. Travelers' diarrhea and toxigenic Escherichia coli.

    PubMed

    Gorbach, S L; Kean, B H; Evans, D G; Evans, D J; Bessudo, D

    1975-05-01

    In a group of 133 United States students studied for 18 days after arriving in Mexico, diarrhea developed in 38 (29 per cent). Diarrhea rarely began before the fourth day, and the mean onset was 13 days after arrival. Symptoms lasted an average of 3.4 days but persisted in 21 per cent of sick students. Heat-labile enterotoxin-producing Escheria coli was found in the stools of 72 per cent of sick and 15 per cent of healthy students. None had heat-labile Esch. coli when they entered Mexico. The incubation period was short, generally 24 to 48 hours, and the carrier state was five days or less in 82 per cent of students surveyed. Entamoeba histolytica was found in 6 per cent of cases of diarrhea, but not salmonella, shigella or penetrating Esch. coli. These studies suggest that approximately 70 per cent of travelers' diarrhea in Mexico is associated with heat-labile toxigenic strains of Esch. coli.

  1. Regulation of Staphylococcal Enterotoxin Biosynthesis

    DTIC Science & Technology

    1981-02-05

    1969. J. Bacteriol. 98:351-358. 26. Clewell, D . B., and D . R. Helinski. 1970. Biochemistry 9:4428-4432. 27. Shalita, Z., I. Hertman, and S. Sarid . J.977...TS;~ z3%S ARE IMPSI mJ IS D ~I SCLOSURE L A 0 4,.. t~ I4 1.0 II. 111111.25 ll14 II1. At’ MICROCOPY RESOLUTION TEST CHART NAT:ONAT. BUREAU OF STANDAROS...Background .................... ........................ 6 c. Approach to the p-:oblem ............ .................. 10 d . Results and discussion

  2. Regulation of Staphylococcal Enterotoxin Biosynthesis

    DTIC Science & Technology

    1981-05-01

    incubation mixture (1001 Ai was precipitated with sary to) prepare t.5N2. Potritv(if all itlasouit preparak- I ml of 20’, t richloroacet ic acid and hoiled...deoxyribonucleic acid (DNA) species. Additional studies demonstrated that the toxin phenotype and by implication, the toxin gene, could be transmitted to...to the amino acid sequence of SEB do not reside on the majority (approx. 2/3) of pSN2. However, if the structural SEB gene is present in pSN2, it

  3. Promoting La Cultura Hispana

    ERIC Educational Resources Information Center

    Pluviose, David

    2007-01-01

    Launched in 1985 at Arizona State University, the Hispanic Research Center's (HRC) efforts to promote Latino and Chicano art and issues have flourished in recent years. In 2004, the HRC hosted the Arizona International Latina/o Arts Festival in collaboration with the Mesa Southwest Museum. The HRC has also founded a mentoring institute for…

  4. The Ideal Promotion Effort.

    ERIC Educational Resources Information Center

    Morris, Edward L.

    The ideal promotional effort for an educational television (ETV) station is dependent on a professional approach to the problem. This means that each ETV station should employ a public relations manager and should keep him informed about all major station decisions. The Public Broadcasting Service (PBS) has a campaign of its own to bring attention…

  5. Health Promotion Interventions.

    ERIC Educational Resources Information Center

    Jason, Leonard A.; Curie, Carrie J.; Townsend, Stephanie M.; Pokorny, Steven B.; Katz, Richard B.; Sherk, Joseph L.

    2002-01-01

    Reviews four areas from the prevention science field, including: promoting healthy behavior; preventing substance abuse; preventing high-risk sexual behaviors; and preventing child abuse and sexual abuse. Recommendations are made regarding strategies for implementing empirically validated programs, supplementing school programs with ecological…

  6. Does Paid Promotion Pay?

    ERIC Educational Resources Information Center

    Scherer, Chris

    1980-01-01

    A study to determine the cost effectiveness of newspaper display advertising for cooperative extension programs showed such publicity to be more effective in large metropolitan areas. Though not a technique for increasing enrollment, this method provided long-range benefits in the overall promotion of extension educational services. (SK)

  7. Promoting Healthy Dietary Behaviors.

    ERIC Educational Resources Information Center

    Perry, Cheryl L.; Story, Mary; Lytle, Leslie A.

    This chapter reviews the research on promoting healthy dietary behaviors in all youth, not just those who exhibit problems such as obesity or eating disorders. The first section of this chapter presents a rationale for addressing healthy dietary behavior with children and adolescents, on the basis of the impact of these behaviors on short- and…

  8. Partners: Promoting Accessible Recreation.

    ERIC Educational Resources Information Center

    Sable, Janet; Gravink, Jill

    1995-01-01

    The Promoting Accessible Recreation through Networking, Education, Resources and Services (PARTNERS) Project, a partnership between Northeast Passage, the University of New Hampshire, and Granite State Independent Living Foundation, helps create barrier-free recreation for individuals with physical disabilities. The paper describes PARTNERS and…

  9. Promoting Continuing Education Programs.

    ERIC Educational Resources Information Center

    Hendrickson, Gayle A.

    This handbook is intended for use by institutions in marketing their continuing education programs. A section on "Devising Your Strategy" looks at identifying a target audience, determining the marketing approach, and developing a marketing plan and promotional techniques. A discussion of media options looks at the advantages and…

  10. 50 Practical Promotion Ideas.

    ERIC Educational Resources Information Center

    Madeyski, Tom

    1997-01-01

    Includes 50 cost-effective ideas for promoting camp in the areas of recruiting new campers, encouraging returning campers, advertising strategies, printing brochures and other written materials, using photographs, targeting groups for camp facility rental, and effectively using the media. (LP)

  11. Diagnosis and Treatment of Acute or Persistent Diarrhea

    PubMed Central

    Pawlowski, Sean W; Warren, Cirle Alcantara; Guerrant, Richard

    2009-01-01

    Studies of microbial pathogens and the toxins they produce are important for determining the mechanisms by which they cause disease and spread throughout a population. Some bacteria produce secretory enterotoxins (such as choleratoxin or the heat-labile or stable enterotoxins produced by E. coli) that invade cells directly. Others produce cytotoxins (such as those produced by Shigella, enteroinvasive E. coli, or C. difficile) that damage cells or trigger host responses that cause small or large bowel diseases (such as enteroaggregative or enteropathogenic E. coli or Salmonella). Viruses (such as noroviruses and rotaviruses) and protozoa (such as Cryptosporidium, Giardia or Entameba histolytica) disrupt cell functions and cause short- or long-term disease. Much epidemiological data about these pathogens have been collected from community- and hospital-acquired settings, as well from patients with traveler’s or persistent diarrhea. These studies have led to practical approaches for prevention, diagnosis and treatment. PMID:19457416

  12. Diarrhoeagenic Escherichia coli and salmonellae in calves and lambs in Kashmir absence, prevalence and antibiogram.

    PubMed

    Wani, S A; Hussain, I; Beg, S A; Rather, M A; Kabli, Z A; Mir, M A; Nishikawa, Y

    2013-12-01

    Polymerase chain reaction assays and culture were used to investigate 728 faecal samples from 404 calves (286 diarrhoeic, 118 healthy) and 324 lambs (230 diarrhoeic, 94 healthy) in Kashmir, India, for the presence of enterotoxigenic Escherichia coli (ETEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC) and salmonellae. Antimicrobial sensitivity patterns were also investigated. In total, 23 ETEC isolates were obtained from the diarrhoeic calves and 12 from diarrhoeic lambs. Most (74%) of the isolates from calves harboured the gene encoding heat-labile enterotoxin I, whereas 75% of the isolates from lambs possessed only the gene encoding for heat-stable enterotoxin a. The ETEC isolates belonged to 20 serogroups, among which serogroups O15 (five isolates) and O8 (four isolates) were the most frequent. Salmonella Typhimurium or S. Enteritidis was identified in three samples from diarrhoeic lambs. The ETEC isolates and the salmonellae showed multidrug resistance. No EAEC or DAEC was detected in any of the samples.

  13. Virulence Genes in Expanded-Spectrum-Cephalosporin-Resistant and -Susceptible Escherichia coli Isolates from Treated and Untreated Chickens.

    PubMed

    Baron, S; Delannoy, S; Bougeard, S; Larvor, E; Jouy, E; Balan, O; Fach, P; Kempf, I

    2015-12-14

    This study investigated antimicrobial resistance, screened for the presence of virulence genes involved in intestinal infections, and determined phylogenetic groups of Escherichia coli isolates from untreated poultry and poultry treated with ceftiofur, an expanded-spectrum cephalosporin. Results show that none of the 76 isolates appeared to be Shiga toxin-producing E. coli or enteropathogenic E. coli. All isolates were negative for the major virulence factors/toxins tested (ehxA, cdt, heat-stable enterotoxin [ST], and heat-labile enterotoxin [LT]). The few virulence genes harbored in isolates generally did not correlate with isolate antimicrobial resistance or treatment status. However, some of the virulence genes were significantly associated with certain phylogenetic groups.

  14. Molecular Detection, Quantification, and Toxigenicity Profiling of Aeromonas spp. in Source- and Drinking-Water.

    PubMed

    Robertson, Boakai K; Harden, Carol; Selvaraju, Suresh B; Pradhan, Suman; Yadav, Jagjit S

    2014-01-01

    Aeromonas is ubiquitous in aquatic environments and has been associated with a number of extra-gastrointestinal and gastrointestinal illnesses. This warrants monitoring of raw and processed water sources for pathogenic and toxigenic species of this human pathogen. In this study, a total of 17 different water samples [9 raw and 8 treated samples including 4 basin water (partial sand filtration) and 4 finished water samples] were screened for Aeromonas using selective culturing and a genus-specific real-time quantitative PCR assay. The selective culturing yielded Aeromonas counts ranging 0 - 2 x 10(3)CFU/ml and 15 Aeromonas isolates from both raw and treated water samples. The qPCR analysis indicated presence of a considerable nonculturable population (3.4 x 10(1) - 2.4 x 10(4) cells/ml) of Aeromonas in drinking water samples. Virulence potential of the Aeromonas isolates was assessed by multiplex/singleplex PCR-based profiling of the hemolysin and enterotoxin genes viz cytotoxic heat-labile enterotoxin (act), heat-labile cytotonic enterotoxin (alt), heat-stable cytotonic enterotoxin (ast), and aerolysin (aerA) genes. The water isolates yielded five distinct toxigenicity profiles, viz. act, alt, act+alt, aerA+alt, and aerA+alt+act. The alt gene showed the highest frequency of occurrence (40%), followed by the aerA (20%), act (13%), and ast (0%) genes. Taken together, the study demonstrated the occurrence of a considerable population of nonculturable Aeromonads in water and prevalence of toxigenic Aeromonas spp. potentially pathogenic to humans. This emphasizes the importance of routine monitoring of both source and drinking water for this human pathogen and role of the developed molecular approaches in improving the Aeromonas monitoring scheme for water.

  15. The promotion of breastfeeding.

    PubMed

    Tuluhungwa, R R; Yung, W

    1979-01-01

    To reverse the current trend of a significant decline worldwide in breast feeding means reeducation of medical and health personnel as well as the general public. Programs to promote breast feeding require the commitment of governments, with support from various ministries including health, education, labor, community development and judiciary. Examples of what 3 developing countries--Jamaica, Colombia and Thailand--are doing to promote breast feeding are reported. A large scale breast feeding campaign was launched in Jamaica in October 1977. The 3 phases of the campaign were: 1) preliminary surveys and research and motivation of professional, voluntary and extension groups through training seminars, panel discussions, and meetings; 2) promotion of breast feeding via mass media and motivation of target groups by trained personnel; and 3) evaluation of the campaign. A survey undertaken in 1978 showed that the breast feeding messages had achieved the desired effect--more mothers practiced breast feeding. In Colombia the breast feeding campaign emphasized non-formal education through the use of games and pictures. A game is used which is usually initiated by a health worker in the waiting room of a health center and involves the mothers, the general public, and sometimes the professional personnel. Through reading and interpreting rhymed breast feeding messages, the participants exchange opinions and experiences. Before starting a campaign to encourage low-income urban and semi-urban mothers to breast feed, the National Food and Nutrition Committee of Thailand pretested slogans and posters designed for the promotion of breast feeding. Posters develpoed in accordance with the suggestions made by the women were tested among 126 pregnant and lactating women. The Committee decided which picture to print for low-income and rural audiences and which to print for middle-class audiences.

  16. Democracy Promotion in Oman

    DTIC Science & Technology

    2013-01-01

    succession process . The “Ruling Family Council” will meet to designate a successor within 3 days of Qaboos’s death. If it fails to select someone, it will...the Gulf is to increase democracy promotion programs designed to encourage timely and peaceful transitions to more representative forms of govern...stronger efforts toward reform supported by the United States could foster a more peaceful political transformation. This process should begin with the

  17. Publishing and academic promotion.

    PubMed

    Dixon, A K

    2009-09-01

    Clearly, academic endeavour has to be the single most important criterion for appointment to an academic position and for subsequent promotion. It is rare for excellence either in teaching or clinical practice to offset a poor publication record. However, the pressure to publish and gain related grant income can lead to problems in the normal academic pursuits of a department or institution. These and other related issues will be explored in this editorial.

  18. Pathogenicity of Vietnamese enterotoxigenic Escherichia coli strains in colostrum-deprived one-day-old piglets.

    PubMed

    Do, T N; Wilkie, I; Driesen, S J; Fahy, V A; Trott, D J

    2006-03-01

    Preweaning colibacillosis is a major cause of economic loss to the swine industry in Vietnam. The aim of this study was to examine the enteropathogenicity of representative enterotoxigenic Escherichia coli (ETEC) strains obtained during an earlier epidemiologic survey conducted in five provinces in North Vietnam. This included isolates belonging to serotype O8 that produced heat-stable and heat-labile enterotoxins but did not produce any of the recognized fimbriae (F4, F5, F6, F41, F18). In vitro hemagglutination (unique mannose-resistant hemagglutination activity with guinea pig, sheep, human, and chicken red blood cells at 37 degrees C, but not at 18 degrees C) and enterocyte brush border attachment assays suggested that the F- ETEC strains produced an unidentified colonization factor that promoted adherence to the intestinal epithelium. Colostrum-deprived 1-day-old piglets challenged with an F- strain (1-2 x 10(9) bacteria) developed acute watery diarrhea within 4 hours of inoculation and suffered up to 20% weight loss, with comparable severity to piglets challenged with conventional F4 and F5 strains. At necropsy, viable counts and histopathologic examination of intestinal sections demonstrated colonization of the duodenum, jejunum, and ileum by F4-positive strains. In comparison, the F- and F5-positive strains attached exclusively to the ileum. Transmission electron micrographs of negatively stained F- cells grown at 37 degrees C demonstrated the presence of fimbriae. These results confirm the presence of a potentially new pathogenic ETEC fimbrial type in piggeries in Vietnam, with a unique hemagglutination property and attachment characteristics similar to ETEC bearing F5 fimbriae.

  19. Adjuvant effect of Japanese herbal medicines on the mucosal type 1 immune responses to human papillomavirus (HPV) E7 in mice immunized orally with Lactobacillus-based therapeutic HPV vaccine in a synergistic manner.

    PubMed

    Taguchi, Ayumi; Kawana, Kei; Yokoyama, Terufumi; Adachi, Katsuyuki; Yamashita, Aki; Tomio, Kensuke; Kojima, Satoko; Oda, Katsutoshi; Fujii, Tomoyuki; Kozuma, Shiro

    2012-08-03

    The Japanese herbal medicines, Juzen-taiho-to (JTT) and Hochu-ekki-to (HET), have been shown to enhance humoral immune responses to vaccine antigen when used as adjuvants for prophylactic vaccines. However, their adjuvant effect on mucosal cellular immune responses remains unstudied. The precursor lesion of cervical cancer, high-grade CIN that expresses HPV E7 oncoprotein ubiquitously is a target for HPV therapeutic vaccines that elicit mucosal E7-specific type 1 T cell responses. We have demonstrated that oral immunization with recombinant Lactobacillus casei expressing HPV16 E7 (LacE7) is more effective in eliciting mucosal E7-specific IFNγ-producing cells than subcutaneous or intramuscular antigen delivery. Here we report the synergistic effect of an oral Lactobacillus-based vaccine and Japanese herbal medicines on mucosal immune responses. Oral immunization of mice with LacE7 plus either a Japanese herbal medicine (JTT or HET) or a mucosal adjuvant, heated-labile enterotoxin T subunit (LTB), promotes systemic E7-specific type 1 T cell responses but not mucosal responses. Administration of LacE7 plus either Japanese herbal medicine and LTB enhanced mucosal E7-specific type 1 T cell response to levels approximately 3-fold higher than those after administration of LacE7 alone. Furthermore, secretion of IFNγ and IL-2 into the intestinal lumen was observed after oral administration of LacE7 and was enhanced considerably by the addition of Japanese herbal medicines and LTB. Our data indicated that Japanese herbal medicines, in synergy with Lactobacillus and LTB, enhance the mucosal type 1 immune responses to orally immunized antigen. Japanese herbal medicines may be excellent adjuvants for oral Lactobacillus-based vaccines and oral immunization of LacE7, HET and LTB may have the potential to elicit extremely high E7-specific mucosal cytotoxic immune response to HPV-associated neoplastic lesions.

  20. Mucosal and systemic immune responses to Mycobacterium tuberculosis antigen 85A following its co-delivery with CpG, MPLA or LTB to the lungs in mice.

    PubMed

    Todoroff, Julie; Lemaire, Muriel M; Fillee, Catherine; Jurion, Fabienne; Renauld, Jean-Christophe; Huygen, Kris; Vanbever, Rita

    2013-01-01

    Pulmonary vaccination is a promising route for immunization against tuberculosis because the lung is the natural site of infection with Mycobacterium tuberculosis. Yet, adjuvants with a suitable safety profile need to be found to enhance mucosal immunity to recombinant antigens. The aim of this study was to evaluate the immunogenicity, the safety and the protective efficacy of a subunit vaccine composed of the immunodominant mycolyl-transferase antigen 85A (Ag85A) and one of three powerful mucosal adjuvants: the oligodeoxynucleotide containing unmethylated cytosine-phosphate-guanine motifs (CpG), the monophosphoryl lipid A of Salmonella minnesota (MPLA) or the B subunit of heat-labile enterotoxin of Escherichia coli (LTB). BALB/c mice were vaccinated in the deep lungs. Our results showed that lung administration of these adjuvants could specifically induce different types of T cell immunity. Both CpG and MPLA induced a Th-1 type immune response with significant antigen-specific IFN-γ production by spleen mononuclear cells in vitro and a tendency of increased IFN-γ in the lungs. Moreover, MPLA triggered a Th-17 response reflected by high IL-17A levels in the spleen and lungs. By contrast, LTB promoted a Th-2 biased immune response, with a production of IL-5 but not IFN-γ by spleen mononuclear cells in vitro. CpG did not induce inflammation in the lungs while LTB and MPLA showed a transient inflammation including a neutrophil influx one day after pulmonary administration. Pulmonary vaccination with Ag85A without or with MPLA or LTB tended to decrease bacterial counts in the spleen and lungs following a virulent challenge with M. tuberculosis H37Rv. In conclusion, CpG and MPLA were found to be potential adjuvants for pulmonary vaccination against tuberculosis, providing Th-1 and Th-17 immune responses and a good safety profile.

  1. Bicycle Promotion Plan

    SciTech Connect

    Simone, G. A.

    1981-03-09

    The objective of this Bicycle Promotion Plan is to outline a set of recommendations and supporting strategies for implementation by the US DOE toward increased use of the bicycle for energy conservation. The recommendations are designed in such a way as to function in concert with: (1) bicycle programs administered by other Federal government agencies; and (2) related programs and activities already sponsored by DOE. The approach to preparation of the Plan involved a review of all current and planned bicycle promotion programs at the Federal level as well as a review of the array of lierature on the subject. The UniWorld project staff also interacted with several DOE program offices, in order to determine the extent to which they might appropriately contribute to the implementation of bicycle promotional efforts. A synthesis of all the information gathered was published in January of 1981 as a part of the project (The Bicycle Program Review). Based upon this information and an examination of the barriers to bicycle use identified by bicycle transportation specialists in the field, UniWorld developed a series of the most potentially effective recommendations and program strategies for implementation by DOE. The recommendations address activities that could be undertaken in conjunction with existing DOE programs, new developments that might be considered to fulfill critical needs in the field, and interagency efforts that DOE could play a role in.

  2. Differences in serological responses and excretion patterns of volunteers challenged with enterotoxigenic Escherichia coli with and without the colonization factor antigen.

    PubMed Central

    Evans, D G; Satterwhite, T K; Evans, D J; DuPont, H L

    1978-01-01

    Double-blind studies were performed to compare the virulence of enterotoxigenic Escherichia coli with and without the fimbriate colonization factor antigen (CFA), using young healthy adults (mean age, 23 years) as volunteers. In the first study one group of volunteers ingested 1 X 10(6) E. coli H-10407, the CFA-positive strain, and another group ingested 1 X 10(6) E. coli H-10407-P, the CFA-negative spontaneous derivative of strain H-10407. The second study was similar except that the test strains were administered at a dose of 1 X 10(8) viable cells. Three parameters of infection were monitored: (i) diarrhea and associated symptoms; (ii) excretion pattern of test strains; and (iii) humoral antibody response to CFA, somatic antigen, and heat-labile enterotoxin. Significant signs of illness occurred only in six of seven volunteers who ingested E. coli H-10407 at a dose of 1 X 10(8). At both doses, E. coli H-10407-P appeared in the stool on day 1 postchallenge and disappeared by day 4. In contrast, strain H-10407 was persistently excreted from the first to the last day of the study. Also, only those volunteers in the H-10407 challenge groups (12 of 13 analyzed) responded with a fourfold antibody titer rise to CFA, somatic antigen, and/or heat-labile enterotoxin. No reversion of H-10407-P to H-10407 was detected. PMID:346488

  3. Evaluation of antisera used for detecting enterotoxigenic Escherichia coli in Sao Paulo.

    PubMed Central

    Guth, B E; Trabulsi, L R

    1985-01-01

    The usefulness of antisera in detecting enterotoxigenic Escherichia coli (ETEC) strains in Sao Paulo was evaluated. Polyvalent antisera detected 49% of ETEC isolates and were more effective in identifying E. coli that produced heat-labile and heat-stable enterotoxins and in strains that produced only heat-stable enterotoxin. ETEC strains not detected by the antisera belonged to different serogroups not isolated in Sao Paulo before; 34% of these strains had undetermined O antigens, and most of them produced only heat-labile toxin. A variation of serogroups over time was especially observed among strains that produced heat-stable toxin. The importance of H-antigen determinations in the effectiveness of ETEC diagnosis by serological methods became evident, as non-ETEC strains were also detected by polyvalent antisera, but their serotypes were different from those of ETEC strains. Although antisera can be used to identify O:H types of ETEC strains with accuracy, serotyping cannot be recommended for routine diagnosis. However, such a procedure may be useful for studying outbreaks of ETEC diarrhea if the involved serotypes are already known. PMID:3908475

  4. The dynamics of mobile promoters: Enhanced stability in promoter regions.

    PubMed

    Rabbani, Mahnaz; Wahl, Lindi M

    2016-10-21

    Mobile promoters are emerging as a new class of mobile genetic elements, first identified by examining prokaryote genome sequences, and more recently confirmed by experimental observations in bacteria. Recent datasets have identified over 40,000 putative mobile promoters in sequenced prokaryote genomes, however only one-third of these are in regions of the genome directly upstream from coding sequences, that is, in promoter regions. The presence of many promoter sequences in non-promoter regions is unexplained. Here we develop a general mathematical model for the dynamics of mobile promoters, extending previous work to capture the dynamics both within and outside promoter regions. From this general model, we apply rigorous model selection techniques to identify which parameters are statistically justified in describing the available mobile promoter data, and find best-fit values of these parameters. Our results suggest that high rates of horizontal gene transfer maintain the population of mobile promoters in promoter regions, and that once established at these sites, mobile promoters are rarely lost, but are commonly copied to other genomic regions. In contrast, mobile promoter copies in non-promoter regions are more numerous and more volatile, experiencing substantially higher rates of duplication, loss and diversification.

  5. Human Promoters Are Intrinsically Directional

    PubMed Central

    Duttke, Sascha H.C.; Lacadie, Scott A.; Ibrahim, Mahmoud M.; Glass, Christopher K.; Corcoran, David L.; Benner, Christopher; Heinz, Sven; Kadonaga, James T.; Ohler, Uwe

    2015-01-01

    Divergent transcription, in which reverse-oriented transcripts occur upstream of eukaryotic promoters in regions devoid of annotated genes, has been suggested to be a general property of active promoters. Here we show that the human basal RNA polymerase II transcriptional machinery and core promoter are inherently unidirectional, and that reverse-oriented transcripts originate from their own cognate reverse-directed core promoters. In vitro transcription analysis and mapping of nascent transcripts in cells revealed that sequences at reverse start sites are similar to those of their forward counterparts. The use of DNase I accessibility to define proximal promoter borders revealed that up to half of promoters are unidirectional and that unidirectional promoters are depleted at their upstream edges of reverse core promoter sequences and their associated chromatin features. Divergent transcription is thus not an inherent property of the transcription process, but rather the consequence of the presence of both forward- and reverse-directed core promoters. PMID:25639469

  6. Organization and ELISA-Based Results of the First Proficiency Testing to Evaluate the Ability of European Union Laboratories to Detect Staphylococcal Enterotoxin Type B (SEB) in Buffer and Milk

    PubMed Central

    Nia, Yacine; Rodriguez, Mélanie; Zeleny, Reinhard; Herbin, Sabine; Auvray, Frédéric; Fiebig, Uwe; Avondet, Marc-André; Munoz, Amalia; Hennekinne, Jacques-Antoine

    2016-01-01

    The aim of this work was to organize the first proficiency test (PT) dedicated to staphylococcal enterotoxin B (SEB) detection in milk and buffer solutions. This paper describes the organization of the PT trial according to EN ISO 17043 requirements. Characterization of the SEB stock solution was performed using SDS-PAGE and SE-specific ELISA, and amino acid analysis was used to assign its protein concentration. The solution was then used to prepare six PT materials (four milk and two buffer batches) at a ng/g toxin level, which included one blank and one SEA-containing milk as specificity control. Suitable material homogeneity and stability were assessed using screening and quantitative ELISAs. Among the methods used by the participants, ELISA-based methods demonstrated their efficiency for the detection of SEB in both simple and complex matrices. The results serve as a basis for further improving the detection capabilities in expert laboratories and can therefore be considered as a contribution to biopreparedness. PMID:27649244

  7. B subunits of cholera toxin and thermolabile enterotoxin of Escherichia coli have similar adjuvant effect as whole molecules on rotavirus 2/6-VLP specific antibody responses and induce a Th17-like response after intrarectal immunization.

    PubMed

    Thiam, Fatou; Charpilienne, Annie; Poncet, Didier; Kohli, Evelyne; Basset, Christelle

    2015-12-01

    The purpose of this study was to evaluate the adjuvant effect of the B subunits of cholera toxin (CT) and the thermolabile enterotoxin of Escherichia coli (LT) by the intrarectal route of immunization and compare them to the whole molecules CT and LT-R192G, a non toxic mutant of LT, using 2/6-VLP as an antigen, in mice. All molecules induced similar antigen specific antibody titers in serum and feces, whereas different T cell profiles were observed. CTB and LTB, conversely to CT and LT-R192G, did not induce detectable production of IL-2 by antigen specific T cells. Moreover, CTB, conversely to LT-R192G, CT and LTB, did not induce antigen specific CD4+CD25+Foxp3- and Foxp3+ T cells, thus showing different effects between the B subunits themselves. However, all molecules induced an antigen specific Th17 response. In conclusion, B subunits are potent adjuvants on B cell responses by the intrarectal route. Although their impact on T cell responses are different, all molecules induce a 2/6-VLP-specific Th17 T cell response that may play a major role in helping B cell responses and thus in adjuvanticity and protection.

  8. Bacteroides fragilis Enterotoxin Upregulates Heme Oxygenase-1 in Intestinal Epithelial Cells via a Mitogen-Activated Protein Kinase- and NF-κB-Dependent Pathway, Leading to Modulation of Apoptosis

    PubMed Central

    Ko, Su Hyuk; Rho, Da Jeong; Jeon, Jong Ik; Kim, Young-Jeon; Woo, Hyun Ae; Lee, Yun Kyung

    2016-01-01

    The Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), interacts with intestinal epithelial cells and can provoke signals that induce mucosal inflammation. Although expression of heme oxygenase-1 (HO-1) is associated with regulation of inflammatory responses, little is known about HO-1 induction in ETBF infection. This study was conducted to investigate the effect of BFT on HO-1 expression in intestinal epithelial cells. Stimulation of intestinal epithelial cells with BFT resulted in upregulated expression of HO-1. BFT activated transcription factors such as NF-κB, AP-1, and Nrf2 in intestinal epithelial cells. Upregulation of HO-1 in intestinal epithelial cells was dependent on activated IκB kinase (IKK)–NF-κB signals. However, suppression of Nrf2 or AP-1 signals in intestinal epithelial cells did not result in significant attenuation of BFT-induced HO-1 expression. HO-1 induction via IKK–NF-κB in intestinal epithelial cells was regulated by p38 mitogen-activated protein kinases (MAPKs). Furthermore, suppression of HO-1 activity led to increased apoptosis in BFT-stimulated epithelial cells. These results suggest that a signaling pathway involving p38 MAPK–IKK–NF-κB in intestinal epithelial cells is required for HO-1 induction during exposure to BFT. Following this induction, increased HO-1 expression may regulate the apoptotic process in responses to BFT stimulation. PMID:27324483

  9. An Outbreak of Diarrhea in Mandera, Kenya, Due to Escherichia coli Serogroup O-Nontypable Strain That Had a Coding Gene for Enteroaggregative E. coli Heat-Stable Enterotoxin 1.

    PubMed

    Ochi, Sadayuki; Shah, Mohammad; Odoyo, Erick; Bundi, Martin; Miringu, Gabriel; Guyo, Sora; Wandera, Ernest; Kathiiko, Cyrus; Kariuki, Samuel; Karama, Mohamed; Tsuji, Takao; Ichinose, Yoshio

    2017-02-08

    In an outbreak of gastroenteritis in December 2009, in Mandera, Kenya, Escherichia coli O-nontypable (ONT) strain was isolated from stool specimens of patients (18/24, 75%). The E. coli ONT organisms could not be assigned to any of the recognized diarrheagenic groups of E. coli However, they possessed the enteroaggregative E. coli heat-stable enterotoxin-1 gene. The cell-free culture filtrates of the E. coli ONT strain isolated from the outbreak cases induced considerable amount of fluid accumulation in suckling mouse intestine, indicating production of an enterotoxic factor(s). These results identify E. coli that did not have any diarrheagenic characteristics except astA as the etiological agent of the diarrheal outbreak in Mandera. It is however considered necessary to characterize the fluid accumulation factor(s) to determine whether any novel toxins were responsible for the fluid accumulation. Moreover, it is important to study dissemination of strains producing the enterotoxic factor(s) to assess their public health significance distribution in the environment.

  10. Claudin-4 Overexpression in Epithelial Ovarian Cancer Is Associated with Hypomethylation and Is a Potential Target for Modulation of Tight Junction Barrier Function Using a C-Terminal Fragment of Clostridium perfringens Enterotoxin1

    PubMed Central

    Litkouhi, Babak; Kwong, Joseph; Lo, Chun-Min; Smedley, James G; McClane, Bruce A; Aponte, Margarita; Gao, Zhijian; Sarno, Jennifer L; Hinners, Jennifer; Welch, William R; Berkowitz, Ross S; Mok, Samuel C; Garner, Elizabeth I O

    2007-01-01

    Background Claudin-4, a tight junction (TJ) protein and receptor for the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), is overexpressed in epithelial ovarian cancer (EOC). Previous research suggests DNA methylation is a mechanism for claudin-4 overexpression in cancer and that C-CPE acts as an absorption-enhancing agent in claudin-4-expressing cells. We sought to correlate claudin-4 overexpression in EOC with clinical outcomes and TJ barrier function, investigate DNA methylation as a mechanism for overexpression, and evaluate the effect of C-CPE on the TJ. Methods Claudin-4 expression in EOC was quantified and correlated with clinical outcomes. Claudin-4 methylation status was determined, and claudin-4-negative cell lines were treated with a demethylating agent. Electric cell-substrate impedance sensing was used to calculate junctional (paracellular) resistance (Rb) in EOC cells after claudin-4 silencing and after C-CPE treatment. Results Claudin-4 overexpression in EOC does not correlate with survival or other clinical endpoints and is associated with hypomethylation. Claudin-4 overexpression correlates with Rb and C-CPE treatment of EOC cells significantly decreased Rb in a dose- and claudin-4-dependent noncytotoxic manner. Conclusions C-CPE treatment of EOC cells leads to altered TJ function. Further research is needed to determine the potential clinical applications of C-CPE in EOC drug delivery strategies. PMID:17460774

  11. PROMOTIONS: PROper MOTION Software

    NASA Astrophysics Data System (ADS)

    Caleb Wherry, John; Sahai, R.

    2009-05-01

    We report on the development of a software tool (PROMOTIONS) to streamline the process of measuring proper motions of material in expanding nebulae. Our tool makes use of IDL's widget programming capabilities to design a unique GUI that is used to compare images of the objects from two epochs. The software allows us to first orient and register the images to a common frame of reference and pixel scale, using field stars in each of the images. We then cross-correlate specific morphological features in order to determine their proper motions, which consist of the proper motion of the nebula as a whole (PM-neb), and expansion motions of the features relative to the center. If the central star is not visible (quite common in bipolar nebulae with dense dusty waists), point-symmetric expansion is assumed and we use the average motion of high-quality symmetric pairs of features on opposite sides of the nebular center to compute PM-neb. This is then subtracted out to determine the individual movements of these and additional features relative to the nebular center. PROMOTIONS should find wide applicability in measuring proper motions in astrophysical objects such as the expanding outflows/jets commonly seen around young and dying stars. We present first results from using PROMOTIONS to successfully measure proper motions in several pre-planetary nebulae (transition objects between the red giant and planetary nebula phases), using images taken 7-10 years apart with the WFPC2 and ACS instruments on board HST. The authors are grateful to NASA's Undergradute Scholars Research Program (USRP) for supporting this research.

  12. Promoting healthy sleep.

    PubMed

    Price, Bob

    2016-03-09

    Nurses are accustomed to helping others with their sleep problems and dealing with issues such as pain that may delay or interrupt sleep. However, they may be less familiar with what constitutes a healthy night's sleep. This article examines what is known about the process and purpose of sleep, and examines the ways in which factors that promote wakefulness and sleep combine to help establish a normal circadian rhythm. Theories relating to the function of sleep are discussed and research is considered that suggests that sleep deficit may lead to metabolic risks, including heart disease, obesity, type 2 diabetes mellitus and several types of cancer.

  13. Students Promoted Despite Test Failure

    ERIC Educational Resources Information Center

    Wakefield, Dara V.

    2012-01-01

    "No Child Left Behind" (NCLB) dictates students in Grades 3, 5, and 8 pass state tests to be promoted. Accordingly, most state education codes require students to pass reading and math exams for promotion. The majority of those who fail, however, appear to be promoted anyway. This article addresses core questions concerning the paradigm…

  14. TWEAK Promotes Peritoneal Inflammation

    PubMed Central

    Sanz, Ana Belen; Aroeira, Luiz Stark; Bellon, Teresa; del Peso, Gloria; Jimenez-Heffernan, Jose; Santamaria, Beatriz; Sanchez-Niño, Maria Dolores; Blanco-Colio, Luis Miguel; Lopez-Cabrera, Manuel; Ruiz-Ortega, Marta; Egido, Jesus; Selgas, Rafael; Ortiz, Alberto

    2014-01-01

    Peritoneal dialysis (PD) is complicated by peritonitis episodes that cause loss of mesothelium and eventually sclerosing peritonitis. An improved understanding of the molecular contributors to peritoneal injury and defense may increase the therapeutic armamentarium to optimize peritoneal defenses while minimizing peritoneal injury. There is no information on the expression and function of the cytokine TWEAK and its receptor Fn14 during peritoneal injury. Fn14 expression and soluble TWEAK levels were measured in human PD peritoneal effluent cells or fluids with or without peritonitis. Fn14 expression was also analyzed in peritoneal biopsies from PD patients. Actions of intraperitoneal TWEAK were studied in mice in vivo. sTWEAK levels were increased in peritoneal effluent in PD peritonitis. Effluent sTWEAK levels correlated with the number of peritoneal macrophages (r = 0.491, p = 0.002). Potential TWEAK targets that express the receptor Fn14 include mesothelial cells and macrophages, as demonstrated by flow cytometry of peritoneal effluents and by analysis of peritoneal biopsies. Peritoneal biopsy Fn14 correlated with mesothelial injury, fibrosis and inflammation, suggesting a potential deleterious effect of TWEAK/Fn14. In this regard, intraperitoneal TWEAK administration to mice promoted peritoneal inflammation characterized by increased peritoneal effluent MCP-1, Fn14 and Gr1+ macrophages, increased mesothelial Fn14, MCP-1 and CCL21 expression and submesothelial tissue macrophage recruitment. Taken together these data suggest that the TWEAK/Fn14 system may promote inflammation and tissue injury during peritonitis and PD. PMID:24599047

  15. Leptin Promotes Glioblastoma

    PubMed Central

    Lawrence, Johnathan E.; Cook, Nicholas J.; Rovin, Richard A.; Winn, Robert J.

    2012-01-01

    The hormone leptin has a variety of functions. Originally known for its role in satiety and weight loss, leptin more recently has been shown to augment tumor growth in a variety of cancers. Within gliomas, there is a correlation between tumor grade and tumor expression of leptin and its receptor. This suggests that autocrine signaling within the tumor microenvironment may promote the growth of high-grade gliomas. Leptin does this through stimulation of cellular pathways that are also advantageous for tumor growth and recurrence: antiapoptosis, proliferation, angiogenesis, and migration. Conversely, a loss of leptin expression attenuates tumor growth. In animal models of colon cancer and melanoma, a decline in the expression and secretion of leptin resulted in a reduction of tumor growth. In these models, positive mental stimulation through environmental enrichment decreased leptin secretion and improved tumor outcome. This review explores the link between leptin and glioblastoma. PMID:22263109

  16. Love promotes health.

    PubMed

    Esch, Tobias; Stefano, George B

    2005-06-01

    Love has consequences for health and well-being. Engaging in joyful activities such as love may activate areas in the brain responsible for emotion, attention, motivation and memory (i.e., limbic structures), and it may further serve to control the autonomic nervous system, i.e., stress reduction. This specific CNS activity pattern appears to exert protective effects, even on the brain itself. Moreover, anxiolytic effects of pleasurable experiences may occur by promotion of an inhibitory tone in specific areas of the brain. Thus, love and pleasure clearly are capable of stimulating health, well-being and (re)productivity: This wonderful biological instrument makes procreation and maintenance of organisms and their species a deeply rewarding and pleasurable experience, thus ensuring survival, health, and perpetuation.

  17. Surface Protonics Promotes Catalysis

    NASA Astrophysics Data System (ADS)

    Manabe, R.; Okada, S.; Inagaki, R.; Oshima, K.; Ogo, S.; Sekine, Y.

    2016-12-01

    Catalytic steam reforming of methane for hydrogen production proceeds even at 473 K over 1 wt% Pd/CeO2 catalyst in an electric field, thanks to the surface protonics. Kinetic analyses demonstrated the synergetic effect between catalytic reaction and electric field, revealing strengthened water pressure dependence of the reaction rate when applying an electric field, with one-third the apparent activation energy at the lower reaction temperature range. Operando–IR measurements revealed that proton conduction via adsorbed water on the catalyst surface occurred during electric field application. Methane was activated by proton collision at the Pd–CeO2 interface, based on the inverse kinetic isotope effect. Proton conduction on the catalyst surface plays an important role in methane activation at low temperature. This report is the first describing promotion of the catalytic reaction by surface protonics.

  18. Surface Protonics Promotes Catalysis

    PubMed Central

    Manabe, R.; Okada, S.; Inagaki, R.; Oshima, K.; Ogo, S.; Sekine, Y.

    2016-01-01

    Catalytic steam reforming of methane for hydrogen production proceeds even at 473 K over 1 wt% Pd/CeO2 catalyst in an electric field, thanks to the surface protonics. Kinetic analyses demonstrated the synergetic effect between catalytic reaction and electric field, revealing strengthened water pressure dependence of the reaction rate when applying an electric field, with one-third the apparent activation energy at the lower reaction temperature range. Operando–IR measurements revealed that proton conduction via adsorbed water on the catalyst surface occurred during electric field application. Methane was activated by proton collision at the Pd–CeO2 interface, based on the inverse kinetic isotope effect. Proton conduction on the catalyst surface plays an important role in methane activation at low temperature. This report is the first describing promotion of the catalytic reaction by surface protonics. PMID:27905505

  19. Sales promotions and food consumption.

    PubMed

    Hawkes, Corinna

    2009-06-01

    Sales promotions are widely used to market food to adults, children, and youth. Yet, in contrast to advertising, practically no attention has been paid to their impacts on dietary behaviors, or to how they may be used more effectively to promote healthy eating. This review explores the available literature on the subject. The objective is to identify if and what literature exists, examine the nature of this literature, and analyze what can be learned from it about the effects of sales promotions on food consumption. The review finds that while sales promotions lead to significant sales increases over the short-term, this does not necessarily lead to changes in food-consumption patterns. Nevertheless, there is evidence from econometric modeling studies indicating that sales promotions can influence consumption patterns by influencing the purchasing choices of consumers and encouraging them to eat more. These effects depend on the characteristics of the food product, sales promotion, and consumer. The complexity of the effects means that sales promotions aiming to encourage consumption of nutritious foods need to be carefully designed. These conclusions are based on studies that use mainly sales data as a proxy for dietary intake. The nutrition (and economics) research communities should add to this existing body of research to provide evidence on the impact of sales promotions on dietary intake and related behaviors. This would help support the development of a sales promotion environment conducive to healthy eating.

  20. Environmental Health Promotion: Bridging Traditional Environmental Health and Health Promotion

    ERIC Educational Resources Information Center

    Howze, Elizabeth H.; Baldwin, Grant T.; Kegler, Michelle Crozier

    2004-01-01

    This article highlights the juncture between environmental health and health promotion and underscores the need for health promotion involvement in environmental health practice. It begins with a synopsis of current issues in environmental public health and deficiencies in environmental public health practice that could be partly ameliorated by an…

  1. 7 CFR 1219.22 - Promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions § 1219.22 Promotion. Promotion means any action to advance the image, desirability, or marketability of Hass...

  2. 7 CFR 1219.22 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions § 1219.22 Promotion. Promotion means any action to advance the image, desirability, or marketability of Hass...

  3. 7 CFR 1219.22 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions § 1219.22 Promotion. Promotion means any action to advance the image, desirability, or marketability of Hass...

  4. 7 CFR 1219.22 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions § 1219.22 Promotion. Promotion means any action to advance the image, desirability, or marketability of Hass...

  5. 7 CFR 1217.22 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOFTWOOD LUMBER RESEARCH, PROMOTION, CONSUMER EDUCATION AND INDUSTRY INFORMATION ORDER Softwood Lumber Research, Promotion, Consumer Education, and Industry Information Order Definitions § 1217.22 Promotion. Promotion means any action...

  6. 7 CFR 1217.22 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOFTWOOD LUMBER RESEARCH, PROMOTION, CONSUMER EDUCATION AND INDUSTRY INFORMATION ORDER Softwood Lumber Research, Promotion, Consumer Education, and Industry Information Order Definitions § 1217.22 Promotion. Promotion means any action...

  7. 7 CFR 1217.22 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOFTWOOD LUMBER RESEARCH, PROMOTION, CONSUMER EDUCATION AND INDUSTRY INFORMATION ORDER Softwood Lumber Research, Promotion, Consumer Education, and Industry Information Order Definitions § 1217.22 Promotion. Promotion means any action...

  8. Health-promoting schools: an opportunity for oral health promotion.

    PubMed Central

    Kwan, Stella Y. L.; Petersen, Poul Erik; Pine, Cynthia M.; Borutta, Annerose

    2005-01-01

    Schools provide an important setting for promoting health, as they reach over 1 billion children worldwide and, through them, the school staff, families and the community as a whole. Health promotion messages can be reinforced throughout the most influential stages of children's lives, enabling them to develop lifelong sustainable attitudes and skills. Poor oral health can have a detrimental effect on children's quality of life, their performance at school and their success in later life. This paper examines the global need for promoting oral health through schools. The WHO Global School Health Initiative and the potential for setting up oral health programmes in schools using the health-promoting school framework are discussed. The challenges faced in promoting oral health in schools in both developed and developing countries are highlighted. The importance of using a validated framework and appropriate methodologies for the evaluation of school oral health projects is emphasized. PMID:16211159

  9. Activation of thermosensitive RNA splicing and production of a heat-labile P85gag-mos kinase by the introduction of a specific deletion in murine sarcoma virus-124 DNA.

    PubMed Central

    de Mars, M; Cizdziel, P E; Murphy, E C

    1988-01-01

    Murine sarcoma virus ts110 (MuSVts110) is a conditionally transformation-defective MuSV mutant lacking 1,487 bases found in its wild-type parent, MuSV-349 (MuSV-124). Expression of the MuSVts110 v-mos gene product, P85gag-mos, requires splicing of the viral transcript to align the gag and mos genes in frame. However, this splice event is restricted to growth temperatures of 33 degrees C or lower. No splicing of the viral RNA, no production of P85gag-mos, and, hence, no cell transformation is observed at growth temperatures above 33 degrees C. To determine whether thermosensitive splicing is an intrinsic property of To determine whether thermosensitive splicing is an intrinsic property of MuSVts110 RNA specified by the 1,487-base deletion or a result of a cellular defect, we examined an "equivalent" or MuSVts110 DNA (designated ts32 DNA) constructed by combining wild-type MuSV-124 DNA fragments with a synthetic oligonucleotide to yield an otherwise wild-type viral DNA containing the same 1,487-base deletion as authentic MuSVts110. As observed in control cells (6m2 cells) infected with the authentic MuSVts110 virus, NIH 3T3 cells transfected with ts32 DNA appeared morphologically transformed when grown at 33 degrees C, but were converted to a more normal, flattened shape within a few hours of a shift to 39 degrees C. In concert with these morphological changes, both the processing of the ts32 RNA transcripts and the production of ts32 p85gag-mos kinase were found to be optimal at growth temperatures from 28 to 33 degrees C, but dramatically reduced at 37 to 41 degrees C. Like authentic P85gag-mos, the ts32 P85gag-mos kinase activity was rapidly inactivated by brief exposure to 39 degrees C. These results suggested that the MuSVts110 equivalent is functionally indistinguishable from authentic MuSVts110 and that the novel temperature-sensitive splicing of MuSVts110 transcripts is specified by an intrinsic property of the viral RNA. Images PMID:2835496

  10. Promoting Leadership in Australian Universities

    ERIC Educational Resources Information Center

    Bradley, Andrew P.; Grice, Tim; Paulsen, Neil

    2017-01-01

    In this paper we review current practices for developing and promoting academic leadership in universities. We consider the forms of leadership that are appropriate for academic organisations, while exploring the types of leadership favoured by recruitment and promotion committees. Using the Australian higher education context as a case study, we…

  11. ACTFL's Accent! on Promoting FL's.

    ERIC Educational Resources Information Center

    Accent on ACTFL, 1975

    1975-01-01

    This insert in "Accent on ACTFL" is devoted to ways of promoting the study of foreign languages. The first section is a comic book, "The Continuing Story of Captain Fore Lang," created as an assignment by education students. The comic points out several benefits of language study. The second section is a language-specific promotion focusing on…

  12. Promoting Reading in Developing Countries.

    ERIC Educational Resources Information Center

    Greaney, Vincent, Ed.

    With the intention of illuminating the many obstacles involved with literacy promotion in the developing nations of Africa, Asia, and South America, the authors of the 10 articles in this collection share their knowledge and experience of literacy promotion in the developing world--including the unique challenges faced by those who publish, print,…

  13. Promoting Creativity in Young Children.

    ERIC Educational Resources Information Center

    Honig, Alice Sterling

    This paper discusses creativity in young children and what teachers can do to support and promote it. Topics addressed in the paper include: (1) teacher interest in promoting creativity; (2) defining creativity; (3) creativity in the socioemotional domain; (4) the relationship between creativity and empathy for others; (4) bibliotherapy; (5)…

  14. Information technology in health promotion.

    PubMed

    Lintonen, T P; Konu, A I; Seedhouse, D

    2008-06-01

    eHealth, the use of information technology to improve or enable health and health care, has recently been high on the health care development agenda. Given the vivid interest in eHealth, little reference has been made to the use of these technologies in the promotion of health. The aim of this present study was to conduct a review on recent uses of information technology in health promotion through looking at research articles published in peer-reviewed journals. Fifteen relevant journals with issues published between 2003 and June 2005 yielded altogether 1352 articles, 56 of which contained content related to the use of information technology in the context of health promotion. As reflected by this rather small proportion, research on the role of information technology is only starting to emerge. Four broad thematic application areas within health promotion were identified: use of information technology as an intervention medium, use of information technology as a research focus, use of information technology as a research instrument and use of information technology for professional development. In line with this rather instrumental focus, the concepts 'ePromotion of Health' or 'Health ePromotion' would come close to describing the role of information technology in health promotion.

  15. Health promotion: an ethical analysis.

    PubMed

    Carter, Stacy M

    2014-04-01

    Thinking and practising ethically requires reasoning systematically about the right thing to do. Health promotion ethics - a form of applied ethics - includes analysis of health promotion practice and how this can be ethically justified. Existing frameworks can assist in such evaluation. These acknowledge the moral value of delivering benefits. But benefits need to be weighed against burdens, harms or wrongs, and these should be minimised: they include invading privacy, breaking confidentiality, restraining liberty, undermining self-determination or people's own values, or perpetuating injustice. Thinking about the ethics of health promotion also means recognising health promotion as a normative ideal: a vision of the good society. This ideal society values health, sees citizens as active and includes them in decisions that affect them, and makes the state responsible for providing all of its citizens, no matter how advantaged or disadvantaged, with the conditions and resources they need to be healthy. Ethicists writing about health promotion have focused on this relationship between the citizen and the state. Comparing existing frameworks, theories and the expressed values of practitioners themselves, we can see common patterns. All oppose pursuing an instrumental, individualistic, health-at-all-costs vision of health promotio