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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. Electron Acceptors Induce Secretion of Enterotoxigenic Escherichia coli Heat-Labile Enterotoxin under Anaerobic Conditions through Promotion of GspD Assembly

    PubMed Central

    Lu, Xi; Fu, Enqing; Xie, Yonghong

    2016-01-01

    Heat-labile enterotoxin (LT), the major virulence factor of enterotoxigenic Escherichia coli (ETEC), can lead to severe diarrhea and promotes ETEC adherence to intestinal epithelial cells. Most previous in vitro studies focused on ETEC pathogenesis were conducted under aerobic conditions, which do not reflect the real situation of ETEC infection because the intestine is anoxic. In this study, the expression and secretion of LT under anaerobic or microaerobic conditions were determined; LT was not efficiently secreted into the supernatant under anaerobic or microaerobic conditions unless terminal electron acceptors (trimethylamine N-oxide dihydrate [TMAO] or nitrate) were available. Furthermore, we found that the restoration effects of TMAO and nitrate on LT secretion could be inhibited by amytal or ΔtorCAD and ΔnarG E. coli strains, indicating that LT secretion under anaerobic conditions was dependent on the integrity of the respiratory chain. At the same time, electron acceptors increase the ATP level of ETEC, but this increase was not the main reason for LT secretion. Subsequently, the relationship between the integrity of the respiratory chain and the function of the type II secretion system was determined. The GspD protein, the secretin of ETEC, was assembled under anaerobic conditions and was accompanied by LT secretion when TMAO or nitrate was added. Our data also demonstrated that TMAO and nitrate could not induce the GspD assembly and LT secretion in ΔtorCAD and ΔnarG strains, respectively. Moreover, GspD assembly under anaerobic conditions was assisted by the pilot protein YghG. PMID:27430271

  3. 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

  4. 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

  5. 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.

  6. Polymyxin B-Induced Release of Low-Molecular-Weight, Heat-Labile Enterotoxin from Escherichia coli

    PubMed Central

    Evans, Doyle J.; Evans, Dolores G.; Gorbach, Sherwood L.

    1974-01-01

    Polymyxin B-induced release of enterotoxin from Escherichia coli strain H-10407 was demonstrated. Incubation of E. coli cells derived from 6-h cultures with polymyxin caused the rapid release of enterotoxin with a molecular weight of approximately 20,000, as estimated by the gel filtration technique. The rapidity of the release of enterotoxin indicates that it probably resides in the periplasmic space of the cell. The low-molecular-weight enterotoxin possessed vascular permeability factor and diarrheagenic activities, both of which were found to be heat-labile. The permeability factor activity of this enterotoxin was neutralized by antisera prepared against crude E. coli enterotoxin, Vibrio cholerae enterotoxin (choleragen), and V. cholerae toxoid (choleragenoid), respectively. Supernatant fluids of 6-h E. coli cultures did not contain this molecular form of enterotoxin but did contain very high-molecular-weight, heat-labile enterotoxin. Incubation of cells derived from older (18 h) cultures with polymyxin caused the release of both low- (20,000) and high-molecular-weight forms of enterotoxin. We concluded that either the 20,000-dalton form of heat-labile enterotoxin is not released by E. coli under in vitro growth conditions or that enterotoxin released in this form is rapidly destroyed or inactivated. PMID:16558081

  7. Comparison of preservation methods for enterotoxigenic Escherichia coli producing heat-labile enterotoxin.

    PubMed Central

    Yoh, M; Narita, I; Honda, T; Miwatani, T; Nishibuchi, M

    1991-01-01

    Ten strains of enterotoxigenic Escherichia coli producing heat-labile enterotoxin (LT) were preserved under 12 different conditions. After 1 month, 9 months, and 3 years of preservation, the cultures were recovered and examined for LT production. Preservation of the cultures on Dorset Egg Medium at 4 degrees C and preservation by freezing the cell suspensions in tryptic soy broth with 20% glycerol were found to be suitable preservation methods; all strains were alive for 3 years and had a minimum loss of LT production. PMID:1939590

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. Analysis of antigenic determinants in cholera enterotoxin and heat-labile enterotoxins from human and porcine enterotoxigenic Escherichia coli.

    PubMed Central

    Takeda, Y; Honda, T; Sima, H; Tsuji, T; Miwatani, T

    1983-01-01

    Antigenic determinants of cholera enterotoxin (CT) and heat-labile enterotoxin from a human strain (LTh) and a porcine strain (LTp) were analyzed by Ouchterlony double-gel diffusion test against anti-CT, anti-LTh, and anti-LTp, which were treated by immunoaffinity column chromatography. The results showed the existence of the following antigenic determinants: (i) antigenic determinants unique to CT, LTh, and LTp, respectively; (ii) an antigenic determinant common to CT, LTh, and LTp; (iii) an antigenic determinant common to CT and LTh, but not LTp; and (iv) an antigenic determinant common to LTh and LTp, but not CT. On the basis of these results, an antigenic scheme for CT, LTh, and LTp is proposed. Images PMID:6190758

  14. Value of passive immune hemolysis for detection of heat-labile enterotoxin produced by enterotoxigenic Escherichia coli.

    PubMed Central

    Tsukamoto, T; Kinoshita, Y; Taga, S; Takeda, Y; Miwatani, T

    1980-01-01

    The method of passive immune hemolysis of Evans and Evans (Infect. Immun. 16:604-609, 1977) for detection of heat-labile enterotoxin produced by enterotoxigenic Escherichia coli was modified. A total of 373 strains of E. coli were tested by this method using materials obtained by treating the cells with polymyxin B and rabbit antiserum against cholera enterotoxin, purified by affinity gel column coupled with purified cholera enterotoxin, in N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (pH 6.7). The results correlated very well with those obtained in an assay with Chinese hamster ovary cells. It is concluded that passive immune hemolysis is useful as a routine clinical method for identifying E. coli strains that produce heat-labile enterotoxin. PMID:7031077

  15. 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

  16. 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.

  17. Nutritional requirements for synthesis of heat-labile enterotoxin by enterotoxigenic strains of Escherichia coli.

    PubMed

    Gilligan, P H; Robertson, D C

    1979-01-01

    Optimal growth conditions have been established for production of heat-labile enterotoxin (LT) by both porcine and human strains of enterotoxigenic (ENT(+)) Escherichia coli. There were no unusual growth factor requirements, and some strains produced fairly high levels of LT in a basal salts medium containing 0.5% glucose if the pH was carefully controlled. Several amino acids markedly stimulated LT synthesis when added to the basal salts-glucose medium. Methionine and lysine were the most stimulatory for both human and porcine strains. Either aspartic acid or glutamic acid further enhanced LT synthesis in the presence of methionine and lysine, with aspartic acid being more stimulatory for porcine strains and glutamic acid more stimulatory for human strains. There were no apparent vitamin requirements and no unusual cations needed for toxin synthesis except that Fe(3+) was slightly stimulatory for porcine strains. The stimulation by Fe(3+) was observed only in the presence of the three amino acids, suggesting that the effect was indirect rather than on toxin synthesis. The carbon source also influenced the yield of LT. Glucose supported maximal synthesis, but other carbon sources which exhibit a high degree of catabolite repression also supported high levels of synthesis. Little or no LT was released below pH 7.0; therefore, because the pH drops during growth from 7.5 to 6.8, even in highly buffered media, it was necessary to adjust the pH to 8.0 to effect complete release of cell-associated toxin. The defined medium containing three amino acids reduced the amount of UV-absorbing material in culture supernatants about fivefold and increased LT activity for various strains from two- to fivefold over a complex Casamino Acids-yeast extract medium. Conditions found to be optimal for synthesis of LT were inhibitory for the heat-stable enterotoxin.

  18. 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

  19. 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.

  20. Isolation and epidemiological characterization of heat-labile enterotoxin-producing Escherichia fergusonii from healthy chickens.

    PubMed

    Oh, Jae-Young; Kang, Min-Su; An, Byung-Ki; Shin, Eun-Gyeong; Kim, Mi-Jin; Kwon, Jun-Hun; Kwon, Yong-Kuk

    2012-11-09

    Escherichia fergusonii has been associated with a wide variety of intestinal and extraintestinal infections in both humans and animals. The aim of this study was to demonstrate the presence of heat-labile enterotoxin (LT)-producing E. fergusonii in healthy chickens and its plasmid-mediated LT toxin gene transfer to other Enterobacteriaceae. We tested faecal samples from 184 chicken flocks (consisting of 109 broilers and 75 layers) of 78 commercial chicken farms for the presence of the LT gene using a polymerase chain reaction-based screening and identified samples from 43 flocks (23.4%) as positive for the LT gene. We subsequently isolated and identified E. fergusonii harboring the LT gene from all LT-positive samples and observed 21 various biochemical types. The plasmids encoding LT in 16 (37.2%) of the 43 isolates were conjugally transferred to the recipient strain Escherichia coli J53. Southern hybridization showed that all plasmids from the transconjugants carried the eltAB gene (Ent plasmid) and belonged to the narrow-host-range IncF type. In addition, all the E. fergusonii strains identified were classified into 17 pulsed-field gel electrophoresis (PFGE) types, and it is likely that there was an association between the PFGE types and geographical location or breed of flocks. In conclusion, this is the first study to demonstrate that LT-producing E. fergusonii strains were present in the faeces of healthy chickens and that plasmid-mediated virulence genes can be transferred to E. coli and may have a great potential to cause human disease. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. 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

  2. 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

  3. 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.

  4. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. Protection of non-obese diabetic mice from autoimmune diabetes by Escherichia coli heat-labile enterotoxin B subunit

    PubMed Central

    Ola, Thomas O; Williams, Neil A

    2006-01-01

    Autoimmune diabetes in the non-obese diabetic (NOD) mouse is associated with development of inflammation around the islets at around 4–5 weeks of age, which may be prolonged until frank diabetes begins to occur around 12 weeks of age. Although many interventions can halt disease progression if administration coincides with the beginning of the anti-β cell response, very few are able to prevent diabetes development once insulitis is established. Here we describe a strategy which blocks cellular infiltration of islets and prevents diabetes. Intranasal treatment with the B-subunit of Escherichia coli heat labile enterotoxin (EtxB), a protein that binds GM1 ganglioside (as well as GD1b, asialo-GM1 and lactosylceramide with lower affinities), protected NOD mice from developing diabetes in a receptor-binding dependent manner. Protection was associated with a significant reduction in the number of macrophages, CD4+ T cells, B cells, major histocompatibility complex class II+ cells infiltrating the islets. Despite this, treated mice showed increased number of interleukin-10+ cells in the pancreas, and a decrease in both T helper 1 (Th1) and Th2 cytokine production in the pancreatic lymph node. Disease protection was also transferred with CD4+ splenocytes from treated mice. Taken together, these results demonstrated that EtxB is a potent immune modulator capable of blocking diabetes. PMID:16423062

  12. 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.

  13. 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.

  14. Context-dependent activation kinetics elicited by soluble versus outer membrane vesicle-associated heat-labile enterotoxin.

    PubMed

    Chutkan, Halima; Kuehn, Meta J

    2011-09-01

    Enterotoxigenic Escherichia coli (ETEC) is the leading cause of traveler's diarrhea and children's diarrhea worldwide. Among its virulence factors, ETEC produces heat-labile enterotoxin (LT). Most secreted LT is associated with outer membrane vesicles that are rich in lipopolysaccharide. The majority of prior studies have focused on soluble LT purified from ETEC periplasm. We investigated the hypothesis that the extracellular vesicle context of toxin presentation might be important in eliciting immune responses. We compared the polarized epithelial cell responses to apically applied soluble LT and LT-containing vesicles (LT(+) vesicles) as well as controls using a catalytically inactive mutant of LT and vesicles lacking LT. Although vesicle treatments with no or catalytically inactive LT induced a modest amount of interleukin-6 (IL-6), samples containing catalytically active LT elicited higher levels. A combination of soluble LT and LT-deficient vesicles induced significantly higher IL-6 levels than either LT or LT(+) vesicles alone. The responses to LT(+) vesicles were found to be independent of the canonical LT pathway, because the inhibition of cyclic AMP response element (CRE)-binding protein (CREB) phosphorylation did not lead to a decrease in cytokine gene expression levels. Furthermore, soluble LT caused earlier phosphorylation of CREB and activation of CRE compared with LT(+) vesicles. Soluble LT also led to the activation of activator protein 1, whereas LT(+) vesicle IL-6 responses appeared to be mediated by NF-κB. In summary, the results demonstrate that soluble LT and vesicle-bound LT elicit ultimately similar cytokine responses through distinct different activation pathways.

  15. 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

  16. 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

  17. Comparison of the carbohydrate-binding specificities of cholera toxin and Escherichia coli heat-labile enterotoxins LTh-I, LT-IIa, and LT-IIb.

    PubMed Central

    Fukuta, S; Magnani, J L; Twiddy, E M; Holmes, R K; Ginsburg, V

    1988-01-01

    The heat-labile enterotoxins of Vibrio cholerae and Escherichia coli are related in structure and function. They are oligomers consisting of A and B polypeptide subunits. They bind to gangliosides, and they activate adenylate cyclase. The toxins form two antigenically distinct groups; members of each group cross-react but are not necessarily identical. Serogroup I includes cholera toxin (CT) and type I heat-labile enterotoxin (LT-I) of E. coli. LTh-I and LTp-I are antigenic variants of LT-I produced by strains of E. coli from humans and pigs, respectively. Serogroup II contains the type II heat-labile enterotoxin (LT-II) of E. coli. Two antigenic variants designated LT-IIa and LT-IIb have been described. The binding of CT, LTh-I, LT-IIa, and LT-IIb to gangliosides was analyzed by immunostaining thin-layer chromatograms and by solid-phase radioimmunoassay. The four toxins have different glycolipid-binding specificities. LTh-I and CT bind strongly to ganglioside GM1 and less strongly to ganglioside GD1b. However, LTh-I, unlike CT, also binds weakly to GM2 and asialo GM1. LTh-I, like CT, probably binds to the terminal sugar sequence Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)Gal . . ., where GalNAc is N-acetylgalactosamine and NeuAc is N-acetylneuraminic acid. LT-IIa probably binds to the same sugar sequence to which CT and LTh-I bind, with the additional contribution to binding of a second NeuAc as in GD1b and GD2. Also, LT-IIa must bind the Gal beta 1-3GalNAc . . . sequence in such a way that its binding is relatively unaffected by attachment of NeuAc to the terminal galactose residue as in GD1a, GT1b, and GQ1b. LT-IIb probably binds to the terminal sugar sequence NeuAc alpha 2-3Gal beta 1-4GalNAc . . ., as it binds to gangliosides GD1a and GT1b but not to GM1. Images PMID:3290106

  18. 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

  19. 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

  20. 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

  1. [Establishment of a triplex real-time PCR for the detection of cholera toxin gene ctx and heat labile enterotoxin gene elt].

    PubMed

    Li, Jie; Kan, Biao; Zhang, Jingyun

    2014-06-01

    To establish a triplex TaqMan real-time PCR system containing internal amplification control (IAC) to detect cholera toxin gene ctxA and enterotoxigenic Escherichia coli (ETEC)heat-labile enterotoxin gene elt. Primers and probes were designed based on the sequences of ctxA, elt and IAC. Both sensitivity and specificity were analyzed and interactions between different reactions were evaluated. This system showed that the sensitivity of ctxA was 94 copies/reaction while the elt 79 copies/reaction and the amplification efficiency were 94.7% and 98.1%, respectively. Under the ratio of copy numbers on gene ctxA to elt as between 1 : 1-1 : 10, when both targets were detected, with impact was less on each other. However, when the amount of elt or ctxA was 100 times of IAC, the amplification of IAC was significantly inhibited. This system showed both satisfactory sensitivity and specificity, thus could be used to detect pathogenic bacteria in diarrhea stools. The detection of IAC could prompt the presence of PCR inhibitors in samples being tested.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. Intranasal Immunization with Pneumococcal Polysaccharide Conjugate Vaccines with Nontoxic Mutants of Escherichia coli Heat-Labile Enterotoxins as Adjuvants Protects Mice against Invasive Pneumococcal Infections

    PubMed Central

    Jakobsen, Håvard; Schulz, Dominique; Pizza, Mariagrazia; Rappuoli, Rino; Jónsdóttir, Ingileif

    1999-01-01

    Host defenses against Streptococcus pneumoniae depend largely on phagocytosis following opsonization by polysaccharide-specific immunoglobulin G (IgG) antibodies and complement. Since colonization of the respiratory mucosa is the first step in pneumococcal pathogenesis, mucosal immune responses may play a significant role. In addition to inducing systemic immune responses, mucosal vaccination with an effective adjuvant has the advantage of inducing mucosal IgA antibodies. The heat-labile enterotoxin (LT) of Escherichia coli is a well-studied mucosal adjuvant, and adjuvant activity of nontoxic LT mutants has been demonstrated for several protein antigens. We investigated the immunogenicity of pneumococcal polysaccharide conjugate vaccines (PNC) of serotypes 1 and 3 in mice after intranasal (i.n.) immunization by using as an adjuvant the nontoxic LT mutant LT-K63 or LT-R72, which has minimal residual toxicity. Pneumococcal serotype-specific antibodies were measured in serum (IgM, IgG, and IgA) and saliva (IgA), and vaccine-induced protection was evaluated by i.n. challenge with virulent pneumococci of the homologous serotype. When administered with LT mutants, i.n. immunization with both conjugates induced systemic and mucosal immune responses, and serum IgG antibody levels were significantly higher than after subcutaneous immunization. All mice immunized i.n. with PNC-1 and LT mutants were protected against bacteremia and cleared the pneumococci from the lung 24 h after i.n. challenge; pneumococcal density correlated significantly with serum IgG antibody levels. Similarly, the survival of mice immunized i.n. with PNC-3 and LT mutants was significantly prolonged. These results demonstrate that i.n. vaccination with PNC and potent adjuvants can protect mice against invasive and lethal pneumococcal infections, indicating that mucosal vaccination with PNC may be an alternative vaccination strategy for humans. PMID:10531245

  7. Mucosal Adjuvanticity and Immunogenicity of LTR72, a Novel Mutant of Escherichia coli Heat-labile Enterotoxin with Partial Knockout of ADP-ribosyltransferase Activity

    PubMed Central

    Giuliani, Marzia Monica; Del Giudice, Giuseppe; Giannelli, Valentina; Dougan, Gordon; Douce, Gill; Rappuoli, Rino; Pizza, Mariagrazia

    1998-01-01

    Heat-labile Escherichia coli enterotoxin (LT) has the innate property of being a strong mucosal immunogen and adjuvant. In the attempt to reduce toxicity and maintain the useful immunological properties, several LT mutants have been produced. Some of these are promising mucosal adjuvants. However, so far, only those that were still toxic maintained full adjuvanticity. In this paper we describe a novel LT mutant with greatly reduced toxicity that maintains most of the adjuvanticity. The new mutant (LTR72), that contains a substitution Ala → Arg in position 72 of the A subunit, showed only 0.6% of the LT enzymatic activity, was 100,000-fold less toxic than wild-type LT in Y1 cells in vitro, and was at least 20 times less effective than wild-type LT in the rabbit ileal loop assay in vivo. At a dose of 1 μg, LTR72 exhibited a mucosal adjuvanticity, similar to that observed with wild-type LT, better than that induced by the nontoxic, enzymatically inactive LTK63 mutant, and much greater than that of the recombinant B subunit. This trend was consistent for both the amounts and kinetics of the antibody induced, and priming of antigen-specific T lymphocytes. The data suggest that the innate high adjuvanticity of LT derives from the independent contribution of the nontoxic AB complex and the enzymatic activity. LTR72 optimizes the use of both properties: the enzymatic activity for which traces are enough, and the nontoxic AB complex, the effect of which is dose dependent. In fact, in dose–response experiments in mice, 20 μg of LTR72 were a stronger mucosal adjuvant than wild-type LT. This suggests that LTR72 may be an excellent candidate to be tested in clinical trials. PMID:9529328

  8. Bile and unsaturated fatty acids inhibit the binding of cholera toxin and Escherichia coli heat-labile enterotoxin to GM1 receptor.

    PubMed

    Chatterjee, Arpita; Chowdhury, Rukhsana

    2008-01-01

    Cholera toxin (CT) is an archetypal bacterial toxin that binds with a high affinity to the receptor ganglioside GM1 on the intestinal epithelial surface and that causes the severe watery diarrhea characteristic of the disease cholera. Blockage of the interaction of CT with the GM1 receptor is an attractive approach for therapeutic intervention. We report here that crude bile prevents the interaction of CT with GM1 and reduces CT-mediated fluid accumulation in the rabbit intestine. The unsaturated fatty acids detected in crude bile, arachidonic, linoleic, and oleic acids, were found to be the most effective. Crude bile and the unsaturated fatty acids interacted with CT but not GM1 to prevent CT-GM1 binding. Neither crude bile nor the unsaturated fatty acids had any effect on the subunit structure of CT. The binding of CT to unsaturated fatty acids resulted in a shift of the apparent pI of CT from 6.8 to 8.2 and a marked decrease in intrinsic fluorescence. The Kd was calculated from fluorescence quenching assays. It was demonstrated by the rabbit ileal loop model that practically no fluid accumulated in the intestinal loops when CT was administered together with inhibitory concentrations of linoleic acid. The bile present in the intestine was sufficient to inhibit the activity of up to 300 ng CT. Bile and unsaturated fatty acids also inhibited the binding of Escherichia coli heat-labile enterotoxin (LT) to GM1, and no fluid accumulation was observed in rabbit ileal loops when LT was administered together with linoleic acid.

  9. 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.

  10. Activation of Escherichia coli heat-labile enterotoxins by native and recombinant adenosine diphosphate-ribosylation factors, 20-kD guanine nucleotide-binding proteins.

    PubMed Central

    Lee, C M; Chang, P P; Tsai, S C; Adamik, R; Price, S R; Kunz, B C; Moss, J; Twiddy, E M; Holmes, R K

    1991-01-01

    Escherichia coli heat-labile enterotoxins (LT) are responsible in part for "traveler's diarrhea" and related diarrheal illnesses. The family of LTs comprises two serogroups termed LT-I and LT-II; each serogroup includes two or more antigenic variants. The effects of LTs result from ADP ribosylation of Gs alpha, a stimulatory component of adenylyl cyclase; the mechanism of action is identical to that of cholera toxin (CT). The ADP-ribosyltransferase activity of CT is enhanced by 20-kD guanine nucleotide-binding proteins, known as ADP-ribosylation factors or ARFs. These proteins directly activate the CTA1 catalytic unit and stimulate its ADP ribosylation of Gs alpha, other proteins, and simple guanidino compounds (e.g., agmatine). Because of the similarities between CT and LTs, we investigated the effects of purified bovine brain ARF and a recombinant form of bovine ARF synthesized in Escherichia coli on LT activity. ARF enhanced the LT-I-, LT-IIa-, and LT-IIb-catalyzed ADP ribosylation of agmatine, as well as the auto-ADP ribosylation of the toxin catalytic unit. Stimulation of ADP-ribosylagmatine formation by LTs and CT in the presence of ARF was GTP dependent and enhanced by sodium dodecyl sulfate. With agmatine as substrate, LT-IIa and LT-IIb exhibited less than 1% the activity of CT and LT-Ih. CT and LTs catalyzed ADP-ribosyl-Gs alpha formation in a reaction dependent on ARF, GTP, and dimyristoyl phosphatidylcholine/cholate. With Gs alpha as substrate, the ADP-ribosyltransferase activities of the toxins were similar, although CT and LT-Ih appeared to be slightly more active than LT-IIa and LT-IIb. Thus, LT-IIa and LT-IIb appear to differ somewhat from CT and LT-Ih in substrate specificity. Responsiveness to stimulation by ARF, GTP, and phospholipid/detergent as well as the specificity of ADP-ribosyltransferase activity are functions of LTs from serogroups LT-I and LT-II that are shared with CT. Images PMID:1902492

  11. Effects of Site-Directed Mutagenesis of Escherichia coli Heat-Labile Enterotoxin on ADP-Ribosyltransferase Activity and Interaction with ADP-Ribosylation Factors

    PubMed Central

    A. Stevens, Linda; Moss, Joel; Vaughan, Martha; Pizza, Mariagrazia; Rappuoli, Rino

    1999-01-01

    Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of Gsα, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction. PMID:9864224

  12. Stepwise transplantation of an active site loop between heat-labile enterotoxins LT-II and LT-I and characterization of the obtained hybrid toxins.

    PubMed

    Feil, I K; Platas, A A; van den Akker, F; Reddy, R; Merritt, E A; Storm, D R; Hol, W G

    1998-11-01

    Members of the cholera toxin family, including Escherichia coli heat-labile enterotoxins LT-I and LT-II, catalyze the covalent modification of intracellular proteins by transfer of ADP-ribose from NAD to a specific arginine of the target protein. The ADP-ribosylating activity of these toxins is located in the A-subunit, for which LT-I and LT-II share a 63% sequence identity. The flexible loop in LT-I, ranging from residue 47 to 56, closes over the active site cleft. Previous studies have shown that point mutations in this loop have dramatic effects on the activity of LT-I. Yet, in LT-II the sequence of the equivalent loop differs at four positions from LT-I. Therefore five mutants of the active site loop were created by a stepwise replacement of the loop sequence in LT-I with virtually all the corresponding residues in LT-II. Since we discovered that LT-II had no activity versus the artificial substrate diethylamino-benzylidine-aminoguanidine (DEABAG) while LT-I does, our active site mutants most likely probe the NAD binding, not the arginine binding region of the active site. The five hybrid toxins obtained (Q49A, F52N, V53T, Q49V/F52N and Q49V/F52N/V53T) show (i) great differences in holotoxin assembly efficiency; (ii) decreased cytotoxicity in Chinese hamster ovary cells; and (iii) increased in vitro enzymatic activity compared with wild type LT-I. Specifically, the three mutants containing the F52N substitution display a greater Vmax for NAD than wild type LT-I. The enzymatic activity of the V53T mutant is significantly higher than that of wild type LT-I. Apparently this subtle variation at position 53 is beneficial, in contrast to several other substitutions at position 53 which previously had been shown to be deleterious for activity. The most striking result of this study is that the active site loop of LT-I, despite great sensitivity for point mutations, can essentially be replaced by the active site loop of LT-II, yielding an active 'hybrid enzyme' as well

  13. Age-Specific Prevalence of Antibody to Rotavirus, Escherichia coli Heat-Labile Enterotoxin, Norwalk Virus, and Hepatitis A Virus in-a Rural Community in Thailand

    PubMed Central

    Echeverria, Peter; Burke, Donald S.; Blacklow, Neil R.; Cukor, George; Charoenkul, Chanin; Yanggratoke, Samreong

    1983-01-01

    A serological survey in rural Thailand demonstrated that inhabitants acquired antibody to rotavirus between the ages of 6 months and 6 years, to Norwalk virus between the ages of 4 and 5 years, and to hepatitis A between the ages of 6 and 35 years. Antibody of Escherichia coli heat-labile toxin was most prevalent between 1 and 4 years and 18 and 25 years of age. PMID:6306049

  14. Attenuated Shigella flexneri 2a Vaccine Strain CVD 1204 Expressing Colonization Factor Antigen I and Mutant Heat-Labile Enterotoxin of Enterotoxigenic Escherichia coli

    PubMed Central

    Koprowski, Hilary; Levine, Myron M.; Anderson, Richard J.; Losonsky, Genevieve; Pizza, Mariagrazia; Barry, Eileen M.

    2000-01-01

    A multivalent live oral vaccine against both Shigella spp. and enterotoxigenic Escherichia coli (ETEC) is being developed based on the hypothesis that protection can be achieved if attenuated shigellae express ETEC fimbrial colonization factors and genetically detoxified heat-labile toxin from a human ETEC isolate (LTh). Two detoxified derivatives of LTh, LThK63 and LThR72, were engineered by substitution—serine to lysine at residue 63, or lysine to arginine at residue 72. The genes encoding these two derivatives were cloned separately on expression plasmids downstream from the CFA/I operon. Following electroporation into S. flexneri 2a vaccine strain CVD 1204, coexpression of CFA/I and LThK63 or LThR72 was demonstrated by Western blot analysis, GM1 binding assays, and agglutination with anti-CFA/I antiserum. Hemagglutination and electron microscopy confirmed surface expression of CFA/I. Guinea pigs immunized intranasally on days 0 and 15 with CVD 1204 expressing CFA/I and LThK63 or LThR72 exhibited high titers of both serum immunoglobulin G (IgG) and mucosal secretory IgA anti-CFA/I; 40% of the animals produced antibodies directed against LTh. All immunized guinea pigs also produced mucosal IgA (in tears) and serum IgG anti-S. flexneri 2a O antibodies. Furthermore, all immunized animals were protected from challenge with wild-type S. flexneri 2a. This prototype Shigella-ETEC hybrid vaccine demonstrates the feasibility of expressing multiple ETEC antigens on a single plasmid in an attenuated Shigella vaccine strain and engendering immune responses against both the heterologous antigens and vector strain. PMID:10948101

  15. 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.

  16. 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.

  17. Evaluation of the immunogenicity of a transgenic tobacco plant expressing the recombinant fusion protein of GP5 of porcine reproductive and respiratory syndrome virus and B subunit of Escherichia coli heat-labile enterotoxin in pigs.

    PubMed

    Chia, Min-Yuan; Hsiao, Shih-Hsuan; Chan, Hui-Ting; Do, Yi-Yin; Huang, Pung-Ling; Chang, Hui-Wen; Tsai, Yi-Chieh; Lin, Chun-Ming; Pang, Victor Fei; Jeng, Chian-Ren

    2011-04-15

    Escherichia coli heat-labile enterotoxin B subunit (LTB) can be used as an adjuvant for co-administered antigens. Our previous study showed that the expression of neutralizing epitope GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) in transgenic tobacco plant (GP5-T) could induce PRRSV-specific immune responses in pigs. A transgenic tobacco plant co-expressing LTB and PRRSV GP5 as a fusion protein (LTB-GP5-T) was further constructed and its immunogenicity was evaluated. Pigs were given orally three consecutive doses of equal concentration of recombinant GP5 protein expressed in leaves of LTB-GP5-T or GP5-T at a 2-week interval and challenged with PRRSV at 7 weeks post-initial immunization. Pigs receiving LTB-GP5-T or GP5-T developed PRRSV-specific antibody- and cell-mediated immunity and showed significantly lower viremia and tissue viral load and milder lung lesions than wild type tobacco plant (W-T). The LTB-GP5-T-treated group had relatively higher immune responses than the GP5-T-treated group, although the differences were not statistically significant. Copyright © 2011 Elsevier B.V. All rights reserved.

  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. 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

  20. Mucosal vaccination against serogroup B meningococci: induction of bactericidal antibodies and cellular immunity following intranasal immunization with NadA of Neisseria meningitidis and mutants of Escherichia coli heat-labile enterotoxin.

    PubMed

    Bowe, Frances; Lavelle, Ed C; McNeela, Edel A; Hale, Christine; Clare, Simon; Arico, Beatrice; Giuliani, Marzia M; Rae, Aaron; Huett, Alan; Rappuoli, Rino; Dougan, Gordon; Mills, Kingston H G

    2004-07-01

    Conjugated polysaccharide vaccines protect against serogroup C meningococci. However, this approach cannot be applied to serogroup B, which is still a major cause of meningitis. We evaluated the immunogenicity of three surface-exposed proteins from serogroup B Neisseria meningitidis (App, NhhA, and NadA) identified during whole-genome sequencing. Mice were immunized intranasally with individual proteins in the presence of wild-type Escherichia coli heat-labile enterotoxin (LTwt), LTR72, a partially inactivated mutant, or LTK63, a completely nontoxic mutant, as the adjuvant. Each of the meningococcal proteins induced significant cellular responses; NhhA and NadA induced strong antibody responses, but only NadA induced bactericidal antibody when administered intranasally with mucosal adjuvants. In addition, immunoglobulin A and bactericidal antibodies were detected in the respiratory tract following intranasal delivery of NadA. Analysis of antigen-specific cytokine production by T cells from immunized mice revealed that intranasal immunization with NadA alone failed to generate detectable cellular immune responses. In contrast, LTK63, LTR72, and LTwt significantly augmented NadA-specific gamma interferon, interleukin-4 (IL-4), IL-5, and IL-10 production by spleen and lymph node cells, suggesting that both Th1 and Th2 cells were induced in vivo. The strongest cellular responses and highest bactericidal antibody titers were generated with LTR72 as the adjuvant. These findings demonstrate that the quality and magnitude of the immune responses generated by mucosal vaccines are influenced by the antigen as well as the adjuvant and suggest that nasal delivery of NadA with mucosal adjuvants has considerable potential in the development of a mucosal vaccine against serogroup B meningococci.

  1. Mucosal Vaccination against Serogroup B Meningococci: Induction of Bactericidal Antibodies and Cellular Immunity following Intranasal Immunization with NadA of Neisseria meningitidis and Mutants of Escherichia coli Heat-Labile Enterotoxin

    PubMed Central

    Bowe, Frances; Lavelle, Ed C.; McNeela, Edel A.; Hale, Christine; Clare, Simon; Arico, Beatrice; Giuliani, Marzia M.; Rae, Aaron; Huett, Alan; Rappuoli, Rino; Dougan, Gordon; Mills, Kingston H. G.

    2004-01-01

    Conjugated polysaccharide vaccines protect against serogroup C meningococci. However, this approach cannot be applied to serogroup B, which is still a major cause of meningitis. We evaluated the immunogenicity of three surface-exposed proteins from serogroup B Neisseria meningitidis (App, NhhA, and NadA) identified during whole-genome sequencing. Mice were immunized intranasally with individual proteins in the presence of wild-type Escherichia coli heat-labile enterotoxin (LTwt), LTR72, a partially inactivated mutant, or LTK63, a completely nontoxic mutant, as the adjuvant. Each of the meningococcal proteins induced significant cellular responses; NhhA and NadA induced strong antibody responses, but only NadA induced bactericidal antibody when administered intranasally with mucosal adjuvants. In addition, immunoglobulin A and bactericidal antibodies were detected in the respiratory tract following intranasal delivery of NadA. Analysis of antigen-specific cytokine production by T cells from immunized mice revealed that intranasal immunization with NadA alone failed to generate detectable cellular immune responses. In contrast, LTK63, LTR72, and LTwt significantly augmented NadA-specific gamma interferon, interleukin-4 (IL-4), IL-5, and IL-10 production by spleen and lymph node cells, suggesting that both Th1 and Th2 cells were induced in vivo. The strongest cellular responses and highest bactericidal antibody titers were generated with LTR72 as the adjuvant. These findings demonstrate that the quality and magnitude of the immune responses generated by mucosal vaccines are influenced by the antigen as well as the adjuvant and suggest that nasal delivery of NadA with mucosal adjuvants has considerable potential in the development of a mucosal vaccine against serogroup B meningococci. PMID:15213150

  2. 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.

  3. LT-IIb(T13I), a Non-Toxic Type II Heat-Labile Enterotoxin, Augments the Capacity of a Ricin Toxin Subunit Vaccine to Evoke Neutralizing Antibodies and Protective Immunity

    PubMed Central

    Greene, Christopher J.; Chadwick, Chrystal M.; Mandell, Lorrie M.; Hu, John C.; O’Hara, Joanne M.; Brey, Robert N.; Mantis, Nicholas J.; Connell, Terry D.

    2013-01-01

    Currently, there is a shortage of adjuvants that can be employed with protein subunit vaccines to enhance protection against biological threats. LT-IIb(T13I) is an engineered nontoxic derivative of LT-IIb, a member of the type II subfamily of heat labile enterotoxins expressed by Escherichia coli, that possesses potent mucosal adjuvant properties. In this study we evaluated the capacity of LT-IIb(T13I) to augment the potency of RiVax, a recombinant ricin toxin A subunit vaccine, when co-administered to mice via the intradermal (i.d.) and intranasal (i.n.) routes. We report that co-administration of RiVax with LT-IIb(T13I) by the i.d. route enhanced the levels of RiVax-specific serum IgG antibodies (Ab) and elevated the ratio of ricin-neutralizing to non-neutralizing Ab, as compared to RiVax alone. Protection against a lethal ricin challenge was also augmented by LT-IIb(T13I). While local inflammatory responses elicited by LT-IIb(T13I) were comparable to those elicited by aluminum salts (Imject®), LT-IIb(T13I) was more effective than aluminum salts at augmenting production of RiVax-specific serum IgG. Finally, i.n. administration of RiVax with LT-IIb(T13I) also increased levels of RiVax-specific serum and mucosal Ab and enhanced protection against ricin challenge. Collectively, these data highlight the potential of LT-IIb(T13I) as an effective next-generation i.d., or possibly i.n. adjuvant for enhancing the immunogenicity of subunit vaccines for biodefense. PMID:23936344

  4. 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.

  5. HEAT LABILE OPSONINS TO PNEUMOCOCCUS

    PubMed Central

    Smith, Mary Ruth; Wood, W. Barry

    1969-01-01

    Heat labile opsonins (HLO) in normal rat serum to both encapsulated and unencapsulated pneumococci (a) have the same heat lability as complement (C); (b) are active at 37°C but not at 0°C; (c) are inactivated proportionately to hemolytic C by the addition of immune aggregates to the serum; (d) are adsorbed from serum nonspecifically by bacteria at 37°C but not at 0°C; (e) are Ca++- and/or Mg++-dependent in their action; and (f) are inactivated by zymosan and a purified cobra venom factor, and in the case of encapsulated pneumococci, at least, by NH4OH. Like other opsonins, HLO to pneumococci act primarily on the bacteria rather than on the phagocytes. Their combined properties indicate that they involve multiple components of the hemolytic C system. Since HLO are immunologically polyspecific, they presumably play a broad protective role in the early (preantibody) phase of acute bacterial infections. PMID:4390899

  6. 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

  7. 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, ...

  8. 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

  9. Cholera toxin, E. coli heat-labile toxin, and non-toxic derivatives induce dendritic cell migration into the follicle-associated epithelium of Peyer's patches

    PubMed Central

    Anosova, NG; Chabot, S; Shreedhar, V; Borawski, JA; Dickinson, BL; Neutra, MR

    2008-01-01

    The follicle-associated epithelium (FAE) of Peyer's patches (PPs) transports antigens and microorganisms into mucosal lymphoid tissues where they are captured by subepithelial dendritic cells (DCs). Feeding of cholera toxin (CT) induced migration of subepithelial DCs to interfollicular T-cell areas within 24 h. This study investigated short-term effects of CT, Escherichia coli heat-labile toxin, and non-toxic derivatives on DC migration. CT or CTB injected into ligated intestinal loops induced significant increase in CD11c + DCs within the FAE within 90 min. In mice fed CT intragastrically, DC numbers in the FAE increased by 1 h, were maximal by 2 h, declined between 8 and 12 h, and were reversed by 24 h. Feeding of native LT, recombinant CTB, dibutyryl cyclic AMP, and to a lesser extent mutated CT(E29H) or mutated LT(R192G) had the same effect. Thus, both A and B subunits of enterotoxins, presumably acting through distinct signaling pathways, may promote capture of incoming antigens and pathogens by PP DCs. PMID:19079161

  10. Stimulation of cyclic AMP secretion in Vero cells by enterotoxins of Escherichia coli and Vibrio cholerae.

    PubMed Central

    Stavric, S; Speirs, J I; Konowalchuk, J; Jeffrey, D

    1978-01-01

    The morphological response of Vero cells to Escherichia coli heat-labile enterotoxin was similar to that of cholera toxin and was accompanied by increases in the intracellular level of cyclic AMP. The effects of both enterotoxins were enhanced by the presence of phosphodiesterase inhibitor and inhibited by heat or specific antisera. Accumulation of cyclic AMP preceded the morphological response. PMID:80381

  11. 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

  12. 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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. Four Foodborne Disease Outbreaks Caused by a New Type of Enterotoxin-Producing Clostridium perfringens

    PubMed Central

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

    2015-01-01

    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. PMID:25568432

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. Impaired autonomic responsiveness of the cardiovascular system of the rat induced by a heat-labile component of Bordetella pertussis vaccine.

    PubMed Central

    de Wildt, D J; Kreeftenberg, H G; Nijkamp, F P

    1983-01-01

    Bordetella pertussis vaccination induces severe impairment of the autonomic responsiveness of the cardiovascular system in rats. The vasodilation after beta 2-adrenoceptor stimulation with salbutamol as well as the negative chronotropic action induced by the muscarinic receptor stimulant arecoline were inhibited 4 days after vaccination. Moreover, basal blood pressure values appeared to be significantly lower in B. pertussis-vaccinated rats compared with control animals. These effects were dependent upon the bacterial strain used. Differences in pharmacological activity due to strain differences paralleled variations in the content of lymphocytosis-promoting factor of the vaccine. The inhibitory effects were absent after the administration of vaccine heated for 1 h at 80 degrees C, implicating an important role for a heat-labile component, e.g., lymphocytosis-promoting factor, and not for a heat-stable constituent, e.g., endotoxin (lipopolysaccharide). Previous studies indicate that some early biological effects elicited by B. pertussis vaccine can be attributed to lipopolysaccharide, whereas late induced effects are mainly brought about by lymphocytosis-promoting factor. For that reason a role for lipopolysaccharide might be excluded because 5 h after vaccination no disturbances of the autonomic nervous system were observed. We conclude that B. pertussis vaccination induces autonomic hyporesponsiveness due to a heat-labile component that is assumed to be lymphocytosis-promoting factor. PMID:6307869

  20. Different assay conditions for detecting the production and release of heat-labile and heat-stable toxins in enterotoxigenic Escherichia coli isolates.

    PubMed

    Rocha, Letícia B; Ozaki, Christiane Y; Horton, Denise S P Q; Menezes, Caroline A; Silva, Anderson; Fernandes, Irene; Magnoli, Fabio C; Vaz, Tania M I; Guth, Beatriz E C; Piazza, Roxane M F

    2013-12-02

    Enterotoxigenic Escherichia coli (ETEC) produce heat-labile (LT) and/or heat-stable enterotoxins (ST). Despite that, the mechanism of action of both toxins are well known, there is great controversy in the literature concerning the in vitro production and release of LT and, for ST, no major concerns have been discussed. Furthermore, the majority of published papers describe the use of only one or a few ETEC isolates to define the production and release of these toxins, which hinders the detection of ETEC by phenotypic approaches. Thus, the present study was undertaken to obtain a better understanding of ST and LT toxin production and release under laboratory conditions. Accordingly, a collection of 90 LT-, ST-, and ST/LT-producing ETEC isolates was used to determine a protocol for toxin production and release aimed at ETEC detection. For this, we used previously raised anti-LT antibodies and the anti-ST monoclonal and polyclonal antibodies described herein. The presence of bile salts and the use of certain antibiotics improved ETEC toxin production/release. Triton X-100, as chemical treatment, proved to be an alternative method for toxin release. Consequently, a common protocol that can increase the production and release of LT and ST toxins could facilitate and enhance the sensitivity of diagnostic tests for ETEC using the raised and described antibodies in the present work.

  1. 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.

  2. Isolation of hybridoma cell lines and characterization of monoclonal antibodies against cholera enterotoxin and its subunits.

    PubMed

    Robb, M; Nichols, J C; Whoriskey, S K; Murphy, J R

    1982-10-01

    Hybridoma cell lines which produced monoclonal antibodies against cholera toxin were isolated. These cell lines were detected with an enzyme-linked immunosorbent assay screening procedure with purified cholera toxin or subunit A of cholera toxin. Seven cell lines were characterized with respect to their reactivity with cholera toxin subunits by Western blot analysis. Five clones produced antibodies which were directed against subunit A, and two clones produced antibodies which reacted with subunit B. These antibodies were also characterized by Western blot analysis for reactivity with the heat-labile enterotoxin produced by porcine and human enterotoxinogenic strains of Escherichia coli. Monoclonal antibodies which reacted with subunit A of cholera toxin also reacted with subunit A of both porcine and human heat-labile enterotoxins. In contrast, monoclonal antibodies to subunit B of cholera toxin did not react with subunit B of the heat-labile enterotoxin. Antibodies directed against subunit B neutralized the biological activity of cholera toxin in vitro in the S49 mouse lymphosarcoma assay. In contrast to polyclonal anti-subunit A antisera, monoclonal anti-subunit A from four of five clones had small but measurable neutralizing capacities in vitro.

  3. Isolation of hybridoma cell lines and characterization of monoclonal antibodies against cholera enterotoxin and its subunits.

    PubMed Central

    Robb, M; Nichols, J C; Whoriskey, S K; Murphy, J R

    1982-01-01

    Hybridoma cell lines which produced monoclonal antibodies against cholera toxin were isolated. These cell lines were detected with an enzyme-linked immunosorbent assay screening procedure with purified cholera toxin or subunit A of cholera toxin. Seven cell lines were characterized with respect to their reactivity with cholera toxin subunits by Western blot analysis. Five clones produced antibodies which were directed against subunit A, and two clones produced antibodies which reacted with subunit B. These antibodies were also characterized by Western blot analysis for reactivity with the heat-labile enterotoxin produced by porcine and human enterotoxinogenic strains of Escherichia coli. Monoclonal antibodies which reacted with subunit A of cholera toxin also reacted with subunit A of both porcine and human heat-labile enterotoxins. In contrast, monoclonal antibodies to subunit B of cholera toxin did not react with subunit B of the heat-labile enterotoxin. Antibodies directed against subunit B neutralized the biological activity of cholera toxin in vitro in the S49 mouse lymphosarcoma assay. In contrast to polyclonal anti-subunit A antisera, monoclonal anti-subunit A from four of five clones had small but measurable neutralizing capacities in vitro. Images PMID:6183210

  4. 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.

  5. Identification of Enterotoxin E

    PubMed Central

    Bergdoll, Merlin S.; Borja, Concordia R.; Robbins, Ruth N.; Weiss, Karl F.

    1971-01-01

    Identification of a new enterotoxin was accomplished by purification of the enterotoxin produced by staphylococcal strain FRI-326 and by preparation of specific antitoxin to the enterotoxin. Toxicity of the preparations was determined in rhesus monkeys, and specificity of the enterotoxin-antitoxin reaction was determined in gel diffusion plates. The enterotoxin was designated enterotoxin E. PMID:5005309

  6. Cloning of enterotoxin gene from Aeromonas hydrophila provides conclusive evidence of production of a cytotonic enterotoxin.

    PubMed Central

    Chakraborty, T; Montenegro, M A; Sanyal, S C; Helmuth, R; Bulling, E; Timmis, K N

    1984-01-01

    Culture filtrates of two Aeromonas hydrophila strains which were isolated from patients with diarrhea and assumed to be causative agents of the infections were shown to contain enterotoxic, cytotoxic, and hemolytic activities. Modest heat treatment of the filtrates inactivated the cytotoxic and cytolytic activities, but not the enterotoxic activity. The construction of cosmid gene banks in Escherichia coli of DNA from both A. hydrophila strains demonstrated that the determinants of the three activities are located on three different segments of the A. hydrophila chromosome. Both heated culture filtrates of A. hydrophila and nonheated filtrates of an E. coli clone containing the A. hydrophila enterotoxin gene provoked fluid accumulation in the rabbit ileal loop and suckling mouse models and caused elongation of Chinese hamster ovary cells. Differences in the responses of the models to the A. hydrophila enterotoxin and to the heat-labile and heat-stabile toxins of E. coli indicated that the former is distinct from the latter two types of toxin. These results constitute conclusive evidence for the production by A. hydrophila of a cytotonic enterotoxin that is distinct from the A. hydrophila cytotoxin and hemolysin and known E. coli enterotoxins. Images PMID:6500697

  7. 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.

  8. 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

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

    PubMed

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

    2010-08-27

    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 degrees C or 65 degrees 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. Copyright 2010 Elsevier Inc. All rights reserved.

  10. CodY Promotes Sporulation and Enterotoxin Production by Clostridium perfringens Type A Strain SM101.

    PubMed

    Li, Jihong; Freedman, John C; Evans, Daniel R; McClane, Bruce A

    2017-03-01

    Clostridium perfringens type D strains cause enterotoxemia and enteritis in livestock via epsilon toxin production. In type D strain CN3718, CodY was previously shown to increase the level of epsilon toxin production and repress sporulation. C. perfringens type A strains producing C. perfringens enterotoxin (CPE) cause human food poisoning and antibiotic-associated diarrhea. Sporulation is critical for C. perfringens type A food poisoning since spores contribute to transmission and resistance in the harsh food environment and sporulation is essential for CPE production. Therefore, the current study asked whether CodY also regulates sporulation and CPE production in SM101, a derivative of C. perfringens type A food-poisoning strain NCTC8798. An isogenic codY-null mutant of SM101 showed decreased levels of spore formation, along with lower levels of CPE production. A complemented strain recovered wild-type levels of both sporulation and CPE production. When this result was coupled with the earlier results obtained with CN3718, it became apparent that CodY regulation of sporulation varies among different C. perfringens strains. Results from quantitative reverse transcriptase PCR analysis clearly demonstrated that, during sporulation, codY transcript levels remained high in SM101 but rapidly declined in CN3718. In addition, abrB gene expression patterns varied significantly between codY-null mutants of SM101 and CN3718. Compared to the levels in their wild-type parents, the level of abrB gene expression decreased in the CN3718 codY-null mutant strain but significantly increased in the SM101 codY-null mutant strain, demonstrating CodY-dependent regulation differences in abrB expression between these two strains. This difference appears to be important since overexpression of the abrB gene in SM101 reduced the levels of sporulation and enterotoxin production, supporting the involvement of AbrB repression in regulating C. perfringens sporulation. Copyright © 2017

  11. 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

  12. 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.

  13. 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

  14. Enterotoxigenic Escherichia coli heat-labile toxin seroconversion in US travelers to Mexico.

    PubMed

    Flores, Jose; DuPont, Herbert L; Jiang, Zhi-Dong; Belkind-Gerson, Jaime; Mohamed, Jamal A; Carlin, Lily G; Padda, Ranjit S; Paredes, Mercedes; Martinez-Sandoval, Jose Francisco; Villa, Nicolas A; Okhuysen, Pablo C

    2008-01-01

    Enterotoxigenic Escherichia coli (ETEC) is the most common bacterial pathogen isolated from travelers suffering of diarrhea. Exposure to heat-labile toxin (LT) produces a high rate of seroconversion. However, the role of LT-producing ETEC (LT-ETEC) as a cause of diarrhea is controversial. We conducted a cohort study in US students traveling to Mexico to assess the ETEC-LT seroconversion rate after natural exposure. Participants provided a serum sample on arrival and departure and a stool sample when ill. ETEC-LT immunoglobulin G antibodies were measured by enzyme-linked immunosorbent assay, and LT-ETEC were detected by means of polymerase chain reaction done on fecal DNA. A total of 422 participants with a mean age of 34.5 years were followed a mean of 19.9 days; 304 were females (72.0%), and 319 (75.6%) traveled during the summer months. In total, 177 individuals (41.9%) developed travelers' diarrhea and 33.9% had LT-ETEC identified in their stools. Among individuals having an LT-ETEC strain, 74% seroconverted compared to 11% of those not having diarrhea (p < 0.0001). When analyzed with a logistic regression model, the odds of seroconversion were significantly reduced in participants not having LT-ETEC in their stool (odds ratio = 0.1, p < 0.0001) after adjusting for season, length of stay, age, gender, race, and ethnicity. In US young adults traveling to Mexico, ETEC-LT seroconversion reliably identifies individuals naturally exposed to ETEC and correlates with symptomatic illness, length and season of travel.

  15. 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.

  16. 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

  17. 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.

  18. Enterotoxin production and serogroups of Campylobacter jejuni and Campylobacter coli from patients with diarrhea and from healthy laying hens.

    PubMed Central

    Lindblom, G B; Kaijser, B; Sjögren, E

    1989-01-01

    Enterotoxin production, a possible virulence factor, was determined in Campylobacter jejuni and Campylobacter coli by two different techniques, the CHO cell test and the GM1 enzyme-linked immunosorbent assay. The frequency of enterotoxigenic Campylobacter strains was 32% in strains from both humans with acute enteritis and healthy laying hens, as measured by the CHO cell test. The CHO cell test was significantly more sensitive than the GM1 enzyme-linked immunosorbent assay in the detection of enterotoxigenic strains. Enterotoxin production was compared with the presence of heat-stable and heat-labile antigens. There was no significant correlation between enterotoxin production and serogroups for C. jejuni or C. coli. The difference in enterotoxigenicity between C. jejuni (34.1%) and C. coli (21.9%) was not significant. PMID:2754001

  19. Nonimmunoglobulin fraction of human milk inhibits bacterial adhesion (hemagglutination) and enterotoxin binding of Escherichia coli and Vibrio cholerae.

    PubMed Central

    Holmgren, J; Svennerholm, A M; Ahrén, C

    1981-01-01

    Human milk and colostrum samples were divided into an immunoglobulin and a nonimmunoglobulin fraction by immunosorbent chromatography. The ability of these fractions to inhibit bacterial cell adhesion and enterotoxin receptor binding of Vibrio cholerae and various Escherichia coli isolates was then tested by in vitro assays. The strongest effect was generally seen with the nonimmunoglobulin fractions, which were shown to significantly inhibit E. coli cell adhesion (hemagglutination) mediated by CFA/I, CFA/II, or K88 fimbriae (but not type 1 pili) and V. cholerae hemagglutination, as well as the binding of cholera toxin and E. coli heat-labile enterotoxin to GM1 ganglioside. Also, the immunoglobulin fractions had significant inhibitory activity in some of these systems. The results are interpreted to suggest that human milk and colostrum may contain secreted structure analogs of the cell receptors for some bacterial adhesions and enterotoxins; this might contribute to the protective effect of milk against enteric infections. PMID:7021421

  20. 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.

  1. Identification of a New Enterotoxin as Enterotoxin C

    PubMed Central

    Bergdoll, Merlin S.; Borja, Concordia R.; Avena, Remedios M.

    1965-01-01

    Bergdoll, Merlin S. (University of Chicago, Chicago, Ill.), Concordia R. Borja, and Remedios M. Avena. Identification of a new enterotoxin as enterotoxin C. J. Bacteriol. 90:1481–1485. 1965.—Identification of a new enterotoxin was accomplished by purification of the enterotoxins produced by staphylococcal strains 137 and 361 and by the preparation of specific antitoxin to the enterotoxin. Toxicity of the preparations was determined in rhesus monkeys, and specificity of the enterotoxin-antitoxin reaction was determined in gel-diffusion plates. The enterotoxin has been designated enterotoxin C, and staphylococcal strain 137 (ATCC 19095) was selected as the prototype strain. Images PMID:4954560

  2. TLR2-dependent modulation of dendritic cells by LT-IIa-B5, a novel mucosal adjuvant derived from a type II heat-labile enterotoxin

    PubMed Central

    Lee, Chang Hoon; Masso-Welch, Patricia; Hajishengallis, George; Connell, Terry D.

    2011-01-01

    A host of human pathogens invades the body at mucosal surfaces. Yet, strong, protective mucosal immune responses directed against those pathogens routinely cannot be induced without the use of adjuvants. Although the strongest mucosal adjuvants are members of the family of HLTs, the inherent toxicities of HLT holotoxins preclude their clinical use. Herein, it is shown that LT-IIa-B5 enhances mucosal immune responses by modulating activities of DCs. i.n. immunization of mice with OVA in the presence of LT-IIa-B5 recruited DCs to the NALT and significantly increased uptake of OVA by those DCs. Furthermore, LT-IIa-B5 increased expression of CCR7 by DCs, which mediated enhanced migration of the cells from the NALT to the draining CLNs. LT-IIa-B5 also enhanced maturation of DCs, as revealed by increased surface expression of CD40, CD80, and CD86. Ag-specific CD4+ T cell proliferation was augmented in the CLNs of mice that had received i.n. LT-IIa-B5. Finally, when used as an i.n. adjuvant, LT-IIa-B5 dramatically increased the levels of OVA-specific salivary IgA and OVA-specific serum IgG. Strikingly, each of the activities induced by LT-IIa-B5 was strictly TLR2-dependent. The data strongly suggest that the immunomodulatory properties of LT-IIa-B5 depend on the productive modulation of mucosal DCs. Notably, this is the first report for any HLT to demonstrate in vivo the elicitation of strong, TLR2-dependent modulatory effects on DCs with respect to adjuvanticity. PMID:21791597

  3. Enterotoxins of Staphylococci

    DTIC Science & Technology

    1988-01-01

    Enterotoxin Genes 148 B. Membrane-Bound Toxin Precursor 148 C. Plasmid Involvement in Enterotoxin B Production 149 D. Genetic Analysis of Enterotoxin A...N-Z Amines manufactured by Sheffield and/or Protein Hydrolysate Powder prepared by Meade Johnson. Some of the enterotoxins also require...1.4 50-70 80-90 Hydroxyapatite 500-600 0.75-1.2 90-95 >99 4. About 1 lb of the strong carboxylic acid exchanger, CG-50 (Rohm and Haas), is stirred into

  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. Transient Facial Nerve Paralysis (Bell's Palsy) following Intranasal Delivery of a Genetically Detoxified Mutant of Escherichia coli Heat Labile Toxin

    PubMed Central

    Lewis, David J. M.; Huo, Zhiming; Barnett, Susan; Kromann, Ingrid; Giemza, Rafaela; Galiza, Eva; Woodrow, Maria; Thierry-Carstensen, Birgit; Andersen, Peter; Novicki, Deborah; Del Giudice, Giuseppe; Rappuoli, Rino

    2009-01-01

    Background An association was previously established between facial nerve paralysis (Bell's palsy) and intranasal administration of an inactivated influenza virosome vaccine containing an enzymatically active Escherichia coli Heat Labile Toxin (LT) adjuvant. The individual component(s) responsible for paralysis were not identified, and the vaccine was withdrawn. Methodology/Principal Findings Subjects participating in two contemporaneous non-randomized Phase 1 clinical trials of nasal subunit vaccines against Human Immunodeficiency Virus and tuberculosis, both of which employed an enzymatically inactive non-toxic mutant LT adjuvant (LTK63), underwent active follow-up for adverse events using diary-cards and clinical examination. Two healthy subjects experienced transient peripheral facial nerve palsies 44 and 60 days after passive nasal instillation of LTK63, possibly a result of retrograde axonal transport after neuronal ganglioside binding or an inflammatory immune response, but without exaggerated immune responses to LTK63. Conclusions/Significance While the unique anatomical predisposition of the facial nerve to compression suggests nasal delivery of neuronal-binding LT–derived adjuvants is inadvisable, their continued investigation as topical or mucosal adjuvants and antigens appears warranted on the basis of longstanding safety via oral, percutaneous, and other mucosal routes. PMID:19756141

  6. 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.

  7. Genetically Detoxified Mutants of Heat-Labile Toxin from Escherichia coli Are Able To Act as Oral Adjuvants

    PubMed Central

    Douce, Gill; Giannelli, Valentina; Pizza, Mariagrazia; Lewis, David; Everest, Paul; Rappuoli, Rino; Dougan, Gordon

    1999-01-01

    Detoxified mutants of the Escherichia coli heat-labile toxin (LT) act as mucosal adjuvants to intranasally presented coadministered antigens. Here, we compare the adjuvant activity of a panel of detoxified derivatives of LT, using both intranasal (i.n.) and oral (p.o.) routes of administration. The mutants used as adjuvants varied in sensitivity to proteases and toxicity. With keyhole limpet hemocyanin (KLH) as the bystander antigen, the immune responses to i.n. immunizations were consistently higher than the equivalent p.o.-delivered proteins. LT-G192, a mutant which demonstrates a 10-fold reduction in toxicity in vitro, demonstrated wild-type adjuvant activity both i.n. and p.o., inducing similar titers of KLH specific antibody in the sera and immunoglobulin A in local mucosal secretions as wild-type LT. In line with previous data, the nontoxic holotoxoid LT-K63 induced intermediate immune responses in both the serum and mucosal secretions which were lower than those achieved with wild-type LT but at least 10-fold higher than those measured when the antigen was administered with LT-B. Although significant levels of local and systemic anti-KLH antibodies were induced following p.o. immunization with LT-K63, cellular proliferative responses to KLH was poor or undetectable. In contrast, LT and LT-G192 induced significant T-cell responses to KLH following p.o. immunization. These proliferating cells secreted both gamma interferon and interleukin-5, suggesting that the type of immune response induced following p.o. coimmunization with LT and purified protein is a mixed Th1/Th2 response. PMID:10456880

  8. 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

  9. 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.

  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. 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

  12. 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.

  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. 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

  15. 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

  16. Signaling of Escherichia coli enterotoxin on supramolecular redox bilayer vesicles

    SciTech Connect

    Cheng, Q.; Peng, T.; Stevens, R.C.

    1999-07-21

    Electron transport in supramolecular assemblies containing redox centers has been a subject of great interest. Depending on spatial arrangement of redox moieties in macromolecular structures, transport of electrons may occur via a diffusion mechanism or electron hopping between the neighboring redox sites. While research has largely dealt with 3-D redox polymers, some 2-D systems such as self-assembled and Langmuir-Blodgett monolayers have been exploited as well. The authors describe here a new interfacial architecture that combines the high redox concentration in 3-D polymers and controllable structure and functionality of the 2-D monolayer systems. The new interface utilizes structurally defined redox liposomes engineered with biomolecular recognition capability by incorporating cell surface receptor G{sub M1} into the bilayer membrane. The design allows for direct inspection of the dependency of electron transport on the state and extent of biomolecular recognition that has taken place on the vesicles and, thus, provides a method for direct measurement of E. coli heat-labile enterotoxin binding by electrochemistry.

  17. Modulation of the immune response by the cholera-like enterotoxins.

    PubMed

    Plant, Andrea; Williams, Neil A

    2004-01-01

    Cholera toxins and heat labile enterotoxin from E. coli differ from most soluble proteins in eliciting systemic immunity both against themselves and unrelated admixed antigens, rather than tolerance following administration to a mucosal surface. Several reports have also demonstrated preferential induction of Th2-type responses when these molecules are used as adjuvants. Conversely, these proteins and their non-toxic derivatives, including the B sub-units are also able prevent and alleviate autoimmune diseases in naïve and systemically immune hosts demonstrating wide-ranging effects on the immune system. The recent observation that amelioration of autoimmune disease is associated with the generation of regulatory T cells which inhibit pathogenic Th1 responses may also help to consolidate these two apparently contradictory outcomes of exposure to the cholera-like enterotoxins. Furthermore, the observation that EtxB is able to alleviate autoimmune disease in the absence of conjugation to autoantigen highlights its potential for use in the clinical setting where the target antigen is often unknown. Direct effects on T cells, B cells and APC have been demonstrated in vitro which have provided insights into how these molecules may elicit these diverse effects. Further investigation is required for elucidation of the mechanisms of action of adjuvanticity and tolerance induction by these molecules to realise their potential for use in vaccines and therapies for autoimmune disease in humans.

  18. 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.

  19. Efficacy and safety of a patch vaccine containing heat-labile toxin from Escherichia coli against travellers' diarrhoea: a phase 3, randomised, double-blind, placebo-controlled field trial in travellers from Europe to Mexico and Guatemala.

    PubMed

    Behrens, Ronald H; Cramer, Jakob P; Jelinek, Tomas; Shaw, Hilary; von Sonnenburg, Frank; Wilbraham, Darren; Weinke, Thomas; Bell, David J; Asturias, Edwin; Pauwells, Hermann L Enkerlin; Maxwell, Roberto; Paredes-Paredes, Mercedes; Glenn, Gregory M; Dewasthaly, Shailesh; Stablein, Donald M; Jiang, Zhi-Dong; DuPont, Herbert L

    2014-03-01

    Enterotoxigenic Escherichia coli (ETEC) is a major cause of travellers' diarrhoea. We investigated the efficacy and safety of a skin-patch vaccine containing the pathogen's heat-labile toxin (LT) in a population of travellers to Mexico and Guatemala. In this phase 3, randomised, double-blind, placebo-controlled field trial, healthy adults (aged 18-64 years) travelling from Germany or the UK to Mexico or Guatemala were assigned in a 1:1 ratio by a dynamic electronic randomisation system to receive transcutaneous immunisation with a patch containing 37.5 μg of ETEC LT or a placebo patch. Participants, site staff, and the investigators who did the analyses were masked to group assignment. Participants were vaccinated before travel, with two patches given 14 days apart. In the destination country, participants tracked stool output in a diary and provided stool samples for pathogen identification if diarrhoea occurred. The primary endpoint was the proportion of participants with at least one episode of moderate-to-severe diarrhoea (defined as four or more unformed stools in a 24 h period) in which either or both ETEC enterotoxins (LT and heat-stable toxin [ST]) were detected. The study is registered at ClinicalTrials.gov, number NCT00993681. 2036 participants were recruited and randomly assigned between Oct 14, 2009, and Aug 13, 2010, with 1016 allocated to receive the LT patch and 1020 the placebo patch. 821 participants in the LT-patch group and 823 in the placebo group received both vaccinations and were analysed in the per-protocol population. 30 (3.7%, 95% CI 2.5-5.2) participants in the LT-patch group and 46 (5.6%, 4.1-7.4) in the placebo group had moderate or severe ETEC diarrhoea (vaccine efficacy 34.6%, -2.2 to 58.9; p=0.0621). 9333 local (ie, patch-site) adverse events (including erythema, rash, pruritus, hyperpigmentation, pain, hypopigmentation, and oedema) occurred in 943 (93%) of 1015 participants in the LT-patch group, compared with 1444 local adverse

  20. Development of a candidate stabilizing formulation for bulk storage of a double mutant heat labile toxin (dmLT) protein based adjuvant.

    PubMed

    Toprani, Vishal M; Sahni, Neha; Hickey, John M; Robertson, George A; Middaugh, C Russell; Joshi, Sangeeta B; Volkin, David B

    2017-10-04

    This work describes the formulation design and development of a novel protein based adjuvant, a double mutant of heat labile toxin (dmLT), based on knowledge of the protein's structural integrity and physicochemical degradation pathways. Various classes of pharmaceutical excipients were screened for their stabilizing effect on dmLT during exposure to thermal and agitation stresses as monitored by high throughput analytical assays for dmLT degradation. Sucrose, phosphate, sodium chloride, methionine and polysorbate-80 were identified as potential stabilizers that protected dmLT against either conformational destabilization, aggregation/particle formation or chemical degradation (e.g., Met oxidation and Lys glycation). Different combinations and concentrations of the selected stabilizers were then evaluated to further optimize dmLT stability while maintaining pharmaceutically acceptable ranges of solution pH and osmolality. The effect of multiple freeze-thaw (FT) cycles on the physical stability of candidate bulk formulations was also examined. Increasing the polysorbate-80 concentration to 0.1% in the lead candidate bulk formulation mitigated the loss of protein mass during FT. This formulation development study enabled the design of a new bulk formulation of the dmLT adjuvant and provides flexibility for future use in combination with a variety of different vaccine dosage forms with different antigens. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. 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.

  2. 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

  3. Influence of heat-labile serum components in the presence of OmpA on the outer membrane of Salmonella gallinarum.

    PubMed

    Vega-Manriquez, X; Huerta-Ascencio, L; Martínez-Gómez, D; López-Vidal, Y; Verdugo-Rodríguez, A

    2016-03-01

    Salmonella gallinarum is the causative agent of fowl typhoid. Being a Gram-negative bacteria, its outer membrane proteins (OMP) can be regulated by different microenvironments. S. gallinarum was cultured under the following conditions: nutrient broth (NB), NB supplemented with serum from specific pathogen-free birds (NBS) and NB with serum incubated at 56 °C prior to incubation with the bacteria (NBSD); OMP were subsequently extracted. Several changes were observed in the apparent expression of OMP, mainly a decrease in an OMP with a size of 30 kDa, approximately, under the NBS condition. In contrast, the same event was not observed in NB and NBSD when using one- and two-dimensional polyacrylamide gels (SDS-PAGE). Using the OMP with a size of 30 kDa, approximately, as antigen in indirect ELISA, we were able to differentiate serum from healthy and vaccinated birds, as well as birds infected with S. gallinarum and S. enteritidis. The amino-terminal of this protein was sequenced, showing 100 % identity with OmpA of S. typhimurium. Subsequently, we designed primers to amplify the gene by PCR. The partial sequence of the amplified gene showed 100 % identity with OmpA of S. gallinarum. (1) Heat-labile serum components influence the presence of OmpA in the OM of S. gallinarum; (2) by the way of ELISA, OmpA allows to specifically differentiate healthy from diseased birds.

  4. 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

  5. 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.

  6. Chicken serum albumin (Gal d 5*) is a partially heat-labile inhalant and food allergen implicated in the bird-egg syndrome.

    PubMed

    Quirce, S; Marañón, F; Umpiérrez, A; de las Heras, M; Fernández-Caldas, E; Sastre, J

    2001-08-01

    Chicken serum albumin (alpha-livetin) has been implicated as the causative allergen of the bird-egg syndrome. However, the clinical relevance of sensitization to this allergen has not been confirmed by specific challenge tests and environmental sampling. We investigated whether chicken albumin can be detected in air samples collected in a home with birds, and whether sensitization to this protein may cause respiratory and food allergy symptoms. The heat resistance of chicken albumin and the possible cross-reactivity with conalbumin were also investigated. We studied eight patients with food allergy to egg yolk who also suffered from respiratory symptoms (rhinitis and/or asthma) caused by exposure to birds. Sensitization to egg yolk and bird antigens was investigated by skin and serologic tests. Hypersensitivity to chicken albumin was confirmed by specific bronchial, conjunctival, and oral provocation tests. All patients had positive skin tests and serum IgE against egg yolk, chicken serum, chicken meat, bird feathers, and chicken albumin. The presence of airborne chicken albumin in the domestic environment was confirmed. Specific bronchial challenge to chicken albumin elicited early asthmatic responses in six patients with asthma. An oral challenge with chicken albumin provoked digestive and systemic allergic symptoms in the two patients challenged. IgE reactivity to chicken albumin was reduced by 88% after heating at 90 degrees C for 30 min. ELISA inhibition demonstrated only partial cross-reactivity between chicken albumin and conalbumin. Chicken albumin (Gal d 5) is a partially heat-labile allergen that may cause both respiratory and food-allergy symptoms in patients with the bird-egg syndrome.

  7. 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.

  8. 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

  9. Non-recombinant display of the B subunit of the heat labile toxin of Escherichia coli on wild type and mutant spores of Bacillus subtilis

    PubMed Central

    2013-01-01

    Background Mucosal infections are a major global health problem and it is generally accepted that mucosal vaccination strategies, able to block infection at their entry site, would be preferable with respect to other prevention approaches. However, there are still relatively few mucosal vaccines available, mainly because of the lack of efficient delivery systems and of mucosal adjuvants. Recombinant bacterial spores displaying a heterologous antigen have been shown to induce protective immune responses and, therefore, proposed as a mucosal delivery system. A non-recombinant approach has been recently developed and tested to display antigens and enzymes. Results We report that the binding subunit of the heat-labile toxin (LTB) of Escherichia coli efficiently adsorbed on the surface of Bacillus subtilis spores. When nasally administered to groups of mice, spore-adsorbed LTB was able to induce a specific immune response with the production of serum IgG, fecal sIgA and of IFN-γ in spleen and mesenteric lymph nodes (MLN) of the immunized animals. Dot blotting experiments showed that the non-recombinant approach was more efficient than the recombinant system in displaying LTB and that the efficiency of display could be further increased by using mutant spores with an altered surface. In addition, immunofluorescence microscopy experiments showed that only when displayed on the spore surface by the non-recombinant approach LTB was found in its native, pentameric form. Conclusion Our results indicate that non-recombinant spores displaying LTB pentamers can be administered by the nasal route to induce a Th1-biased, specific immune response. Mutant spores with an altered coat are more efficient than wild type spores in adsorbing the antigen, allowing the use of a reduced number of spores in immunization procedures. Efficiency of display, ability to display the native form of the antigen and to induce a specific immune response propose this non-recombinant delivery system as

  10. Non-recombinant display of the B subunit of the heat labile toxin of Escherichia coli on wild type and mutant spores of Bacillus subtilis.

    PubMed

    Isticato, Rachele; Sirec, Teja; Treppiccione, Lucia; Maurano, Francesco; De Felice, Maurilio; Rossi, Mauro; Ricca, Ezio

    2013-10-29

    Mucosal infections are a major global health problem and it is generally accepted that mucosal vaccination strategies, able to block infection at their entry site, would be preferable with respect to other prevention approaches. However, there are still relatively few mucosal vaccines available, mainly because of the lack of efficient delivery systems and of mucosal adjuvants. Recombinant bacterial spores displaying a heterologous antigen have been shown to induce protective immune responses and, therefore, proposed as a mucosal delivery system. A non-recombinant approach has been recently developed and tested to display antigens and enzymes. We report that the binding subunit of the heat-labile toxin (LTB) of Escherichia coli efficiently adsorbed on the surface of Bacillus subtilis spores. When nasally administered to groups of mice, spore-adsorbed LTB was able to induce a specific immune response with the production of serum IgG, fecal sIgA and of IFN-γ in spleen and mesenteric lymph nodes (MLN) of the immunized animals. Dot blotting experiments showed that the non-recombinant approach was more efficient than the recombinant system in displaying LTB and that the efficiency of display could be further increased by using mutant spores with an altered surface. In addition, immunofluorescence microscopy experiments showed that only when displayed on the spore surface by the non-recombinant approach LTB was found in its native, pentameric form. Our results indicate that non-recombinant spores displaying LTB pentamers can be administered by the nasal route to induce a Th1-biased, specific immune response. Mutant spores with an altered coat are more efficient than wild type spores in adsorbing the antigen, allowing the use of a reduced number of spores in immunization procedures. Efficiency of display, ability to display the native form of the antigen and to induce a specific immune response propose this non-recombinant delivery system as a powerful mucosal vaccine

  11. 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

  12. An Enterotoxin-like Binary Protein from Pseudomonas protegens with Potent Nematicidal Activity.

    PubMed

    Wei, Jun-Zhi; Siehl, Daniel L; Hou, Zhenglin; Rosen, Barbara; Oral, Jarred; Taylor, Christopher G; Wu, Gusui

    2017-07-21

    Soil microbes are a major food source for free-living soil nematodes. It is known that certain soil bacteria have evolved systems to combat predation. We identified the nematode-antagonistic Pseudomonas protegens strain 15G2 from screening of microbes. Through protein purification we identified a binary protein, designated as Pp-ANP, that is responsible for the nematicidal activity. This binary protein inhibits Caenorhabditis elegans growth and development by arresting larvae at L1 stage and killing older staged worms. The two subunits, Pp-ANP1a and Pp-ANP2a are active when reconstituted from separate expression in Escherichia coli The binary toxin also shows strong nematicidal activity against three other free-living nematodes, Pristionchus pacificus, Panagrellus redivivus, and Acrobeloides sp., but no activity was found for insects and fungi under test conditions, indicating specificity for nematodes. Pp-ANP1a has no significant homology to any known proteins, while Pp-ANP2a shows ∼30% homology to E. coli heat-labile enterotoxin (LT) subunit A and cholera toxin (CT) subunit A. Protein modeling indicates Pp-ANP2a is structurally similar to CT/LT and likely acts as a ADP-ribosyltransferase. Despite the similarity, Pp-ANP shows several characteristics distinct from CT/LT toxins. Our results indicate that Pp-ANP is a new enterotoxin-like binary toxin with potent and specific activity to nematodes. The potency and specificity of Pp-ANP suggests applications in controlling parasitic nematodes and opens an avenue for further research on its mechanism of action and role in bacterium-nematode interaction.IMPORTANCE This study reports the discovery of a new enterotoxin-like binary protein, Pp-ANP from a Pseudomonas protegens strain. Pp-ANP shows strong nematicidal activity against Caenorhabditis elegans larvae and older staged worms. It also shows strong activity on other free-living nematodes, Pristionchus pacificus, Panagrellus redivivus, and Acrobeloides sp. The two

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

    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

  20. 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

  1. 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

  2. Regulation of staphylococcal enterotoxin B.

    PubMed

    Morse, S A; Mah, R A; Dobrogosz, W J

    1969-04-01

    Several factors influenced the formation of enterotoxin B by Staphylococcus aureus strain S-6. In the standard casein hydrolysate medium, toxin was not produced in detectable quantities during exponential growth; it was produced during the post-exponential phase when total protein synthesis was arithmetic. The rate of toxin synthesis was much greater than the rate of total protein synthesis. The appearance of enterotoxin was inhibited by chloramphenicol; thus, the presence of toxin was dependent on de novo protein synthesis. When low concentrations of glucose (<0.30%) were added to the casein hydrolysate medium, growth was diauxic; glucose was completely metabolized during the first growth period. During the second growth period, enterotoxin was synthesized. In unbuffered casein hydrolysate medium containing excess glucose, toxin synthesis was completely repressed. The absence of toxin production under such conditions might be explained by the low (4.6) pH resulting from the acid end products of glucose metabolism. At pH <5.0, little or no toxin was produced. Toxin synthesis was initiated in the presence of glucose when the medium were buffered at any pH above 5.6. In such media, the differential rates of toxin synthesis, with respect to the rates of total protein synthesis, were lower than the differential rates in casein hydrolysate medium alone. Addition of glucose to a culture synthesizing toxin resulted in an immediate decrease in the differential rate without any change in pH. Thus, toxin synthesis appeared to be regulated by catabolite repression.

  3. 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.

  4. Staphylococcal Enterotoxin C: Solid-Phase Radioimmunoassay

    PubMed Central

    Bukovic, Joann A.; Johnson, Howard M.

    1975-01-01

    A solid-phase radioimmunoassay test employing 125I-labeled enterotoxin C and polystyrene tubes coated with specific antibody was used for the detection and quantitation of enterotoxin C in condensed milk, cheddar cheese, custard, and ham salad. The assay was sensitive to 1 to 10 ng of toxin per g of food; nonspecific inhibitions were 16% or less. PMID:1190765

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. [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.

  10. Bacterial Toxins-Staphylococcal Enterotoxin B.

    PubMed

    Fries, Bettina C; Varshney, Avanish K

    2013-12-01

    Staphylococcal enterotoxin B is one of the most potent bacterial superantigens that exerts profound toxic effects upon the immune system, leading to stimulation of cytokine release and inflammation. It is associated with food poisoning, nonmenstrual toxic shock, atopic dermatitis, asthma, and nasal polyps in humans. Currently, there is no treatment or vaccine available. Passive immunotherapy using monoclonal antibodies made in several different species has shown significant inhibition in in vitro studies and reduction in staphylococcal enterotoxin B-induced lethal shock in in vivo studies. This should encourage future endeavors to develop these antibodies as therapeutic reagents.

  11. Digestion of staphylococcal enterotoxin by Bacillus natto.

    PubMed

    Osawa, R; Matsumoto, K

    1997-05-01

    Cooked rice contaminated with staphylococcal enterotoxin A (SEA) was mixed with 'natto', a Bacillus natto fermented soybean food, and the mixture was incubated at 37 degrees C for 1 h. Reversed passive latex agglutination (RPLA) tests performed on the mixture revealed that the RPLA titer against SEA was significantly reduced after incubation. Subsequent analytical tests showed that the SEA protein molecule was fragmented to small peptides by an extracellular protease, subtilisin, produced by B. natto. The proteolytic activity of B. natto was also found to be effective against other types of staphylococcal enterotoxins.

  12. 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

  13. Staphylococcal enterotoxin and thermonuclease production during induced bovine mastitis and the clinical reaction of enterotoxin in udders.

    PubMed Central

    Niskanen, A; Koranen, L; Roine, K

    1978-01-01

    Enterotoxin A- and C-producing strains of Staphylococcus aureus and partially and extensively purified enterotoxin A were inoculated into the udder quarters of cows. In the course of experimentally induced mastitis caused by the inoculated S. aureus strain, enterotoxin C but not A was detected in the infected udder. Enterotoxin C was observed in mastitic milk samples at very low S. aureus population levels (10(2) to 10(3) colony-forming units per ml). The results suggest that either the synthesis of enterotoxin C is stimulated in vitro or that growth of S. aureus cells in udders was, in fact, higher than the colony-forming unit values indicated. Thermonuclease was shown to be excreted into mastitic milk at a slower rate than was enterotoxin. An inoculation of 1 microgram of enterotoxin A in autogenic milk returned to the udder caused clinical reactions (swelling, palpation sensitivity, and increase in the level of somatic cells) within 6 h. PMID:631886

  14. Rapid differentiation of enterotoxigenic Escherichia coli that produce heat-stable and heat-labile toxins by frequency-pulsed electron capture gas-liquid chromatography analysis of diarrheal stool specimens.

    PubMed Central

    Brooks, J B; Basta, M T; el Kholy, A M; Moss, C W

    1984-01-01

    Thirty-three stool specimens from infants in the village of Tamooh near Cairo, Egypt, were studied by frequency-pulsed electron capture gas-liquid chromatography (FPEC-GLC). In 13 of the diarrheal cases, the suspected causative agent isolated was Escherichia coli which produced heat-stable toxin (ST), and in 10 other cases E. coli that produced heat-labile toxin (LT) were isolated. Ten control stool samples, collected from infants from whom no pathogenic organisms were isolated, were analyzed at the same time. Comparisons also were made against healthy control stools from individuals in the United States who had been previously analyzed by FPEC-GLC (Brooks et al., J. Clin. Microbiol. 20:549-560, 1984). The stools were suspended in water and centrifuged, and the supernatant was extracted with organic solvents and derivatized to form electron-capturing derivatives of carboxylic acids, hydroxy acids, alcohols, and amines. Results from the study showed distinct differences among the FPEC-GLC profiles of E. coli ST-positive stools, of E. coli LT-positive stools, and of the control stool samples. An unidentified compound appearing in the ether-soluble hydroxy acid fraction from E. coli ST-positive stools was tentatively identified by mass spectrometry as 6-methoxy-2-hydroxyhexanoic acid. 6-Methoxy-2-hydroxyhexanoic acid was found in all stools that contained E. coli ST but was not present either in stools from which E. coli LT was isolated or in control samples. 6-Methoxy-2-hydroxyhexanoic acid may prove to be an important marker for use in the identification of E. coli ST. In addition to 6-methoxy-2-hydroxyhexanoic acid, the carboxylic acid, alcohol, and amine FPEC-GLC profiles obtained from stools were very different between these two organisms. The data indicate that FPEC-GLC analysis of diarrheal stool specimens might be a rapid way to distinguish diarrhea caused by E. coli ST, E. coli LT, Clostridium difficile, and rotavirus. PMID:6394617

  15. Ganglioside-binding specificities of E. coli enterotoxin LT-IIc: Importance of long-chain fatty acyl ceramide

    PubMed Central

    Berenson, Charles S; Nawar, Hesham F; Kruzel, Ragina L; Mandell, Lorrie M; Connell, Terry D

    2013-01-01

    Bacterial heat-labile (LT) enterotoxins signal through tightly regulated interactions with host cell gangliosides. LT-IIa and LT-IIb of Escherichia coli bind preferentially to gangliosides with a NeuAcα2-3Galβ1-3GalNAc terminus, with key distinctions in specificity. LT-IIc, a newly discovered E. coli LT, is comprised of an A polypeptide with high homology, and a B polypeptide with moderate homology, to LT-IIa and LT-IIb. LT-IIc is less cytotoxic than LT-IIa and LT-IIb. We theorized that LT-IIc–host cell interaction is regulated by specific structural attributes of immune cell ganglioside receptors and designed experiments to test this hypothesis. Overlay immunoblotting to a diverse array of neural and macrophage gangliosides indicated that LT-IIc bound to a restrictive range of gangliosides, each possessing a NeuAcα2-3Galβ1-3GalNAc with a requisite terminal sialic acid. LT-IIc did not bind to GM1a with short-chain fatty acyl ceramides. Affinity overlay immunoblots, constructed to a diverse array of known ganglioside structures of murine peritoneal macrophages, established that LT-IIc bound to GM1a comprised of long-chain fatty acyl ceramides. Findings were confirmed with LT-IIc also binding to GM1a of RAW264.7 cells, comprised of a long-chain fatty acyl ceramide. Thus, LT-IIc-ganglioside binding differs distinctly from that of LT-IIa and LT-IIb. LT-IIc binding is not just dependent on carbohydrate composition, but also upon the orientation of the oligosaccharide portion of GM1a by the ceramide moiety. These studies are the first demonstration of LT-ganglioside dependence upon ceramide composition and underscore the contribution of long-chain fatty acyl ceramides to host cell interactions. PMID:22917572

  16. Ganglioside-binding specificities of E. coli enterotoxin LT-IIc: Importance of long-chain fatty acyl ceramide.

    PubMed

    Berenson, Charles S; Nawar, Hesham F; Kruzel, Ragina L; Mandell, Lorrie M; Connell, Terry D

    2013-01-01

    Bacterial heat-labile (LT) enterotoxins signal through tightly regulated interactions with host cell gangliosides. LT-IIa and LT-IIb of Escherichia coli bind preferentially to gangliosides with a NeuAcα2-3Galβ1-3GalNAc terminus, with key distinctions in specificity. LT-IIc, a newly discovered E. coli LT, is comprised of an A polypeptide with high homology, and a B polypeptide with moderate homology, to LT-IIa and LT-IIb. LT-IIc is less cytotoxic than LT-IIa and LT-IIb. We theorized that LT-IIc-host cell interaction is regulated by specific structural attributes of immune cell ganglioside receptors and designed experiments to test this hypothesis. Overlay immunoblotting to a diverse array of neural and macrophage gangliosides indicated that LT-IIc bound to a restrictive range of gangliosides, each possessing a NeuAcα2-3Galβ1-3GalNAc with a requisite terminal sialic acid. LT-IIc did not bind to GM1a with short-chain fatty acyl ceramides. Affinity overlay immunoblots, constructed to a diverse array of known ganglioside structures of murine peritoneal macrophages, established that LT-IIc bound to GM1a comprised of long-chain fatty acyl ceramides. Findings were confirmed with LT-IIc also binding to GM1a of RAW264.7 cells, comprised of a long-chain fatty acyl ceramide. Thus, LT-IIc-ganglioside binding differs distinctly from that of LT-IIa and LT-IIb. LT-IIc binding is not just dependent on carbohydrate composition, but also upon the orientation of the oligosaccharide portion of GM1a by the ceramide moiety. These studies are the first demonstration of LT-ganglioside dependence upon ceramide composition and underscore the contribution of long-chain fatty acyl ceramides to host cell interactions.

  17. 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

  18. 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.

  19. 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

  20. Toward the development of a stable, freeze-dried formulation of Helicobacter pylori killed whole cell vaccine adjuvanted with a novel mutant of Escherichia coli heat-labile toxin.

    PubMed

    Summerton, Nancy A; Welch, Richard W; Bondoc, Laureano; Yang, Huei-Hsiung; Pleune, Brett; Ramachandran, Naryaswamy; Harris, Andrea M; Bland, Desiree; Jackson, W James; Park, Sukjoon; Clements, John D; Nabors, Gary S

    2010-02-03

    No vaccine exists for the prevention of infection with the ubiquitous gastric pathogen Helicobacter pylori, and drug therapy for the infection is complicated by poor patient compliance, the high cost of treatment, and ineffectiveness against drug-resistant strains. A new medical advancement is required to reduce the incidence of peptic ulcer disease and stomach cancer, two conditions caused by infection with H. pylori. Clinical trials have been performed with a formalin-inactivated H. pylori whole cell (HWC) vaccine, given orally in combination with the mucosal adjuvant mLT(R192G), a mutant of Escherichia coli heat-labile toxin. Following the initial dose of this vaccine, some subjects experienced gastrointestinal side effects. To reduce side effects and potentially further increase the amount of adjuvant that can safely be administered with the HWC vaccine, experiments were performed with a form of LT that carried two mutations in the A subunit, a substitution of G for R at position 192, and A for L at position 211. The double mutant LT (dmLT) adjuvant stimulated immune responses as effectively as the single mutant LT in mice. Additionally, following a challenge infection, the dmLT-adjuvanted vaccine was as effective as single mutant LT in reducing gastric urease levels (diagnostic for H. pylori infection), and H. pylori colonization in the stomach as assessed by quantitative analysis of stomach homogenates. A lyophilized formulation of HWC was developed to improve stability and to potentially reduce reliance on cold chain maintenance. It was observed that a dmLT-adjuvanted lyophilized vaccine was equally as protective in the mouse model as the liquid formulation as assessed by gastric urease analysis and analysis of stomach homogenates for viable H. pylori. No readily detectable effect of tonicity or moisture content was observed for the lyophilized vaccine within the formulation limits evaluated. In an accelerated stability study performed at 37 degrees C the

  1. 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.

  2. Chromatofocusing in the purification of staphylococcal enterotoxin D.

    PubMed Central

    Lei, Z; Reiser, R F; Bergdoll, M S

    1988-01-01

    A chromatofocusing procedure for the purification of staphylococcal enterotoxin D was developed. The purification included the removal of the toxic protein from culture supernatant fluids of Staphylococcus aureus 1151m by batch adsorption with CG-50 resin, chromatofocusing on Polybuffer Exchanger 94, and gel permeation chromatography on Sephacryl S-200. The purity of the staphylococcal enterotoxin D obtained was approximately 98%. PMID:3384936

  3. Chromatofocusing in the purification of staphylococcal enterotoxin D.

    PubMed

    Lei, Z; Reiser, R F; Bergdoll, M S

    1988-06-01

    A chromatofocusing procedure for the purification of staphylococcal enterotoxin D was developed. The purification included the removal of the toxic protein from culture supernatant fluids of Staphylococcus aureus 1151m by batch adsorption with CG-50 resin, chromatofocusing on Polybuffer Exchanger 94, and gel permeation chromatography on Sephacryl S-200. The purity of the staphylococcal enterotoxin D obtained was approximately 98%.

  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. 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

  6. 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

  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 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

  8. 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.

  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. 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

  11. Stability of the Encoding Plasmids and Surface Expression of CS6 Differs in Enterotoxigenic Escherichia coli (ETEC) Encoding Different Heat-Stable (ST) Enterotoxins (STh and STp)

    PubMed Central

    Tobias, Joshua; Von Mentzer, Astrid; Loayza Frykberg, Patricia; Aslett, Martin; Page, Andrew J.; Sjöling, Åsa; Svennerholm, Ann-Mari

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC), one of the most common reasons of diarrhea among infants and children in developing countries, causes disease by expression of either or both of the enterotoxins heat-labile (LT) and heat-stable (ST; divided into human-type [STh] and porcine-type [STp] variants), and colonization factors (CFs) among which CS6 is one of the most prevalent ETEC CFs. In this study we show that ETEC isolates expressing CS6+STh have higher copy numbers of the cssABCD operon encoding CS6 than those expressing CS6+STp. Long term cultivation of up to ten over-night passages of ETEC isolates harboring CS6+STh (n = 10) or CS6+STp (n = 15) showed instability of phenotypic expression of CS6 in a majority of the CS6+STp isolates, whereas most of the CS6+STh isolates retained CS6 expression. The observed instability was a correlated with loss of genes cssA and cssD as examined by PCR. Mobilization of the CS6 plasmid from an unstable CS6+STp isolate into a laboratory E. coli strain resulted in loss of the plasmid after a single over-night passage whereas the plasmid from an CS6+STh strain was retained in the laboratory strain during 10 passages. A sequence comparison between the CS6 plasmids from a stable and an unstable ETEC isolate revealed that genes necessary for plasmid stabilization, for example pemI, pemK, stbA, stbB and parM, were not present in the unstable ETEC isolate. Our results indicate that stable retention of CS6 may in part be affected by the stability of the plasmid on which both CS6 and STp or STh are located. PMID:27054573

  12. Stability of the Encoding Plasmids and Surface Expression of CS6 Differs in Enterotoxigenic Escherichia coli (ETEC) Encoding Different Heat-Stable (ST) Enterotoxins (STh and STp).

    PubMed

    Tobias, Joshua; Von Mentzer, Astrid; Loayza Frykberg, Patricia; Aslett, Martin; Page, Andrew J; Sjöling, Åsa; Svennerholm, Ann-Mari

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC), one of the most common reasons of diarrhea among infants and children in developing countries, causes disease by expression of either or both of the enterotoxins heat-labile (LT) and heat-stable (ST; divided into human-type [STh] and porcine-type [STp] variants), and colonization factors (CFs) among which CS6 is one of the most prevalent ETEC CFs. In this study we show that ETEC isolates expressing CS6+STh have higher copy numbers of the cssABCD operon encoding CS6 than those expressing CS6+STp. Long term cultivation of up to ten over-night passages of ETEC isolates harboring CS6+STh (n = 10) or CS6+STp (n = 15) showed instability of phenotypic expression of CS6 in a majority of the CS6+STp isolates, whereas most of the CS6+STh isolates retained CS6 expression. The observed instability was a correlated with loss of genes cssA and cssD as examined by PCR. Mobilization of the CS6 plasmid from an unstable CS6+STp isolate into a laboratory E. coli strain resulted in loss of the plasmid after a single over-night passage whereas the plasmid from an CS6+STh strain was retained in the laboratory strain during 10 passages. A sequence comparison between the CS6 plasmids from a stable and an unstable ETEC isolate revealed that genes necessary for plasmid stabilization, for example pemI, pemK, stbA, stbB and parM, were not present in the unstable ETEC isolate. Our results indicate that stable retention of CS6 may in part be affected by the stability of the plasmid on which both CS6 and STp or STh are located.

  13. 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

  14. Enterotoxin production by staphylococci isolated from foods in France.

    PubMed

    Rosec, J P; Guiraud, J P; Dalet, C; Richard, N

    1997-04-15

    Two hundred and thirteen Staphylococcus aureus and 51 other staphylococcal strains were isolated from 121 foodstuffs of current consumption and two cutaneous samples. Their ability to produce staphylococcal enterotoxins was tested and S. aureus strains were biotyped. The S. aureus strains (30.5%) produced at least one of the five known staphylococcal enterotoxins whereas coagulase negative staphylococci did not produce any of them. The raw milk cheeses analysed were primarily contaminated by strains belonging to animal or unspecified biovars. Only 15.9% of the S. aureus strains isolated from these products produced enterotoxins whereas 43% were found to be enterotoxigenic amongst the S. aureus strains isolated from the other foodstuffs. The scheme of biotyping used seems to be reliable, allowing the classification of 73.7% of the strains. S. aureus strains of human biovar origin were most often enterotoxigenic and enterotoxin C was the predominant type identified. It was produced by 66% of the enterotoxigenic strains, singly or in combination with other enterotoxins. Approximately 77% of the human strains also produced enterotoxin C, which is an amazing epidemiological distinctive feature of the strains studied. Moreover ELISA tests used in this work exhibit problems of specificity.

  15. A high molecular weight protein from Staphylococcus intermedius cross-reacts with Staphylococcus aureus enterotoxin antibodies.

    PubMed

    Laffan, J J; Petras, P; Ferguson, K P; Lambe, D W

    1996-01-01

    Enterotoxin production by Staphylococcus species other than Staphylococcus aureus has been reported. Staphylococcus strains (104 in toto) representing twelve species and subspecies were examined for enterotoxins using a commercial staphylococcal enterotoxin ELISA immunoassay (TECRA, International Bioproducts). Staphylococcus intermedius (24 strains) and S. aureus (7 strains) were positive with this test. Western blots of S. aureus exoproteins demonstrated proteins of approximately 30 kD, consistent with known staphylococcal enterotoxins. The major antigen in all S. intermedius strains, a 75 kD protein, was not analogous to previously described staphylococcal enterotoxins. This protein was unique to S. intermedius. Gel filtration data indicate that the protein is a subunit of a larger protein in vivo. The 75 kD protein cross-reacts with several enterotoxin antibodies. It is unclear whether the protein is a toxin, but its homology with S. aureus enterotoxins may indicate a shared toxic region, or this protein may create false positive results in screening for enterotoxin.

  16. 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

  17. Enterotoxin production by strains of Staphylococcus aureus isolated from foods and human beings

    PubMed Central

    Wieneke, Antonnette A.

    1974-01-01

    Enterotoxin production by strains of Staphylococcus aureus isolated from routine samples of foods and from human beings was investigated. Twenty-one to 26% of 112 strains isolated from raw meat, sausages and poultry and 32-36% of 183 strains isolated from cooked foods, e.g. meat, chicken and frozen seafoods, produced enterotoxins A, B, C, D or E. Staph. aureus isolated from raw meat and chicken less frequently produced enterotoxins A, B, C or E and more frequently enterotoxin D, than those from cooked meat and seafoods. Of the 113 strains isolated from cheese and raw milk 6-11% produced enterotoxin and most of these produced enterotoxin D. Only a few strains isolated from foods produced enterotoxin E. Results of enterotoxin tests on Staph. aureus from human beings resembled those on strains from cooked foods. PMID:4278965

  18. 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

  19. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  1. Staphylococcus aureus isolates from Irish domestic refrigerators possess novel enterotoxin and enterotoxin-like genes and are clonal in nature.

    PubMed

    Smyth, Davida S; Kennedy, Jean; Twohig, Jane; Miajlović, Helen; Bolton, Declan; Smyth, Cyril J

    2006-03-01

    A previous study carried out by the National Food Centre in Dublin on bacterial contamination of Irish domestic refrigeration systems revealed that 41% were contaminated with Staphylococcus aureus. One hundred fifty-seven S. aureus isolates were screened by multiplex PCR analysis for the presence of 15 staphylococcal enterotoxin and enterotoxin-like genes (sea-see, seg-sei, selj-selo, and selq) and the toxic shock toxin superantigen tst gene. Of the refrigerator isolates, 64.3% possessed more than one staphylococcal enterotoxin or staphylococcal enterotoxin-like gene. All bar one of the 101 staphylococcal enterotoxin or staphylococcal enterotoxin-like gene-positive strains possessed the egc locus bearing the seg, sei, selm, seln, and selo genes. Twelve random amplified polymorphic DNA (RAPD) types accounted for 119 (75.8%) of the strains, two of these types accounting for 25 (RAPD type 1, 15.9%) and 52 (RAPD type 5, 33.1%), respectively. All of the RAPD type 5 isolates possessed the egc gene cluster only. The RAPD type 5 amplicon profile was identical to that of S. aureus isolates associated with osteomyelitis in broiler chickens in Northern Ireland that also possessed the egc locus only. However, the RAPD type 5 domestic refrigerator and chicken isolates differed in penicillin G sensitivity, production of Protein A and staphylokinase, and crystal violet agar growth type. These findings highlight that the average Irish household refrigerator harbors potential enterotoxin-producing S. aureus that may or may not be of animal origin and, accordingly, is a potential reservoir for staphylococcal food poisoning.

  2. 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.

  3. Single radial immunodiffusion method for screening Staphylococcal isolates for enterotoxin.

    PubMed Central

    Meyer, R F; Palmieri, M J

    1980-01-01

    A direct system for screening large numbers of staphylococcal isolates for enterotoxin production has been developed. The system employs polyvalent (serotypes A, B, C, D, and E) immunodiffusion assay slides in conjunction with a multiple-culturing system for toxin production. With the combined system, as many as 50 cultures can be screened simultaneously on a single assay slide having a sensitivity of about 0.3 microgram/ml. The system should be useful for detecting potential enterotoxin in foods containing a predominance of non-enterotoxigenic strains. Images PMID:6779703

  4. Rapid Solid-Phase Radioimmunoassay for Staphylococcal Enterotoxin A

    PubMed Central

    Collins, William S.; Johnson, Anna D.; Metzger, Joseph F.; Bennett, Reginald W.

    1973-01-01

    A rapid solid-phase radioimmunoassay for staphylococcal enterotoxin A is described. The assay procedure requires 3 to 4 h for completion by using a competitive inhibition system in which the antibody is attached to bromacetyl cellulose particles. It is accurate to a level of 0.01 μg of enterotoxin A/ml in a variety of media such as ham, milk products, crab meat, custard, etc. No significant interference was found with any media or food product tested. PMID:4715556

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. Staphylococcal Enterotoxins A and B: Solid-Phase Radioimmunoassay in Food

    PubMed Central

    Johnson, H. M.; Bukovic, J. A.; Kauffmann, P. E.

    1973-01-01

    An immunoassay employing 125I labeled enterotoxins A and B and polystyrene tubes coated with specific antibodies was used for detection and quantitation of enterotoxin in food. Ham salad, cheddar cheese, custard, condensed milk, and salami were studied. Enterotoxin was successfully determined in all the foods by simple extraction procedures. The assay was sensitive to 1 to 10 ng of toxin per g of food; nonspecific inhibitions were 15% or less. PMID:4796162

  15. [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.

  16. Immunofluorescent Detection of Enterotoxin B in Food and a Culture Medium1

    PubMed Central

    Stark, Raymond L.; Middaugh, P. R.

    1969-01-01

    Both Staphylococcus aureus strains 243 and S-6 cells producing enterotoxin B and free enterotoxin in food and culture medium were rapidly demonstrated by using the fluorescent-antibody technique. Comparison of cell fluorescence and enterotoxin B production determined by double gel diffusion showed that an estimation of enterotoxin production could be made by observing the degree of cell fluorescence. The fluorescent-antibody technique was used to determine whether cells were producing enterotoxin under varying nutritional and environmental conditions: NaCl concentration, culture aeration, and time and temperature of incubation in Brain Heart Infusion broth and shrimp slurries. At the various NaCl concentrations, the fluorescence of cells was found positively associated with enterotoxin B production only during the first 12 hr of growth. As the NaCl concentration was increased from 0 to 10%, the fluorescence of cells and toxin production decreased. Maximum for cell fluorescence and enterotoxin production was observed at 37 C. Little or no difference in cell fluorescence and enterotoxin production with both strains was found between Brain Heart Infusion broth and shrimp slurry cultures. All results obtained with the fluorescent-antibody technique were verified with double gel diffusion for enterotoxin detection and quantitation. PMID:4983957

  17. 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

  18. 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.

  19. Therapeutic Human Hyperimmune Polyclonal Antibodies Against Staphylococcal Enterotoxin B

    DTIC Science & Technology

    2008-05-01

    and specific human anti-SEB antibodies purified from IVIG was used as a surrogate for hyperimmune globulin . It was demonstrated that human polyclonal...AD_________________ Award Number: W81XWH-08-C-0004 TITLE: Therapeutic Human Hyperimmune Polyclonal Antibodies against Staphylococcal...Human Hyperimmune Polyclonal Antibodies against Staphylococcal Enterotoxin B 5b. GRANT NUMBER W81XWH-08-C-0004 5c. PROGRAM ELEMENT NUMBER 6

  20. 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.

  1. 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.

  2. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  4. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  7. Chromatofocusing: a new method for purification of staphylococcal enterotoxins B and C1.

    PubMed Central

    Ende, I A; Terplan, G; Kickhöfen, B; Hammer, D K

    1983-01-01

    A new chromatographic procedure was developed which obtained highly purified preparations of staphylococcal enterotoxins B and C1 in yields of 60% from cultures of Staphylococcus aureus and which is faster than any of the separation methods used previously. The procedure involves chromatography on carboxymethylcellulose, removal of alpha-toxin by adsorption to rabbit erythrocyte membranes, and finally, chromatofocusing as the fundamental new step. Enterotoxins were obtained in highly purified form and behaved in a homogeneous manner as determined by ultracentrifugation and electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, with molecular weights of 34,000 for staphylococcal enterotoxin B and 30,000 for staphylococcal enterotoxin C1. Using chromatofocusing as the final purification step, we isolated three B and six C1 distinct but immunologically identical enterotoxin fractions, which were found to be devoid of any impurities and to possess a marked degree of toxicity in monkeys. Images PMID:6660872

  8. Chromatofocusing: a new method for purification of staphylococcal enterotoxins B and C1.

    PubMed

    Ende, I A; Terplan, G; Kickhöfen, B; Hammer, D K

    1983-12-01

    A new chromatographic procedure was developed which obtained highly purified preparations of staphylococcal enterotoxins B and C1 in yields of 60% from cultures of Staphylococcus aureus and which is faster than any of the separation methods used previously. The procedure involves chromatography on carboxymethylcellulose, removal of alpha-toxin by adsorption to rabbit erythrocyte membranes, and finally, chromatofocusing as the fundamental new step. Enterotoxins were obtained in highly purified form and behaved in a homogeneous manner as determined by ultracentrifugation and electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, with molecular weights of 34,000 for staphylococcal enterotoxin B and 30,000 for staphylococcal enterotoxin C1. Using chromatofocusing as the final purification step, we isolated three B and six C1 distinct but immunologically identical enterotoxin fractions, which were found to be devoid of any impurities and to possess a marked degree of toxicity in monkeys.

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. Regulation of the cytotoxic enterotoxin gene in Aeromonas hydrophila: characterization of an iron uptake regulator.

    PubMed

    Sha, J; Lu, M; Chopra, A K

    2001-10-01

    The cytotoxic enterotoxin Act from a diarrheal isolate, SSU, of Aeromonas hydrophila is aerolysin related and crucial to the pathogenesis of Aeromonas infections. To elucidate the role of environmental signals which influence the expression of the cytotoxic enterotoxin gene (act), a portion of the act gene, including the putative promoter region, was fused in frame to a truncated alkaline phosphatase gene (phoA) of Escherichia coli. The act::phoA reporter gene was then introduced into the chromosome of A. hydrophila by using the suicide vector pJQ200SK, allowing the fusion protein to be secreted out into the culture medium. Western blot analysis demonstrated the presence of a correctly size 110-kDa fusion protein in the culture supernatant, which reacted with both anti-Act and anti-alkaline phosphatase antibodies. Based on alkaline phosphatase (PhoA) activity in the culture supernatant, we demonstrated that calcium significantly increased the activity of the act promoter but that glucose and iron repressed its activity in a dose-dependent fashion. The act promoter exhibited optimal activity at pH 7.0 and at 37 degrees C, and maximal PhoA activity was noted when the culture was aerated. Using a Vibrio cholerae iron uptake regulator gene (fur) as a probe, a 2.6-kb SalI/HindIII DNA fragment from an A. hydrophila chromosome was cloned and sequenced. The DNA sequence revealed a 429-bp open reading frame that exhibited 69% homology at the DNA level with the fur gene and 79% homology at the amino acid level with the iron uptake regulator (Fur) protein of V. cholerae. Complementation experiments demonstrated that the A. hydrophila fur gene could restore iron regulation in an E. coli fur-minus mutant. Using the suicide vector pDMS197, we generated a fur isogenic mutant of wild-type A. hydrophila SSU. Northern blot analysis data indicated that the repression in the transcription of the act gene by iron was relieved in the fur isogenic mutant. Further, iron regulation in the

  14. 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.

  15. Novel Platform for the Detection of Staphylococcus aureus Enterotoxin B in Foods

    PubMed Central

    DeGrasse, Jeffrey A.; Wang, Ningyan; Mattis, Daiva M.; Kranz, David M.

    2013-01-01

    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. PMID:23241982

  16. 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

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

    PubMed Central

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

    2016-01-01

    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. PMID:26999202

  18. 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.

  19. beta Structure of aqueous staphylococcal enterotoxin B by spectropolarimetry and sequence-based conformational predictions.

    PubMed

    Muñoz, P A; Warren, J R; Noelken, M E

    1976-10-19

    Conformations of the globular protein staphylococcal enterotoxin B have been examined experimentally by ultraviolet circular dichroism (CD) and visible optical rotatory dispersion (ORD). Chen-Yang-Chau analysis (Chen, Y.-H., Yang, J.T., and Chau, K. H. (1974), Biochemistry 13, 3350) of the far-ultraviolet CD spectrum of native enterotoxin B revealed (assuming an average helix length of 11 residues) 9% alpha helix, 38% beta structure, and 53% random coil. A fourfold increase in alpha-helix was observed for enterotoxin exposed to 0.2% sodium dodecyl sulfate, behavior typical for globular proteins of low helical content. Values of -40 to -50 for the Moffitt-Yang parameter b0 calculated from visible ORD suggested 6-13% alpha helix in native enterotoxin. Application of a new predictive model (Chou, P. Y., and Fasman, G. D. (1974), Biochemistry 13,222) to the amino acid sequence of enterotoxin B indicated 11% alpha helix, 34% beta structure, and 55% coil in native enterotoxin. The excellent agreement for the amount of alpha and beta conformation utilizing different optical and predictive methods indicates beta structure as the dominant secondary structure in native enterotoxin B. Most of the beta structure is predicted by Chou-Fasman analysis to reside in two large regions of antiparallel beta sheet involving residues 81-148 and residues 184-217. Such highly cooperative regions of anti-parallel beta sheet account for the slow unfolding of enterotoxin B in concentrated guanidine hydrochloride and rapid folding of guanidine hydrochloride denatured enterotoxin B to native conformation(s) (Warren, J.R., Spero, L., and Metzger, J. F. (1974), Biochemistry 13, 1678). A more than twofold increase in alpha-helix content with a small diminution in beta structure was detected by CD and ORD upon acidification of aqueous enterotoxin to pH 2.5. Thus, the beta structure of enterotoxin B appears to resist isothermal denaturation and constitutes a stable interior core of structure in the

  20. 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.

  1. 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.

  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. 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.

  4. Serological detection of enterotoxin in foods implicated in staphylococcal food poisoning

    PubMed Central

    Gilbert, R. J.; Wieneke, Antonnette A.; Lanser, Janice; Šimkovičová, Magda

    1972-01-01

    Two methods are described for the extraction of enterotoxin from foods incriminated in incidents of staphylococcal food poisoning. Enterotoxin was detected serologically in 12 of 24 food samples from 20 separate incidents: eight samples contained enterotoxin A, three contained D and one both A and B. The amount of enterotoxin in nine foods, based on 100% recovery, varied from 0·02 to 0·09 μg./g. Data are also given on the numbers of Staphylococcus aureus isolated from samples of food from 39 food poisoning incidents. Colony counts varied between 7·5 × 105 and 9 × 109/g. with a median value of 7 × 107/g. PMID:4630605

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. Effect of Sodium Chloride and pH on Enterotoxin C Production

    PubMed Central

    Genigeorgis, Constantin; Foda, Mohamed S.; Mantis, Antony; Sadler, Walter W.

    1971-01-01

    Growth and production of enterotoxin C by Staphylococcus aureus strain 137 in 3% + 3% protein hydrolysate powder N-Z Amine NAK broths with 0 to 12% NaCl and an initial pH of 4.00 to 9.83 were studied during an 8-day incubation period at 37 C. Growth was initiated at pH values as low as 4.00 and as high as 9.83 at 0% salt level as long as the inoculum contained at least 108 cells per ml. Rate of growth decreased as the NaCl concentration was increased gradually to 12%. Enterotoxin C was produced in broths inoculated with 108 cells per ml and above and having initial pH ranges of 4.00 to 9.83, 4.40 to 9.43, 4.50 to 8.55 and respective NaCl concentrations of 0, 4, and 8%. In the presence of 10% NaCl, the pH range supporting enterotoxin C production was 5.45 to 7.30 for an inoculum level of 108 cells per ml and 6.38 to 7.30 for 3.6 × 106 cells per ml. In repeated experiments in which the inoculum contained 108 cells per ml, we failed to demonstrate enterotoxin C production in broths with 12% NaCl and a pH range of 4.50 to 8.55 and concentrated up to 14 times. The effect of NaCl on enterotoxin C production followed the same pattern as its effect on enterotoxin B production. As the concentration of NaCl increased from 0 to 10%, yields of enterotoxin B and C decreased to undetectable amounts. PMID:5574320

  12. 75 FR 39667 - Availability for Non-Exclusive or Partially Exclusive Licensing of a U.S. Patent Application

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-12

    ... CFAE) and the B Subunit of Heat- Labile Enterotoxin (LTB) From Enterotoxigenic E. Coli,'' filed May 19... vector constructs which effectively express the cfaB, cfaE and LTB proteins in Shigella spp. without...

  13. 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

  14. 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.

  15. 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.

  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. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  19. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  1. The interaction of Clostridium perfringens enterotoxin with receptor claudins

    PubMed Central

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

    2016-01-01

    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. PMID:27090847

  2. 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.

  3. 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

  4. Sub-inhibitory stress with essential oil affects enterotoxins production and essential oil susceptibility in Staphylococcus aureus.

    PubMed

    Turchi, Barbara; Mancini, Simone; Pistelli, Luisa; Najar, Basma; Cerri, Domenico; Fratini, Filippo

    2017-06-08

    Fourteen wild strains of Staphylococcus aureus positive for gene sea were tested for enterotoxins production and the minimum inhibitory concentration of Leptospermum scoparium, Origanum majorana, Origanum vulgare, Satureja montana and Thymus vulgaris essential oils (EOs) were determined. After this trial, bacteria stressed with sub-inhibitory concentration of each EO were tested for enterotoxins production by an immunoenzymatic assay and resistance to the same EO. Oregano oil exhibited the highest antibacterial activity followed by manuka and thyme oils. After the exposure to a sub-inhibitory concentration of EOs, strains displayed an increased sensitivity in more than 95% of the cases. After treatment with oregano and marjoram EOs, few strains showed a modified enterotoxins production, while 43% of the strains were no longer able to produce enterotoxins after treatment with manuka EO. The results obtained in this study highlight that exposure to sub-inhibitory concentration of EO modifies strains enterotoxins production and EOs susceptibility profile.

  5. [Influence of various pectins on production of staphylococcal enterotoxins types A and B].

    PubMed

    Fluer, F S; Men'shikov, D D; Lazareva, E B; Prokhorov, V Ia; Vesnin, A V

    2007-01-01

    Experimental in vitro study of influence of 2% solution of pectins (red beet, apple, citrus, manufactured by "Vitaline" company, citrus high- and low-etherified pectins, manufactured by "Hercules" company, Unipectine OB 700, and biologically active supplement "Pecto") on growth of staphylococci and production by them of type A and B enterotoxins was performed. It was shown that red beet, citrus high- and low-etherified pectins, as well as biologically active supplement "Pecto" render bactericidal effect on staphylococci and inhibit synthesis of types A and B staphylococcal enterotoxins. Citrus pectin "Vitaline" and Unipectine OB 700 don't have such influence. The most effective pectins, which were able to inhibit synthesis of types A and B staphylococcal enterotoxins, were red beet, apple, and citrus low-etherified pectins as well as biologically active supplement "Pecto".

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. Role of local allergic inflammation and Staphylococcus aureus enterotoxins in Chinese patients with chronic rhinosinusitis with nasal polyps.

    PubMed

    Cheng, K-J; Xu, Y-Y; Zhou, M-L; Zhou, S-H; Wang, S-Q

    2017-08-01

    To investigate the role of local allergic inflammation and Staphylococcus aureus enterotoxins in chronic rhinosinusitis with nasal polyps. This study included 36 patients with chronic rhinosinusitis with nasal polyps and 18 controls. Total immunoglobulin E, eosinophil cationic protein, staphylococcal enterotoxin types A and B specific immunoglobulin E, staphylococcal enterotoxin types A and B, and myeloperoxidase levels were determined. Four patients with chronic rhinosinusitis with nasal polyps had a local allergy. All chronic rhinosinusitis with nasal polyps patients tested negative for staphylococcal enterotoxin types A and B specific immunoglobulin E. The chronic rhinosinusitis with nasal polyps group had significantly elevated staphylococcal enterotoxin types A and B levels in the supernatant. Fourteen patients belonged to the eosinophilic chronic rhinosinusitis with nasal polyps group and the others were characterised as having non-eosinophilic chronic rhinosinusitis with nasal polyps. Local allergy may play a role in chronic rhinosinusitis with nasal polyps, independent of staphylococcal enterotoxin superantigens. Staphylococcal enterotoxins may be important in the pathogenesis of chronic rhinosinusitis with nasal polyps; however, their roles as superantigens were not confirmed in this study. In Chinese subjects, chronic rhinosinusitis with nasal polyps usually manifests as a neutrophilic inflammation.

  20. 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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. 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.

  2. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  4. 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

  5. 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

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

    USDA-ARS?s Scientific Manuscript database

    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. 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.

  8. 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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  10. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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. 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

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

    USDA-ARS?s Scientific Manuscript database

    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, ...

  14. 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.

  15. 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...

  16. IL-2 secretion by T cells for detection of biologically active Staphylococcal enterotoxin type E

    USDA-ARS?s Scientific Manuscript database

    Staphylococcus aureus is a significant worldwide source of clinical infections and foodborne illnesses acting through the synthesis of a group of enterotoxins (SEs) which cause gastroenteritis and also function as superantigens that activate T cells resulting in massive cytokine production yielding ...

  17. 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

  18. 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.

  19. 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

  20. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  2. 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.

  3. 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.

  4. 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

  5. Staphylococcus aureus-derived enterotoxins enhance house dust mite-induced patch test reactions in atopic dermatitis.

    PubMed

    Langer, Katja; Breuer, Kristine; Kapp, Alexander; Werfel, Thomas

    2007-02-01

    Up to 65% of Staphylococcus aureus strains isolated from the skin of patients with atopic dermatitis (AD) produce exotoxins with superantigenic properties that may also act as allergens leading to an induction of exotoxin-specific IgE antibodies. Staphylococcal enterotoxin B (SEB) applied epicutaneously in a concentration of 10 micro g/cm(2), i.e. 200 micro g/ml, under occlusion induces cutaneous inflammation in patients with AD and healthy individuals. We performed patch tests in 32 adult patients with AD using different concentrations (i.e. 2-200 micro g/ml) of SEA, SEB and house dust mite (HDM) extract (500 micro g/ml). Furthermore, the respective enterotoxins and HDM extract were applied simultaneously to the same patch test site. Specific IgE levels to SEA, SEB and HDM were measured with the CAP FEIA. The rates of patch test reactions to SEA and SEB increased with rising enterotoxin concentrations. There were no differences in the rates of patch test reactions to SEA and SEB between patients sensitized to the corresponding enterotoxin and non-IgE-sensitized patients. The number of patch test reactions to the mixture of enterotoxin and HDM extract was higher than the number of patch test reactions to either the enterotoxins or HDM extract. We identified 11 patients with AD who reacted neither to the enterotoxins nor to HDM extract, but who showed patch test reactions to the mixture. These reactions were not predicted by the presence of enterotoxin- or HDM-specific IgE. Colonization with exotoxin-producing S. aureus may influence the outcome of patch tests in patients with AD.

  6. [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.

  7. Production of Diarrheal Enterotoxins and Other Potential Virulence Factors by Veterinary Isolates of Bacillus Species Associated with Nongastrointestinal Infections

    PubMed Central

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

    2003-01-01

    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. PMID:12676723

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. Clostridium and bacillus binary enterotoxins: bad for the bowels, and eukaryotic being.

    PubMed

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

    2014-09-05

    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.

  16. Modification of Lethality Induced by Staphylococcal Enterotoxin B in Dutch Rabbits

    DTIC Science & Technology

    1980-03-01

    1016, 1965. Cardiovascular and hepatic responses of rhesus coccal enterotoxin B. J Infect Dis 117:273-284, 30. Liu CT, Overman RR: Effect of toxic...Grogan E: Hemodynamic changes in response to staph - Biochim Biophys Acta 322:251-257, 1973. EW, et at: The influence of specific antibody on... Infect Dis 135:646-648, 1977. functions and survival in x-irradiated rhesus ver in infectious disease. Biomedicine 28:305- 8. Beisel WR: Pathophysiology

  17. 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

  18. (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

  19. 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.

  20. Stress-caused Anergy of Leukocytes towards Staphylococcal enterotoxin B and Exposure Transcriptome Signatures

    DTIC Science & Technology

    2015-05-28

    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...algorithms on our and four other ( Gene Expression Omnibus) data sets. Cell adhesion, coagulation, hypoxia and vascular endothelial growth factor-mediated

  1. 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

  2. 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.

  3. 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.

  4. Amendments to the theory underlying Ussing chamber data of chloride ion secretion after bacterial enterotoxin exposure.

    PubMed

    Lucas, M L

    2005-05-07

    Bacterial enterotoxins may cause life-threatening diarrhoeal fluid loss in part because they stimulate enterocytes to secrete fluid into the small intestine as well as preventing normal fluid uptake. Abnormal chloride ion secretion is believed to provide the osmotic driving force for the inappropriate fluid movement. Evidence for enhanced chloride secretion consists of isotopic flux measurements in Ussing chambers, the standard apparatus for permeation studies. Flux from the lumen of the intestine is assumed to be determined solely by absorptive processes and flux towards the lumen solely by secretory processes. Bacterial enterotoxin increased flux towards the lumen is taken as an evidence of enhanced secretion. Examination of the flux equation solutions shows that the existing theoretical treatment of the Ussing chamber consists of the super-imposition of two contradictory unidirectional models. In contrast, the present analysis shows that a measured 'unidirectional' flux contains information both about absorptive and secretory processes, regardless of which flux is measured. Reciprocity is predicted for the fluxes, as decreases in the absorptive processes will cause increases in apparent secretory flux. Data from the literature show that mucosal-to-serosal chloride ion flux in rabbit ileum after exposure to secretagogues correlates inversely and highly significantly (r=0.74, n=17, p<0.001) with increases in serosal-to-mucosal chloride ion flux. As a category of evidence, flux data do not provide conclusive evidence of enhanced chloride secretion after exposure to enterotoxins, since an apparently enhanced serosal-to-mucosal flux would also be noted after inhibition of the mucosal-to-serosal flux. As interruption of absorptive processes can be misinterpreted as enhanced secretion in the Ussing chamber, this is a serious deficiency in the evidence for direct enterotoxin enhancement of the intestinal chloride ion channel as a basis for diarrhoeal disease.

  5. Staphylococcal Enterotoxin A Detection from Rheumatoid Arthritis Patients' Blood and Synovial Fluid.

    PubMed

    Ataee, Ramezan Ali; Kahani, Mahboobeh Sadat; Alishiri, Gholam Hossein; Ahamadi, Zyenab

    2016-02-01

    Direct detection of microbial super antigens in synovial fluid of patients with rheumatoid arthritis may be able to guide to the design of cost-effective therapies. The purpose of this study was to assess the existence of Staphylococcal enterotoxin A (superantigen A) in the synovial fluid of patients with RA by the PCR and ELISA methods. This experimental study was conducted on the synovial fluid of 103 RA patients from Baqiyatallah University of Medical Sciences' Rheumatology Clinic in Tehran, Iran in 2011-2014. Bacterial cultures, polymerase chain reaction with specific primer pairs and enzyme-linked immunosorbent assay (ELISA) methods were used. The PCR products were subjected to sequence as a confirmatory molecular method results. The data were descriptively analyzed by SPSS Version 19. The bacteriological study result indicated that, in four cases (3.8%) of the patients, bacterial strains were isolated. The result of PCR molecular method for staphylococcal enterotoxin A gene showed that, 42 of the patients (40.7%) tested positive for the ent A gene. The results of ELISA were positive for staphylococcal enterotoxin A (superantigen A) in 51 cases (49.51%) of the patients' synovial fluids. The results indicated that the possibility of detecting superantigen A in the SF of RA patients, but the origin of the enterotoxin A gene remained unknown. The findings of this study may be able to alter the actual theory on the pathogenesis, diagnosis, and treatment of RA patients. In addition, the results have shown the probability of an endogenous origin for the involved superantigen A in RA patients' synovial fluids.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. Staphylococcal Enterotoxin H Induced Apoptosis of Bovine Mammary Epithelial Cells in Vitro

    PubMed Central

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

    2014-01-01

    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. PMID:25533519

  11. 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

  12. 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.

  13. 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.

  14. Relationship of sporulation, enterotoxin formation, and spoilage during growth of Clostridium perfringens type A in cooked chicken.

    PubMed Central

    Craven, S E; Blankenship, L C; McDonel, J L

    1981-01-01

    Sporulation and enterotoxin formation were determined for 17 strains of Clostridium perfringens type A in autoclaved chicken dark meat and in Duncan-Strong sporulation medium. The mean numbers of heat-resistant spores detected after 24 h at 37 degrees C were log10 1.13 to log10 7.64/ml in Duncan-Strong medium and log10 4.93 to log10 6.59/g in chicken. Of 17 strains, 7 formed enterotoxin in Duncan-Strong culture supernatant (1.0 to 60 microgram/ml) and 8 produced enterotoxin in chicken (0.21 to 24 microgram/g). Additional studies with chicken were conducted with C. perfringens NCTC 8239. With an inoculum of 10(6) cells per g, greater than log10 7.99 vegetative cells per g were detected by 4 h in chicken at 37 degrees C. Heat-resistant spores occurred by 4 and 6 h and enterotoxin occurred by 8 and 6 h in autoclaved chicken dark meat and barbecued chicken drumsticks, respectively. Enterotoxin was detected in autoclaved dark meat after incubation at 45 degrees C for 1.5 h followed by 37 degrees C for 4.5 h, but not after incubation at 45 degrees C for 1.5 to 8 h. With an inoculum of 10(2) cells per g in oven-cooked or autoclaved chicken, greater than log10 8.00 vegetative cells per g were detected by 6 to 8 h at 37 degrees C, heat-resistant spores were detected by 8 h, and enterotoxin was detected by 12 h. A statistical analysis of odor determinants of chicken after growth of C. perfringens indicated that, at the 95% confidence level, the product was considered spoiled (off or unwholesome odor) by the time spores or enterotoxin were formed. PMID:6266336

  15. Influence of elevated temperature on starch hydrolysis by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens type A.

    PubMed Central

    García-Alvarado, J S; Rodriguez, M A; Labbé, R G

    1992-01-01

    Enterotoxin-positive (Ent+) and enterotoxin-negative (Ent-) strains of Clostridium perfringens were cultured in Duncan-Strong sporulation medium containing starch at 37 and 46 degrees C. At 37 degrees C, all strains degraded starch and sporulated well. However, only Ent- strains could hydrolyze starch, grow extensively, and sporulate at 46 degrees C. Growth, sporulation, and starch hydrolysis by Ent+ strains at 46 degrees C were equivalent to those obtained at 37 degrees C when alpha-amylase was added to the cultures during growth. The total amount of extracellular plus intracellular amylase in cultures of Ent+ strains was significantly less in cells incubated at 46 degrees C than in cells incubated at 37 degrees C. These results contradict an earlier report that Ent+ strains cannot sporulate well near their optimal growth temperature (R. G. Labbe and C. L. Duncan, Can. J. Microbiol. 20:1493-1501, 1974) and suggest that synthesis of alpha-amylase in Ent+ strains is regulated by temperature. Images PMID:1371660

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. Exposure to inorganic mercury in vivo attenuates extrinsic apoptotic signaling in Staphylococcal aureus enterotoxin B stimulated T-cells

    SciTech Connect

    Laiosa, Michael D.; Eckles, Kevin G.; Langdon, Margaret; Rosenspire, Allen J.; McCabe, Michael J.

    2007-12-15

    The heavy metal mercury (Hg) is known to have immunomodulatory properties affecting lymphocyte signal transduction, death receptor signaling and autoimmunity. In this study we tested the hypothesis that Hg exposure would attenuate T-cell activation and caspase 8 and 3 activity in response to antigenic stimuli. To test this hypothesis, BALB/cJ mice were exposed to 10 mg/l mercuric chloride (HgCl{sub 2}) in their drinking water for 2 weeks followed by injection with 20 {mu}g of the Staphylococcal aureus enterotoxin B (SEB) superantigen. Eighteen hours after SEB challenge, there was a statistically significant reduction in caspase 8 and caspase 3 enzyme activity in the SEB reactive V{beta}8+ T-cells. The attenuated caspase activity in Hg-exposed mice persisted for 48 h after exposure. Moreover, activation of caspase 8 and caspase 3 was reduced by more than 60% in CD95 deficient MRL/MpJ-Fas{sup lpr} mice demonstrating that caspase 8 and 3 activation in response to SEB is CD95 dependent. In addition to the effects of Hg on caspase activity, expression of the T-cell activation marker CD69 was also attenuated in SEB reactive V{beta}8 T-cells in Hg-exposed mice. Moreover, CD69 expression in MRL/MpJ-Fas{sup lpr} mice was also reduced. Taken together the caspase and CD69 data support a role for CD95 in promoting a proapoptotic and activated state in SEB responsive T-lymphocytes and this state is attenuated by the autoimmune potentiating environmental agent mercury.

  4. Staphylococcal enterotoxin A (SEA) stimulates STAT3 activation and IL-17 expression in cutaneous T-cell lymphoma

    PubMed Central

    Willerslev-Olsen, Andreas; Krejsgaard, Thorbjørn; Lindahl, Lise M.; Litvinov, Ivan V.; Fredholm, Simon; Petersen, David L.; Nastasi, Claudia; Gniadecki, Robert; Mongan, Nigel P.; Sasseville, Denis; Wasik, Mariusz A.; Bonefeld, Charlotte M.; Geisler, Carsten; Woetmann, Anders; Iversen, Lars; Kilian, Mogens; Koralov, Sergei B.

    2016-01-01

    Cutaneous T-cell lymphoma (CTCL) is characterized by proliferation of malignant T cells in a chronic inflammatory environment. With disease progression, bacteria colonize the compromised skin barrier and half of CTCL patients die of infection rather than from direct organ involvement by the malignancy. Clinical data indicate that bacteria play a direct role in disease progression, but little is known about the mechanisms involved. Here, we demonstrate that bacterial isolates containing staphylococcal enterotoxin A (SEA) from the affected skin of CTCL patients, as well as recombinant SEA, stimulate activation of signal transducer and activator of transcription 3 (STAT3) and upregulation of interleukin (IL)-17 in immortalized and primary patient–derived malignant and nonmalignant T cells. Importantly, SEA induces STAT3 activation and IL-17 expression in malignant T cells when cocultured with nonmalignant T cells, indicating an indirect mode of action. In accordance, malignant T cells expressing an SEA-nonresponsive T-cell receptor variable region β chain are nonresponsive to SEA in monoculture but display strong STAT3 activation and IL-17 expression in cocultures with SEA-responsive nonmalignant T cells. The response is induced via IL-2 receptor common γ chain cytokines and a Janus kinase 3 (JAK3)-dependent pathway in malignant T cells, and blocked by tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross talk–dependent activation of STAT3 and expression of IL-17 in malignant T cells, suggesting a mechanism whereby SEA-producing bacteria promote activation of an established oncogenic pathway previously implicated in carcinogenesis. PMID:26738536

  5. 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.

  6. 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. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  7. Integrins α1β1 and α2β1 are receptors for the rotavirus enterotoxin

    PubMed Central

    Seo, Neung-Seon; Zeng, Carl Q.-Y.; Hyser, Joseph M.; Utama, Budi; Crawford, Sue E.; Kim, Kate J.; Höök, Magnus; Estes, Mary K.

    2008-01-01

    Rotavirus NSP4 is a viral enterotoxin capable of causing diarrhea in neonatal mice. This process is initiated by the binding of extracellular NSP4 to target molecule(s) on the cell surface that triggers a signaling cascade leading to diarrhea. We now report that the integrins α1β1 and α2β1 are receptors for NSP4. NSP4 specifically binds to the α1 and α2 I domains with apparent Kd = 1–2.7 μM. Binding is mediated by the I domain metal ion-dependent adhesion site motif, requires Mg2+ or Mn2+, is abolished with EDTA, and an NSP4 point mutant, E120A, fails to bind α2 integrin I domain. NSP4 has two distinct integrin interaction domains. NSP4 amino acids 114–130 are essential for binding to the I domain, and NSP4 peptide 114–135 blocks binding of the natural ligand, collagen I, to integrin α2. NSP4 amino acids 131–140 are not associated with the initial binding to the I domain, but elicit signaling that leads to the spreading of attached C2C12-α2 cells, mouse myoblast cells stably expressing the human α2 integrin. NSP4 colocalizes with integrin α2 on the basolateral surface of rotavirus-infected polarized intestinal epithelial (Caco-2) cells as well as surrounding noninfected cells. NSP4 mutants that fail to bind or signal through integrin α2 were attenuated in diarrhea induction in neonatal mice. These results indicate that NSP4 interaction with integrin α1 and α2 is an important component of enterotoxin function and rotavirus pathogenesis, further distinguishing this viral virulence factor from other microbial enterotoxins. PMID:18587047

  8. Biological activity of recombinant accessory cholerae enterotoxin (ace) on rabbit ileal loops and antibacterial assay.

    PubMed

    Anvari, Shaghayegh; Najar Peerayeh, Shahin; Behmanesh, Mehrdad; Boustanshenas, Mina

    2012-01-01

    Vibrio cholerae (V. cholerae) causes a potentially lethal disease named cholera. The cholera enterotoxin (CT) is a major virulence factor of V. cholerae. In addition to CT, V. cholerae produces other putative toxins, such as the zonula occludens toxin (Zot) and accessory cholera enterotoxin (Ace). The ace gene is the third gene of the V. cholerae virulence cassette. The Ace toxin alters ion transport, causes fluid accumulation in ligated rabbit ileal loops, and is a cause of mild diarrhea. The aim of this study is the cloning and overexpression of the ace gene into Escherichia coli (E. coli) and determination of some characteristics of the recombinant Ace protein. In this experimental study, the ace gene was amplified from V. cholerae strain 62013, then cloned in a pET28a expression vector and transformed into an E. coli (DH5 α) host strain. Subsequently, the recombinant vector was retransformed into E. coli BL21 for expression, induced by isopropythio-β-D-galctoside (IPTG) at a different concentration, and examined by SDS-PAGE and Western blot. A rabbit ileal loop experiment was conducted. Antibacterial activity of the Ace protein was assessed for E. coli, Stapylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa). The recombinant Ace protein with a molecular weight of 18 kDa (dimeric form) was expressed in E. coli BL21. The Ace protein showed poor staining with Coomassie blue stain, but stained efficiently with silver stain. Western blot analysis showed that the recombinant Ace protein reacted with rabbit anti-V. cholerae polyclonal antibody. The Ace protein had antibacterial activity at a concentration of ≥200 µg/ml and caused significant fluid accumulation in the ligated rabbit ileal loop test. This study described an E. coli cloning and expression system (E. coli BL21- pET-28a-ace) for the Ace protein of V. cholerae. We confirmed the antibacterial properties and enterotoxin activity of the resultant recombinant Ace protein.

  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. Anti-angiogenic effects of the superantigen staphylococcal enterotoxin B and bacillus Calmette-Guérin immunotherapy for nonmuscle invasive bladder cancer.

    PubMed

    Reis, Leonardo O; Ferreira, Ubirajara; Billis, Athanase; Cagnon, Valéria H A; Fávaro, Wagner J

    2012-02-01

    We compared and characterized the effects of intravesical bacillus Calmette-Guérin and/or staphylococcal enterotoxin B for nonmuscle invasive bladder cancer. A total of 75 female Fisher 344 rats were anesthetized. Of the rats 15 received 0.3 ml saline (control) and 60 received 1.5 mg/kg MNU (N-methyl-n-nitrosourea) intravesically every other week for 6 weeks. The rats were divided into 5 groups. The MNU and control groups received 0.3 ml saline. The bacillus Calmette-Guérin group received 10(6) cfu bacillus Calmette-Guérin. The staphylococcal enterotoxin B group received 10 μg/ml staphylococcal enterotoxin B. The bacillus Calmette-Guérin plus staphylococcal enterotoxin B group received the 2 treatments simultaneously. Each group was treated intravesically for 6 weeks. At 15 weeks all bladders were collected for histopathological and immunological evaluation, and Western blot. Papillary carcinoma (pTa) and high grade intraepithelial neoplasia (carcinoma in situ) were more common in the MNU group. Papillary hyperplasia was more common in the bacillus Calmette-Guérin and enterotoxin groups. Flat hyperplasia was more common in the bacillus Calmette-Guérin plus enterotoxin group. No significant toxicity was observed. The apoptosis and cellular proliferation indexes decreased in the bacillus Calmette-Guérin, enterotoxin and bacillus Calmette-Guérin plus enterotoxin groups compared to the MNU group. Intensified vascular endothelial growth factor, matrix metalloproteinase-9, Ki-67 and insulin-like growth factor receptor-1 immunoreactivity was verified in the MNU group, moderate in the bacillus Calmette-Guérin and enterotoxin groups, and weak in the bacillus Calmette-Guérin plus enterotoxin and control groups. In contrast, intense endostatin immunoreactivity was verified in the control and bacillus Calmette-Guérin plus enterotoxin groups. Bacillus Calmette-Guérin and staphylococcal enterotoxin B showed similar anti-angiogenic effects. Bacillus Calmette

  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. 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.

  13. Freshwater Suspended Sediments and Sewage Are Reservoirs for Enterotoxin-Positive Clostridium perfringens▿

    PubMed Central

    Mueller-Spitz, Sabrina R.; Stewart, Lisa B.; Klump, J. Val; McLellan, Sandra L.

    2010-01-01

    The release of fecal pollution into surface waters may create environmental reservoirs of feces-derived microorganisms, including pathogens. Clostridium perfringens is a commonly used fecal indicator that represents a human pathogen. The pathogenicity of this bacterium is associated with its expression of multiple toxins; however, the prevalence of C. perfringens with various toxin genes in aquatic environments is not well characterized. In this study, C. perfringens spores were used to measure the distribution of fecal pollution associated with suspended sediments in the nearshore waters of Lake Michigan. Particle-associated C. perfringens levels were greatest adjacent to the Milwaukee harbor and diminished in the nearshore waters. Species-specific PCR and toxin gene profiles identified 174 isolates collected from the suspended sediments, surface water, and sewage influent as C. perfringens type A. Regardless of the isolation source, the beta2 and enterotoxin genes were common among isolates. The suspended sediments yielded the highest frequency of cpe-carrying C. perfringens (61%) compared to sewage (38%). Gene arrangement of enterotoxin was investigated using PCR to target known insertion sequences associated with this gene. Amplification products were detected in only 9 of 90 strains, which suggests there is greater variability in cpe gene arrangement than previously described. This work presents evidence that freshwater suspended sediments and sewage influent are reservoirs for potentially pathogenic cpe-carrying C. perfringens spores. PMID:20581181

  14. Reproducibility study for the detection of Staphylococcal enterotoxins in dairy products between official Italian national laboratories.

    PubMed

    Bianchi, D M; Ingravalle, F; Adriano, D; Gallina, S; Gramaglia, M; Zuccon, F; Astegiano, S; Bellio, A; Macori, G; Ru, G; Decastelli, L

    2014-06-01

    Staphylococcal food poisoning is a common foodborne disease caused by the ingestion of staphylococcal enterotoxins (SEs) produced mainly by enterotoxigenic strains of Staphylococcus aureus. To date, 21 SEs and/or enterotoxin-like types have been identified, several of which represent a potential hazard for consumers. To protect consumer health and to reduce the amount of SE-contaminated food entering the market, European Union legislation regulating food safety requires testing for SEs. The Italian National Reference Laboratory organized a ring trial to test technical and analytical proficiency in the national network of official food laboratories. Twenty-four laboratories took part, and each received and analyzed 24 blind dairy samples. Reproducibility of the results from the laboratories was assessed by the Cohen k index, and accuracy (sensitivity and specificity) was evaluated according to the International Organization for Standardization definition (ISO 16140:2003). Trial results revealed partially satisfactory agreement: 254 of 276 possible paired participants (92%) reached a k value >0.60, which is conventionally recognized as satisfactory. Accuracy was deemed satisfactory; 100% sensitivity and 100% specificity were achieved by 22 and 18 of the 24 laboratories, respectively.

  15. Purification of enterotoxins from Vibrio mimicus that appear to be identical to cholera toxin.

    PubMed Central

    Spira, W M; Fedorka-Cray, P J

    1984-01-01

    Cholera toxin-like (CT-like) enterotoxins produced by two strains of Vibrio mimicus, 61892 and 63616, isolated from diarrhea patients in Bangladesh, were purified, and their physicochemical, biological, and immunological properties were compared with those of CT produced by classical Vibrio cholerae 569B. The CT-like toxins were produced by lincomycin-resistant mutants grown in the presence of lincomycin at 200 micrograms/ml for strain 63616 and 250 micrograms/ml for strain 61892 and were purified by coprecipitation with hexametaphosphate followed by chromatography on phosphocellulose. The pure CT-like toxins were indistinguishable from 569B CT in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rabbit intestinal loop and Y-1 adrenal cell assays, antiserum neutralization and binding inhibition assays, and Ouchterlony immunodiffusion, except that the CT-like toxins appeared to consist almost entirely of A subunit which was proteolytically unnicked. Trypsin nicking, however, resulted in fragments that appeared to be identical to those of 569B CT. These results indicate that at least one species of Vibrio other than V. cholerae can produce enterotoxins which are virtually identical to CT. Images PMID:6469354

  16. Rapid purification of staphylococcal enterotoxin B by high-pressure liquid chromatography.

    PubMed Central

    Strickler, M P; Neill, R J; Stone, M J; Hunt, R E; Brinkley, W; Gemski, P

    1989-01-01

    The Staphylococcus aureus enterotoxins represent a group of proteins that cause emesis and diarrhea in humans and other primates. We have developed a rapid two-step high-pressure liquid chromatography (HPLC) procedure for purification of staphylococcal enterotoxin B (SEB). Sterile filtrates (2.5 liters) of strain 10-275 were adsorbed directly onto a reversed-phase column (50 mm by 30 cm Delta Pak; 300 A [30 nm], 15 microns, C18). SEB was obtained by using a unique sequential gradient system. First, an aqueous ammonium acetate to acetonitrile gradient followed by an aqueous trifluoroacetic acid (TFA) wash was used to remove contaminants. A subsequent TFA to acetonitrile-TFA gradient eluted the bound SEB. Further purification was obtained by rechromatography on a cation-exchange column. From 35 to 45% of the SEB in starting filtrates was recovered. Analysis by immunoblotting of samples separated on sodium dodecyl sulfate-polyacrylamide gels indicated that HPLC-purified SEB exhibited immunological and biochemical properties similar to those of the SEB standard. Induction of an emetic response in rhesus monkeys showed that the HPLC-purified toxin also retained biological activity. Images PMID:2745678

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. Necrotizing enterocolitis and death in a goat kid associated with enterotoxin (CPE)-producing Clostridium perfringens type A

    PubMed Central

    Fernandez Miyakawa, Mariano E.; Saputo, Julian; St. Leger, Judy; Puschner, Birgit; Fisher, Derek J.; McClane, Bruce A.; Uzal, Francisco A.

    2007-01-01

    A goat kid died after being depressed for several days. No significant gross abnormalities were observed at postmortem examination, while histopathological analysis revealed diffuse necrotizing enterocolitis. Isolation of Clostridium perfringens type A secreting enterotoxin (CPE) and presence of CPE in the small intestine suggest that CPE contributed to the death of this kid. PMID:18189049

  2. [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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  4. 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. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  5. Staphylococcal protein A-formulated immune complexes suppress enterotoxin-induced cellular responses in nasal polyps.

    PubMed

    Okano, Mitsuhiro; Fujiwara, Tazuko; Kariya, Shin; Haruna, Takenori; Higaki, Takaya; Noyama, Yasuyuki; Makihara, Sei-ichiro; Kanai, Kengo; Nishizaki, Kazunori

    2015-08-01

    Recent studies have revealed that Staphylococcus aureus and its components participate in the pathogenesis of eosinophilic airway diseases, such as chronic rhinosinusitis with nasal polyps. We sought to determine whether staphylococcal protein A (SpA) from S aureus regulated cellular responses in nasal polyps, especially when coupled to immunoglobulins in immune complexes (ICs). Dispersed nasal polyp cells (DNPCs) or peripheral blood monocytes were cultured in vitro with SpA in the presence or absence of IgG, and IL-5, IL-13, IFN-γ, IL-17A, and IL-10 levels were measured in the supernatants. The effect of SpA exposure on staphylococcal enterotoxin B-induced cytokine production by DNPCs in the presence and absence of IgG, IgA, and autologous serum was also examined. Exposure to SpA induced DNPCs to produce significantly higher IL-10, IL-13, and IL-17A levels than DNPCs without SpA, although the magnitude of the IL-17A increase was less than that of IL-10 and IL-13. SpA induced IL-10 production mainly from adherent DNPCs, and this was significantly enhanced in the presence of IgG; similar results were observed in peripheral blood monocytes. IC formation between SpA and IgG (SpA-IgG ICs) was confirmed by using native polyacrylamide gel electrophoresis. SpA-IgG ICs, but not SpA alone, almost completely suppressed staphylococcal enterotoxin B-induced IL-5, IL-13, IFN-γ, and IL-17A production by DNPCs; similar inhibition was observed in DNPCs treated with SpA in the presence of either IgA or autologous serum. Our results suggest that SpA can regulate the pathogenesis of enterotoxin-induced inflammation in patients with chronic rhinosinusitis with nasal polyps through coupling to immunoglobulins. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  6. Detection of enterotoxin and protease genes among Hungarian clinical Bacteroides fragilis isolates.

    PubMed

    Sárvári, Károly Péter; Sóki, József; Iván, Miklós; Miszti, Cecília; Latkóczy, Krisztina; Melegh, Szilvia Zsóka; Urbán, Edit

    2017-07-25

    Bacteroides fragilis as a commensal bacterium is a member of the human intestinal flora, but as an opportunistic pathogen it can cause serious infections as well. Some of them, harbouring an enterotoxin gene (bft), may cause diarrhoea mainly in young children. Recently it has been shown that a member of C11 proteases called fragipain (fpn) can activate the enterotoxin, while C10 protease (bfp) is suspected of playing an important role in the invasiveness of the B. fragilis isolates. The objective of this study was to investigate the prevalence and distribution of the bft isotypes in 200 Hungarian B. fragilis isolates collected recently; and in a subset of 72 strains, we wanted to determine the prevalence of bfp1-4 and fpn genes in bft-positive and bft-negative strains. Using the MALDI-TOF MS cfiA identification project file, 19 B. fragilis strains belonging to Division II were identified and the presence of the cfiA gene was confirmed by RT-PCR. Twenty six (13.0%) B. fragilis isolates turned out to be bft gene positive by RT-PCR; 20 isolates harboured bft-1 and six bft-2 isotypes, but no bft-3 isotype containing strains were found. A melting curve analysis and the PCR-RFLP were performed to differentiate between the bft-1 and bft-2 isotypes confirmed by sequencing. Thirty eight strains harboured bfp1, 58 isolates contained bfp2 gene, while 17 isolates proved positive for bfp3. Morever, no bfp4 positive isolate was found, and some of the B. fragilis strains tested harboured two or three bfp isotypes simultaneously. Among the 26 bft-positive strains, 24 contained the fpn gene, which confirms the role of fragipain in the activation of B. fragilis enterotoxin. In experiments, a significant negative correlation between fpn and cfiA was demonstrated (p < 0.000), a positive correlation was found between bfp2 and fpn genes (p = 0.0000803), and a negative correlation between bfp2 and cfiA genes (p = 0.011). Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. 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.

  8. 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.

  9. Peracetic Acid: A Practical Agent for Sterilizing Heat-Labile Polymeric Tissue-Engineering Scaffolds

    PubMed Central

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

    2014-01-01

    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. PMID:24341350

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. Effect of shaking speed on the secretion of enterotoxin B by Staphylococcus aureus.

    PubMed

    Dietrich, G G; Watson, R J; Silverman, G J

    1972-10-01

    The concentration of enterotoxin B secreted by four strains of Staphylococcus aureus was dependent upon the shaking speed. For the conditions established, each strain demonstrated an optimal shaking speed, and speeds in excess of the optimum resulted in decreased secretion of toxin. At the optimal shaking speed, maximum secretion occurred at 37 C. At 45 C, both growth and toxin secretion were absent. By using agar containing antienterotoxin B sera, studies with strain S-6 at optimal and suboptimal shaking speeds demonstrated that individual cells vary in their toxin-synthesizing ability and that the relative numbers of high and low producers change during the growth cycle. Although most of the toxin was secreted during the first 12 hr of growth, a portion was secreted during the subsequent 6 hr, even though growth as measured by colony-forming units per milliliter decreased and Klett units increased.

  15. 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

  16. 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

  17. 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.

  18. 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

  19. Identification and characterization of two novel staphylococcal enterotoxins, types S and T.

    PubMed

    Ono, Hisaya K; Omoe, Katsuhiko; Imanishi, Ken'ichi; Iwakabe, Yoshihiro; Hu, Dong-Liang; Kato, Hidehito; Saito, Naoyuki; Nakane, Akio; Uchiyama, Takehiko; Shinagawa, Kunihiro

    2008-11-01

    In addition to two known staphylococcal enterotoxin-like genes (selj and selr), two novel genes coding for two superantigens, staphylococcal enterotoxins S and T (SES and SET), were identified in plasmid pF5, which is harbored by food poisoning-related Staphylococcus aureus strain Fukuoka 5. This strain was implicated in a food poisoning incident in Fukuoka City, Japan, in 1997. Recombinant SES (rSES) specifically stimulated human T cells in a T-cell receptor Vbeta9- and Vbeta16-specific manner in the presence of major histocompatibility complex (MHC) class II(+) antigen-presenting cells (APC). rSET also stimulated T cells in the presence of MHC class II(+) APC, although its Vbeta skewing was not found in reactive T cells. Subsequently, we examined the emetic activity of SES and SET. We also studied SElR to determine emetic activity in primates. This toxin was identified in previous studies but was not examined in terms of possession of emetic activity for primates. rSES induced emetic reactions in two of four monkeys at a dose of 100 microg/kg within 5 h of intragastric administration. In one monkey, rSET induced a delayed reaction (24 h postadministration) at a dose of 100 microg/kg, and in the other one, the reaction occurred 5 days postadministration. rSElR induced a reaction in two of six animals within 5 h at 100 microg/kg. On this basis, we speculate that the causative toxins of vomiting in the Fukuoka case are SES and SER. Additionally, SES, SER, and SET also induced emesis in house musk shrews as in the monkeys.

  20. 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.

  1. 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)

  2. 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.

  3. Toxigenic Escherichia Coli and Childhood Diarrhea

    PubMed Central

    Mundell, Dave H.; Anselmo, Carl R.; Thrupp, Lauri D.; Wishnow, Rodney M.

    1976-01-01

    Stool specimens were examined from 40 children with diarrhea who were under three years of age to determine the incidence of enterotoxigenic Escherichia coli in endemic diarrhea. Heat-labile E. coli enterotoxin was assayed in the very sensitive and reproducible cultured adrenal tumor cell system. Toxigenic E. coli were isolated from only one stool specimen and in this case infection with Shigella dysenteriae was also present. None of the eight classic enteropathogenic E. coli isolates were positive in the adrenal assay. This study suggests that heat-labile enterotoxin-producing E. coli are not an important cause of endemic childhood diarrhea in Southern California. PMID:775792

  4. 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.

  5. [A simple method for the detection of staphylococcal enterotoxin type B in vanilla custard using the ELISA (author's transl)].

    PubMed

    Büning-Pfaue, H; Timmermans, P; Notermans, S

    1981-01-01

    The ELISA for the detection of staphylococcal enterotoxin type B (SEB) was employed to demonstrate SEB in Dutch Vanilla custard. Due to the sensitivity of the ELISA the extraction procedure, which is necessary when the Ouchterlony test is used, can be abbreviated to a great extent. Two successive extractions at pH 7.4 and pH 4.5 followed by a concentration (1:20) was sufficient to detect 0.1 mcg SEB in 100 g custard.

  6. 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

  7. 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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  8. 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

  9. 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.

  10. 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

  11. Characterization of Enterotoxigenic Bacillus cereus sensu lato and Staphylococcus aureus Isolates and Associated Enterotoxin Production Dynamics in Milk or Meat-Based Broth

    PubMed Central

    Walker-York-Moore, Laura; Moore, Sean C.; Fox, Edward M.

    2017-01-01

    Bacillus cereus sensu lato species, as well as Staphylococcus aureus, are important pathogenic bacteria which can cause foodborne illness through the production of enterotoxins. This study characterised enterotoxin genes of these species and examined growth and enterotoxin production dynamics of isolates when grown in milk or meat-based broth. All B. cereus s. l. isolates harboured nheA, hblA and entFM toxin genes, with lower prevalence of bceT and hlyII. When grown at 16 °C, toxin production by individual B. cereus s. l. isolates varied depending on the food matrix; toxin was detected at cell densities below 5 log10(CFU/mL). At 16 °C no staphylococcal enterotoxin C (SEC) production was detected by S. aureus isolates, although low levels of SED production was noted. At 30 °C all S. aureus isolates produced detectable enterotoxin in the simulated meat matrix, whereas SEC production was significantly reduced in milk. Relative to B. cereus s. l. toxin production, S. aureus typically required reaching higher cell numbers to produce detectable levels of enterotoxin. Phylogenetic analysis of the sec and sel genes suggested population evolution which correlated with animal host adaptation, with subgroups of bovine isolates or caprine/ovine isolates noted, which were distinct from human isolates. Taken together, this study highlights the marked differences in the production of enterotoxins both associated with different growth matrices themselves, but also in the behaviour of individual strains when exposed to different food matrices. PMID:28714887

  12. The Potential Therapeutic Agent Mepacrine Protects Caco-2 Cells against Clostridium perfringens Enterotoxin Action.

    PubMed

    Freedman, John C; Hendricks, Matthew R; McClane, Bruce A

    2017-01-01

    Clostridium perfringens enterotoxin (CPE) causes the diarrhea associated with a common bacterial food poisoning and many antibiotic-associated diarrhea cases. The severity of some CPE-mediated disease cases warrants the development of potential therapeutics. A previous study showed that the presence of mepacrine inhibited CPE-induced electrophysiology effects in artificial lipid bilayers lacking CPE receptors. However, that study did not assess whether mepacrine inactivates CPE or, instead, inhibits a step in CPE action. Furthermore, CPE action in host cells is complex, involving the toxin binding to receptors, receptor-bound CPE oligomerizing into a prepore on the membrane surface, and β-hairpins in the CPE prepore inserting into the membrane to form a pore that induces cell death. Therefore, the current study evaluated the ability of mepacrine to protect cells from CPE. This drug was found to reduce CPE-induced cytotoxicity in Caco-2 cells. This protection did not involve mepacrine inactivation of CPE, indicating that mepacrine affects one or more steps in CPE action. Western blotting then demonstrated that mepacrine decreases CPE pore levels in Caco-2 cells. This mepacrine-induced reduction in CPE pore levels did not involve CPE binding inhibition but rather an increase in CPE monomer dissociation due to mepacrine interactions with Caco-2 membranes. In addition, mepacrine was also shown to inhibit CPE pores when already present in Caco-2 cells. These in vitro studies, which identified two mepacrine-sensitive steps in CPE-induced cytotoxicity, add support to further testing of the therapeutic potential of mepacrine against CPE-mediated disease. IMPORTANCEClostridium perfringens enterotoxin (CPE) causes the gastrointestinal (GI) symptoms of a common bacterial food poisoning and several nonfoodborne human GI diseases. A previous study showed that, via an undetermined mechanism, the presence of mepacrine blocks CPE-induced electrophysiologic activity in artificial

  13. 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.

  14. 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

  15. The Type III Secretion System and Cytotoxic Enterotoxin Alter the Virulence of Aeromonas hydrophila

    PubMed Central

    Sha, Jian; Pillai, Lakshmi; Fadl, Amin A.; Galindo, Cristi L.; Erova, Tatiana E.; Chopra, Ashok K.

    2005-01-01

    Many gram-negative bacteria use a type III secretion system (TTSS) to deliver effector proteins into host cells. Here we report the characterization of a TTSS chromosomal operon from the diarrheal isolate SSU of Aeromonas hydrophila. We deleted the gene encoding Aeromonas outer membrane protein B (AopB), which is predicted to be involved in the formation of the TTSS translocon, from wild-type (WT) A. hydrophila as well as from a previously characterized cytotoxic enterotoxin gene (act)-minus strain of A. hydrophila, thus generating aopB and act/aopB isogenic mutants. The act gene encodes a type II-secreted cytotoxic enterotoxin (Act) that has hemolytic, cytotoxic, and enterotoxic activities and induces lethality in a mouse model. These isogenic mutants (aopB, act, and act/aopB) were highly attenuated in their ability to induce cytotoxicity in RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells. The act/aopB mutant demonstrated the greatest reduction in cytotoxicity to cultured cells after 4 h of infection, as measured by the release of lactate dehydrogenase enzyme, and was avirulent in mice, with a 90% survival rate compared to that of animals infected with Act and AopB mutants, which caused 50 to 60% of the animals to die at a dose of three 50% lethal doses. In contrast, WT A. hydrophila killed 100% of the mice within 48 h. The effects of these mutations on cytotoxicity could be complemented with the native genes. Our studies further revealed that the production of lactones, which are involved in quorum sensing (QS), was decreased in the act (32%) and aopB (64%) mutants and was minimal (only 8%) in the act/aopB mutant, compared to that of WT A. hydrophila SSU. The effects of act and aopB gene deletions on lactone production could also be complemented with the native genes, indicating specific effects of Act and the TTSS on lactone production. Although recent studies with other bacteria have indicated TTSS regulation by QS, this is the first

  16. 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

  17. Concerted Action of Sphingomyelinase and Non-Hemolytic Enterotoxin in Pathogenic Bacillus cereus

    PubMed Central

    Doll, Viktoria M.

    2013-01-01

    Bacillus cereus causes food poisoning and serious non-gastrointestinal-tract infections. Non-hemolytic enterotoxin (Nhe), which is present in most B. cereus strains, is considered to be one of the main virulence factors. However, a B. cereus ΔnheBC mutant strain lacking Nhe is still cytotoxic to intestinal epithelial cells. In a screen for additional cytotoxic factors using an in vitro model for polarized colon epithelial cells we identified B. cereus sphingomyelinase (SMase) as a strong inducer of epithelial cell death. Using single and double deletion mutants of sph, the gene encoding for SMase, and nheBC in B. cereus we demonstrated that SMase is an important factor for B. cereus cytotoxicity in vitro and pathogenicity in vivo. SMase substantially complemented Nhe induced cytotoxicity in vitro. In addition, SMase but not Nhe contributed significantly to the mortality rate of larvae in vivo in the insect model Galleria mellonella. Our study suggests that the role of B. cereus SMase as a secreted virulence factor for in vivo pathogenesis has been underestimated and that Nhe and SMase complement each other significantly to cause full B. cereus virulence hence disease formation. PMID:23613846

  18. Conformation of protein secreted across bacterial outer membranes: a study of enterotoxin translocation from Vibrio cholerae

    SciTech Connect

    Hirst, T.R.; Holmgren, J.

    1987-11-01

    The secretion of enterotoxin by Vibrio cholerae is punctuated by the transient entry of the toxin subunits into the periplasm. In this paper, the authors show that the subunits oligomerize into an assembled holotoxin within the periplasm prior to their secretion across the outer membrane. The rate of toxin assembly was studied by pulse-labeling cells with (/sup 35/S)-methionine and then monitoring the turnover of radiolabeled subunits as they assembled within the periplasm. The subunits entered the periplasm as monomers and assembled into oligomers with a half-time of approx. = 1 min. Since assembly was a rapid event compared to the rate of toxin efflux from the periplasm, which had a half-time of approx. = 13 min, they conclude that all of the subunits that pass through the periplasm assemble before they traverse the outer membrane. The average concentration of subunit monomers and assembled holotoxin within the periplasm was calculated to be approx. = 20 and approx. = 260 ..mu..g/ml, respectively. This indicates that the periplasm is a suitably concentrated milieu where spontaneous toxin assembly can occur. These findings suggest that protein movement across bacterial outer membranes, in apparent contrast to export across other biological membranes, involves translocation of polypeptides that have already folded into tertiary and even quaternary conformations.

  19. Staphylococcal Enterotoxin B-specific monoclonal antibody 20B1 successfully treats diverse Staphylococcus aureus infections.

    PubMed

    Varshney, Avanish K; Wang, Xiaobo; Scharff, Matthew D; MacIntyre, Jennifer; Zollner, Richard S; Kovalenko, Oleg V; Martinez, Luis R; Byrne, Fergus R; Fries, Bettina C

    2013-12-15

    Methicillin-resistant Staphylococcus aureus (MRSA) has become a major health threat in the United States. Staphylococcal enterotoxin B (SEB) is a potent superantigen that contributes to its virulence. High mortality and frequent failure of therapy despite available antibiotics have stimulated research efforts to develop adjunctive therapies. Treatment benefits of SEB-specific monoclonal antibody (mAb) 20B1 were investigated in mice in sepsis, superficial skin, and deep-tissue infection models. Mice challenged with a SEB-producing MRSA strain developed fatal sepsis, extensive tissue skin infection, and abscess-forming deep-seeded thigh muscle infection. Animals preimmunized against SEB or treated passively with mAb 20B1 exhibited enhanced survival in the sepsis model, whereas decrease of bacterial burden was observed in the superficial skin and deep-tissue models. mAb 20B1 bound to SEB in the infected tissue and decreased abscess formation and proinflammatory cytokine levels, lymphocyte proliferation, and neutrophil recruitment. mAb 20B1, an SEB-neutralizing mAb, is effective against MRSA infection. mAb 20B1 protects against lethal sepsis and reduces skin tissue invasion and deep-abscess formation. The mAb penetrates well into the abscess and binds to SEB. It affects the outcome of S. aureus infection by modulating the host's proinflammatory immune response.

  20. 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

  1. 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.

  2. 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.

  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. 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.

  6. 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.

  7. 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.

  8. 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.

  9. [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.

  10. 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

  11. Detection of staphylococcal enterotoxin B in milk and milk products using immunodiagnostic lateral flow devices.

    PubMed

    Boyle, Thomas; Njoroge, Joyce M; Jones, Robert L; Principato, Maryann

    2010-01-01

    Staphylococcal enterotoxin B (SEB) is an extracellular pyrotoxin produced by Staphylococcus aureus, a known etiologic agent of food poisoning in humans. Lateral flow immunochromatographic devices (LFDs) designed for the environmental detection of SEB were adapted for use in this study to detect SEB in milk containing 2% fat, chocolate-flavored milk, and milk-derived products such as yogurt, infant formula, and ice cream. The advantage of using LFDs in these particular food products was its ease and speed of use with no additional extraction methods needed. No false positives were observed with any of the products used in this study. Dilution of the samples overcame the Hook effect and permitted capillary flow into the membrane. Thus, semisolid products such as ice cream and some yogurts, and products containing thickeners needed to be diluted using a phosphate-buffered saline-based buffer, pH 7.2. SEB was easily detected at concentrations of 5 microg/mL and 500 ng/mL when the LFDs were used. SEB was also reliably detected at concentrations below 5 and 0.25 ng/mL, which may induce serious disease.

  12. 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

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

    PubMed Central

    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. PMID:21792355

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

    SciTech Connect

    Soykut, Esra Acar; Dudak, Fahriye Ceyda; Boyaci, Ismail Hakki

    2008-05-23

    In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinity to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 {+-} 0.7 x 10{sup 5} M{sup -1} which indicates a strong binding close to that of antibody.

  2. Addressing bioterrorism concerns: options for investigating the mechanism of action of Staphylococcus aureus enterotoxin B.

    PubMed

    Lindsay, C D; Griffiths, G D

    2013-06-01

    Staphylococcal enterotoxin B (SEB) is of concern to military and civilian populations as a bioterrorism threat agent. It is a highly potent toxin produced by Staphylococcus aureus and is stable in storage and under aerosolisation; it is able to produce prolonged highly incapacitating illness at very low-inhaled doses and death at elevated doses. Concerns regarding SEB are compounded by the lack of effective medical countermeasures for mass treatment of affected populations. This article considers the mechanism of action of SEB, the availability of appropriate experimental models for evaluating the efficacy of candidate medical countermeasures with particular reference to the need to realistically model SEB responses in man and the availability of candidate countermeasures (with an emphasis on commercial off-the-shelf options). The proposed in vitro approaches would be in keeping with Dstl’s commitment to reduction, refinement and replacement of animal models in biomedical research, particularly in relation to identifying valid alternatives to the use of nonhuman primates in experimental studies.

  3. 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

  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. 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.

  6. Inhibition of biological activity of staphylococcal enterotoxin A (SEA) by apple juice and apple polyphenols.

    PubMed

    Rasooly, Reuven; Do, Paula M; Friedman, Mendel

    2010-05-12

    The foodborne pathogen Staphylococcus aureus produces the virulent staphylococcal enterotoxin A (SEA), a single-chain protein that 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, diarrhea, atopic dermatitis, arthritis, and toxic shock) syndromes. Changes of the native structural integrity may inactivate the toxin by preventing molecular interaction with cell membrane receptor sites of their host cells. In the present study, we evaluated the ability of one commercial and two freshly prepared apple juices and a commercial apple polyphenol preparation (Apple Poly) to inhibit the biological activity of SEA. Dilutions of freshly prepared apple juices and Apple Poly inhibited the biological activity of SEA without any significant cytotoxic effect on the spleen cells. Additional studies with antibody-coated immunomagnetic beads bearing specific antibodies against the toxin revealed that SEA added to apple juice appears to be largely irreversibly bound to the juice constituents. The results suggest that food-compatible and safe anti-toxin phenolic compounds can be used to inactivate SEA in vitro and possibly also in vivo, even after induction of T-cell proliferation by long-term exposure to SEA. The significance of the results for microbial food safety and human health is discussed.

  7. 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

  8. [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.

  9. CD1+ human thymocytes proliferate in response to superantigen staphylococcal enterotoxin B1.

    PubMed

    Todd, S C; Baccala, R; Hedrick, J A; Theofilopoulos, A N; Tsoukas, C D

    1994-09-01

    Exposure of human thymocytes to superantigens results in the deletion of thymocytes expressing specific TCR-V beta genes. The factors that contribute to this deletion may relate to the inherent nature of the T cell at a given stage of development. In this paper, we demonstrate that CD1+ human cortical thymocytes are capable of proliferating in response to a bacterial superantigen (staphylococcal enterotoxin B (SEB)) in the presence of autologous CD2-/low thymic APCs. Phenotypic analysis of the responding populations revealed that the majority of the CD1+ cells were CD4+CD8low or CD8+CD4low cells. The response is triggered by low concentrations of SEB, requires the participation of the TCR and IL-2R molecules, and is inhibited by cyclosporin A. Thymocytes that express specific V beta genes are expanded, which results in an engagement profile that parallels that found in PBLs. Additionally, four V beta-chains that have not been reported previously are shown to engage SEB. Once stimulated, the thymocytes failed to respond to additional SEB; however, they could be induced to proliferative with IL-2, which suggests that these expanded populations had become anergic. These data represent the first demonstration of a human cortical thymocyte subpopulation that responds to superantigen by proliferation and subsequent anergy.

  10. 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

  11. 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.

  12. [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.

  13. 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. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. The prevalence of enterotoxin and antibiotic resistance genes in clinical and intestinal Bacteroides fragilis group isolates in Turkey.

    PubMed

    Kangaba, Achille Aime; Saglam, Filiz Yarimcam; Tokman, Hrisi Bahar; Torun, Mert; Torun, Muzeyyen Mamal

    2015-10-01

    This study was conducted to measure the antibiotic susceptibilities, corresponding gene contents, and the enterotoxin gene bft, in 50 Bacteroides fragilis group isolates, 25 of which were clinical and 25 intestinal. The resistance rates to amoxicillin/clavulanic acid, imipenem and metronidazole were low; ampicillin and tetracyclin resistance was high; clindamycin resistance and ermF gene presence was also high. Regarding phenotypical bacterial resistance and the presence of resistance genes, there was not statistically significant difference between clinical and intestinal isolates and bft positive and negative isolates. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Applicability of an immunoblot technique combined with a semiautomated electrophoresis system for detection of staphylococcal enterotoxins in food extracts.

    PubMed Central

    Orden, J A; Goyache, J; Hernández, J; Doménech, A; Suárez, G; Gómez-Lucía, E

    1992-01-01

    We studied the usefulness of an immunoblot technique for the detection of staphylococcal enterotoxins (SEs) in strains and food extracts. Food samples (milk, yogurt, hot dog sausage, cheese, and mayonnaise) were artificially contaminated with SEA through SEE. Protein A did not interfere with the results; it appeared on electrophoresis gels as bands with molecular weights higher than those of the SEs. Other food proteins were not revealed by the technique. The immunoblot technique proved to be fast, specific, and sensitive for the detection of SEs in foods. Images PMID:1476449

  20. 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.

  1. 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.

  2. Nitrate salts suppress sporulation and production of enterotoxin in Clostridium perfringens strain NCTC8239.

    PubMed

    Yasugi, Mayo; Otsuka, Keisuke; Miyake, Masami

    2016-10-01

    Clostridium perfringens type A is a common source of food-borne illness in humans. Ingested vegetative cells sporulate in the small intestinal tract and in the process produce C. perfringens enterotoxin (CPE). Although sporulation plays a critical role in the pathogenesis of food-borne illness, the molecules triggering/inhibiting sporulation are still largely unknown. It has previously been reported by our group that sporulation is induced in C. perfringens strain NCTC8239 co-cultured with Caco-2 cells in Dulbecco's Modified Eagle Medium (DMEM). In contrast, an equivalent amount of spores was not observed when bacteria were co-cultured in Roswell Park Memorial Institute-1640 medium (RPMI). In the present study it was found that, when these two media are mixed, RPMI inhibits sporulation and CPE production induced in DMEM. When a component of RPMI was added to DMEM, it was found that calcium nitrate (Ca[NO3 ]2 ) significantly inhibits sporulation and CPE production. The number of spores increased when Ca(NO3 )2 -deficient RPMI was used. The other nitrate salts significantly suppressed sporulation, whereas the calcium salts used did not. qPCR revealed that nitrate salts increased expression of bacterial nitrate/nitrite reductase. Furthermore, it was found that nitrite and nitric oxide suppress sporulation. In the sporulation stages, Ca(NO3 )2 down-regulated the genes controlled by Spo0A, a master regulator of sporulation, but not spo0A itself. Collectively, these results indicate that nitrate salts suppress sporulation and CPE production by down-regulating Spo0A-regulated genes in C. perfringens strain NCTC8239. Nitrate reduction may be associated with inhibition of sporulation. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

  3. 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. Published by Elsevier B.V.

  4. 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

  5. Staphylococcus aureus enterotoxin sensitization is associated with allergic poly-sensitization and allergic multimorbidity in adolescents.

    PubMed

    Sørensen, M; Klingenberg, C; Wickman, M; Sollid, J U E; Furberg, A-S; Bachert, C; Bousquet, J

    2017-10-01

    Staphylococcus aureus (S. aureus) carriage and sensitization to S. aureus enterotoxins (SEs) have been associated with allergic diseases. From the Tromsø Study Fit Futures 2, we have previously shown an association between S. aureus carriage and severe allergic disease and allergic multimorbidity. However, the role of S. aureus carriage and SE sensitization on allergic multimorbidity and allergic sensitization is unclear. To study associations of both nasal S. aureus carriage and SE sensitization to allergic disease and allergic sensitization. A cross-sectional study of a school-based cohort in late adolescence (aged 18-19 years: The Tromsø Study Fit Futures 2). Self-reported allergic diseases were assessed using the Mechanisms of the Development of ALLergy questionnaire (MeDALL). Participants were tested for nasal S. aureus carriage, serum total IgE and specific IgE to SEs, and food and inhalant allergens. A total of 868 participants were studied. Sensitization to at least one food or inhalant allergen was found in 319 of 765 (41.7%), and to at least one SE in 173 of 656 (26.2%) participants. SE sensitization, but not S. aureus carriage, was associated with poly-sensitization to food and inhalant allergens. SE-sensitized participants had higher median specific IgE to inhalant allergens (41.4 kUA /L, IQR 10.1-118.4) compared to non-SE-sensitized participants (18.0 kUA /L, IQR 5.5-48.6, P=.004), but not to food allergens. SE sensitization was associated with allergic multimorbidity. Sensitization to SEs may play a role in the development of allergen poly-sensitization and allergic multimorbidity. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. Regulation of staphylococcal enterotoxin B-elicited nitric oxide production by endothelial cells.

    PubMed Central

    LeClaire, R D; Kell, W M; Sadik, R A; Downs, M B; Parker, G W

    1995-01-01

    The effect of staphylococcal enterotoxin B (SEB)-elicited inducible nitric oxide synthase (iNOS) in mouse endothelial cells was investigated. Results showed that SEB stimulated the same level of NO production in gamma interferon (IFN-gamma)-primed cells as did trichloroacetic acid-extracted lipopolysaccharide. The kinetics of induced NO production and expression of mRNA for iNOS differed markedly in endothelial and macrophage cells. Induced endothelial nitrite production was transient and was 15 to 20% of that generated by macrophage cells; mRNA levels peaked by 2 h and then steadily declined, whereas macrophage message levels continually increased. The ability of endothelial cells to produce SEB-induced NO depended on priming with IFN-gamma, although detectable mRNA could be elicited by SEB alone. Induction of endothelial iNOS mRNA was inhibited by cycloheximide, which indicated a requirement for de novo protein synthesis. Niacinamide and interleukin-10 significantly reduced SEB-induced endothelial NO production. Both are reported to affect IFN-gamma-induced class II major histocompatibility complex (MHC) expression on antigen-presenting cells. Niacinamide reduced iNOS mRNA levels and markedly reduced IFN-gamma induction of endothelial class II MHC surface antigen. Interleukin-10 did not consistently reduce iNOS mRNA expression and had no effect on IFN-gamma induction of endothelial class II MHC surface antigen. These results suggest that SEB interacts with IFN-gamma-primed endothelial cells to elicit induced NO and that this induction can be effectively modulated at the receptor or transcriptional level. PMID:7529748

  13. Staphylococcal enterotoxin A: Partial unfolding caused by high pressure or denaturing agents enhances superantigenicity.

    PubMed

    René-Trouillefou, Malika; Benzaria, Amal; Marchal, Stéphane; Lange, Reinhard; Caporiccio, Bertrand; Dumay, Eliane

    2010-06-01

    The effect of transient exposure of Staphylococcus aureus enterotoxin A (SEA) to high pressure and/or denaturing agents was examined by assessing the toxin superantigenicity and immunoreactivity, and by monitoring pressure-induced changes in fluorescence emission spectra. Pressurization of SEA at 600 MPa and 45 degrees C in Tris-HCl buffer (20 mM, pH 7.4) resulted in a marked increase in both T-cell proliferation (superantigenicity) and immunoreactivity. In opposite, pressurization at 20 degrees C did not change significantly SEA superantigenicity and immunoreactivity, indicating some toxin baro-resistance. Exposure of SEA to 8 M urea at atmospheric pressure or at 600 MPa and 20 degrees C, also led to a marked increase of superantigenicity (but not of immunoreactivity). In contrast, exposure of SEA to sodium-dodecylsulfate (30 mM) led to an increase of immunoreactivity with some effect on superantigenicity after pressurization at 45 degrees C only. High pressure up to 600 MPa induced spectral changes which at 20 degrees C were fully reversible upon decompression. At 45 degrees C, however, a sharp break of the centre of spectral mass mainly due to tryptophan residues was observed at 300 MPa, and irreversible spectral changes mainly related to tyrosine residues subsisted after pressure release, indicating a marked protein conformational transition. Urea 8 M further increased SEA structural changes at 600 MPa and 20 degrees C. These results indicate that SEA, under a combination of high pressure and mild temperature, as well as in the presence of urea, partly unfolds to a structure of strongly increased T-cell proliferative ability. Copyright 2010 Elsevier B.V. All rights reserved.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. Phenotypic Profiles of Enterotoxigenic Escherichia coli Associated With Early Childhood Diarrhea in Rural Egypt

    DTIC Science & Technology

    2004-12-01

    excretion of ETEC, either alone (n 729 [78%]) or with one or more copathogens (n 204 [22%]), including Shigella, Salmonella, Campylobacter , rotavirus...A. M., and J. Holmgren. 1978. Identification of Escherichia coli heat-labile enterotoxin by means of a ganglioside immunosorbent assay (GM1- ELISA

  19. 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.

  20. 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).

  1. 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. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. 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. © 2014 Institute of Food Technologists®

  3. Development of a Multiplex PCR Method for Detection of the Genes Encoding 16S rRNA, Coagulase, Methicillin Resistance and Enterotoxins in Staphylococcus aureus

    USDA-ARS?s Scientific Manuscript database

    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 ...

  4. Noncontiguous Finished Genome Sequence of Staphylococcus aureus KLT6, a Staphylococcal Enterotoxin B-Positive Strain Involved in a Food Poisoning Outbreak in Switzerland

    PubMed Central

    Tobes, Raquel; Manrique, Marina; Brozynska, Marta; Stephan, Roger; Pareja, Eduardo

    2013-01-01

    We present the first complete genome sequence of a Staphylococcus aureus strain assigned to clonal complex 12. The strain was isolated in a food poisoning outbreak due to contaminated potato salad in Switzerland in 2009, and it produces staphylococcal enterotoxin B. PMID:23704175

  5. Quantitative analysis of Staphylococcus enterotoxin A by differential expression of IFN-gamma in splenocyte and CD4+ T-cells

    USDA-ARS?s Scientific Manuscript database

    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 ...

  6. 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

  7. Chicken egg yolk immunoglobulin (IgY) developed against fusion protein LTB-STa-STb neutralizes the toxicity of Escherichia coli heat-stable enterotoxins.

    PubMed

    You, J; Xu, Y; Li, H; Wang, L; Wu, F; Xu, F; Jin, L; Li, S; Li, X

    2014-08-01

    To obtain a recombinant enterotoxigenic Escherichia coli (ETEC) fusion enterotoxin protein LTB-STa-STb (Bab) that can express the immunogenicity of the haptens STa and STb and induce their corresponding neutralizing antibodies. The three important ETEC enterotoxin genes coding LTB, STa and STb were PCR-amplified, and the amplified products were fused to construct the trivalent enterotoxin expression vector pET30-Bab. SDS-PAGE and Western blot were used to verify the expression of the fusion protein Bab by E. coli BL21 carrying plasmid pET30-Bab. Laying hens immunized with Bab developed high egg yolk immunoglobulin (IgY) titres specific to LTB, STa and STb, and all were significantly higher than those in the control group (P < 0·01). A suckling mouse assay showed that anti-Bab IgY can neutralize the natural toxicity of STa and STb with the highest dilution of 1/2 and 1/32, respectively. Genetically constructed Bab induced significant antibody responses against STa and STb in chickens, and the resulting IgY had the capacity to neutralize the toxicity of ST. The recombinant Bab protein containing three important ETEC enterotoxins may serve as an effective and convenient polyvalent toxoid that can be used to produce multiple antitoxin IgYs to prevent colibacillosis caused by ETEC with various fimbriae in young animals. © 2014 The Society for Applied Microbiology.

  8. [Multiplex PCR strategy for the simultaneous identification of Staphylococcus aureus and detection of staphylococcal enterotoxins in isolates from food poisoning outbreaks].

    PubMed

    Brizzio, Aníbal A; Tedeschi, Fabián A; Zalazar, Fabián E

    2013-01-01

    Staphylococcal food poisoning is the most frequent type of food poisoning around the world. Staphylococcus aureus enterotoxins cause significant loss of water in the intestinal lumen, followed by vomiting and diarrhea. To report a fast, reliable and inexpensive strategy based on multiplex PCR for the simultaneous identification of S. aureus and detection of five classical S. aureus enterotoxin genes ( sea, seb, sec, sed, see ) in Staphylococcus spp. strains isolated from food poisoning outbreaks. We analyzed isolates from 12 food poisoning outbreaks occurred in Santa Fe province (Argentina). Isolation and phenotypic characterization were carried out by standard procedures. Genotypic analysis was performed by multiplex PCR, using primers for nuc , sea-see and 16S rRNA genes simultaneously. Of all the strains tested, 58% were found to carry toxigenic genes. Sea and seb toxins were found at the same percentage (29%) while sec, sed and see genes were found in a lower and identical proportion (14%). We did not find more than one different type of S. aureus enterotoxin in the isolates analyzed. The multiplex PCR strategy designed in this work has enabled us to identify strains of S. aureus and detect -at the same time- their enterotoxigenic ability. At present, our efforts are devoted to the detection of genes encoding enterotoxins other than the classical ones, in order to know their impact on staphylococcal food poisoning, as well as to investigate their relevance to our country's public health.

  9. Acetic acid increases the phage-encoded enterotoxin A expression in Staphylococcus aureus

    PubMed Central

    2010-01-01

    Background The effects of acetic acid, a common food preservative, on the bacteriophage-encoded enterotoxin A (SEA) expression and production in Staphylococcus aureus was investigated in pH-controlled batch cultures carried out at pH 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5. Also, genomic analysis of S. aureus strains carrying sea was performed to map differences within the gene and in the temperate phage carrying sea. Results The sea expression profile was similar from pH 7.0 to 5.5, with the relative expression peaking in the transition between exponential and stationary growth phase and falling during stationary phase. The levels of sea mRNA were below the detection limit at pH 5.0 and 4.5, confirmed by very low SEA levels at these pH values. The level of relative sea expression at pH 6.0 and 5.5 were nine and four times higher, respectively, in the transitional phase than in the exponential growth phase, compared to pH 7.0 and pH 6.5, where only a slight increase in relative expression in the transitional phase was observed. Furthermore, the increase in sea expression levels at pH 6.0 and 5.5 were observed to be linked to increased intracellular sea gene copy numbers and extracellular sea-containing phage copy numbers. The extracellular SEA levels increased over time, with highest levels produced at pH 6.0 in the four growth phases investigated. Using mitomycin C, it was verified that SEA was at least partially produced as a consequence of prophage induction of the sea-phage in the three S. aureus strains tested. Finally, genetic analysis of six S. aureus strains carrying the sea gene showed specific sea phage-groups and two versions of the sea gene that may explain the different sea expression and production levels observed in this study. Conclusions Our findings suggest that the increased sea expression in S. aureus caused by acetic acid induced the sea-encoding prophage, linking SEA production to the lifecycle of the phage. PMID:20487538

  10. 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

  11. 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

  12. 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

  13. 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

  14. Gold nanoparticle-based localized surface plasmon immunosensor for staphylococcal enterotoxin A (SEA) detection.

    PubMed

    Ben Haddada, Maroua; Hu, David; Salmain, Michèle; Zhang, Lu; Peng, Chen; Wang, Yi; Liedberg, Bo; Boujday, Souhir

    2017-08-16

    We describe the engineering of stable gold nanoparticle (AuNP) bioconjugates for the detection of staphylococcal enterotoxin A (SEA) using localized surface plasmon resonance (LSPR). Two types of AuNP bioconjugates were prepared by covalently attaching anti-SEA antibody (Ab) or SEA to AuNPs. This was achieved by reacting Traut's reagent with lysine residues of both proteins to generate thiol groups that bind to gold atoms on the AuNP surface. These bioconjugates were characterized in-depth by absorption spectroscopy, cryo-transmission electron microscopy, dynamic light scattering, and zeta potential measurements. Their stability over time was assessed after 1 year storage in the refrigerator at 4 °C. Two formats of homogeneous binding assays were set up on the basis of monitoring of LSPR peak shifts resulting from the immunological reaction between the (i) immobilized antibody and free SEA, the direct assay, or (ii) immobilized SEA and free antibody, the competitive assay. In both formats, a correlation between the LSPR band shift and SEA concentration could be established. Though the competitive format did not meet the expected analytical performance, the direct format, the implementation of which was very simple, afforded a specific and sensitive response within a broad dynamic range-nanogram per milliliter to microgram per milliliter. The limit of detection (LOD) of SEA was estimated to equal 5 ng/mL, which was substantially lower than the LOD obtained using a quartz crystal microbalance. Moreover, the analytical performance of AuNP-Ab bioconjugate was preserved after 1 year of storage at 4 °C. Finally, the LSPR biosensor was successfully applied to the detection of SEA in milk samples. The homogeneous nanoplasmonic immunosensor described herein provides an attractive alternative for stable and reliable detection of SEA in the nanogram per milliliter range and offers a promising avenue for rapid, easy to implement, and sensitive biotoxin detection

  15. Staphylococcal enterotoxin IgE sensitization in late-onset severe eosinophilic asthma in the elderly.

    PubMed

    Song, W-J; Sintobin, I; Sohn, K-H; Kang, M-G; Park, H-K; Jo, E-J; Lee, S-E; Yang, M-S; Kim, S-H; Park, H-K; Kwon, Y E; Kim, T-B; Kim, S-H; Park, H-W; Chang, Y-S; Lee, B-J; Jee, Y-K; Choi, B W; Bachert, C; Cho, S-H

    2016-03-01

    Asthma in the elderly (aged ≥ 65 years old) is a significant concern with high morbidity, but the pathophysiology remains unclear particularly in late-onset asthma. Recent studies suggest staphylococcal enterotoxin IgE (SE-IgE) sensitization to be a risk factor for asthma in general populations; however, the associations have not been examined in late-onset elderly asthma. We aimed to examine the associations of SE-IgE sensitization with late-onset asthma in the elderly, using a database of elderly asthma cohort study. A total of 249 elderly patients with asthma and 98 controls were analysed. At baseline, patients were assessed for demographics, atopy, induced sputum profiles and comorbidities including chronic rhinosinusitis (CRS). Serum total IgE and SE-IgE levels were measured. Asthma severity was assessed on the basis of asthma outcomes during a 12-month follow-up period. At baseline, serum SE-IgE concentrations were significantly higher in patients with asthma than in controls [median 0.16 (interquartile range 0.04-0.53) vs. 0.10 (0.01-0.19), P < 0.001]. Elderly asthma patients with high SE-IgE levels had specific characteristics of having more severe asthma, sputum eosinophilia and CRS, compared to those with lower SE-IgE levels. In multivariate logistic regression analyses, the associations between serum SE-IgE concentrations and severe asthma were significant, independently of covariables [SE-IgE-high (≥ 0.35 kU/L) vs. negative (< 0.10 kU/L) group: odds ratio 7.47, 95% confidence interval 1.86-30.03, P = 0.005]. Multiple correspondence analyses also showed that high serum SE-IgE level had close relationships with severe asthma, CRS and sputum eosinophilia together. This is the first report on the significant associations of SE-IgE sensitization with late-onset asthma in the elderly, particularly severe eosinophilic asthma with CRS comorbidity. Our findings indicate a potential implication of SE in the high morbidity burden of elderly asthma and suggest

  16. Rapid eradication of colon carcinoma by Clostridium perfringens Enterotoxin suicidal gene therapy.

    PubMed

    Pahle, Jessica; Menzel, Lutz; Niesler, Nicole; Kobelt, Dennis; Aumann, Jutta; Rivera, Maria; Walther, Wolfgang

    2017-02-13

    Bacterial toxins have evolved to an effective therapeutic option for cancer therapy. The Clostridium perfringens enterotoxin (CPE) is a pore-forming toxin with selective cytotoxicity. The transmembrane tight junction proteins claudin-3 and -4 are known high affinity CPE receptors. Their expression is highly upregulated in human cancers, including breast, ovarian and colon carcinoma. CPE binding to claudins triggers membrane pore complex formation, which leads to rapid cell death. Previous studies demonstrated the anti-tumoral effect of treatment with recombinant CPE-protein. Our approach aimed at evaluation of a selective and targeted cancer gene therapy of claudin-3- and/or claudin-4- expressing colon carcinoma in vitro and in vivo by using translation optimized CPE expressing vector. In this study the recombinant CPE and a translation optimized CPE expressing vector (optCPE) was used for targeted gene therapy of claudin-3 and/or -4 overexpressing colon cancer cell lines. All experiments were performed in the human SW480, SW620, HCT116, CaCo-2 and HT-29 colon cancer and the isogenic Sk-Mel5 and Sk-Mel5 Cldn-3-YFP melanoma cell lines. Claudin expression analysis was done at protein and mRNA level, which was confirmed by immunohistochemistry. The CPE induced cytotoxicity was analyzed by the MTT cytotoxicity assay. In addition patient derived colon carcinoma xenografts (PDX) were characterized and used for the intratumoral in vivo gene transfer of the optCPE expressing vector in PDX bearing nude mice. Claudin-3 and -4 overexpressing colon carcinoma lines showed high sensitivity towards both recCPE application and optCPE gene transfer. The positive correlation between CPE cytotoxicity and level of claudin expression was demonstrated. Transfection of optCPE led to targeted, rapid cytotoxic effects such as membrane disruption and necrosis in claudin overexpressing cells. The intratumoral optCPE in vivo gene transfer led to tumor growth inhibition in colon carcinoma PDX

  17. 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

  18. 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.

  19. Activation of intestinal CFTR Cl- channel by heat-stable enterotoxin and guanylin via cAMP-dependent protein kinase.

    PubMed Central

    Chao, A C; de Sauvage, F J; Dong, Y J; Wagner, J A; Goeddel, D V; Gardner, P

    1994-01-01

    Heat-stable enterotoxins (STa) produced by pathogenic bacteria induce profound salt and water secretion in the gut, leading to diarrhea. Recently, guanylin, an endogenous peptide with properties similar to STa, was identified. While STa and guanylin bind to the same receptor guanylyl cyclase and raise cell cGMP, the signaling mechanism distal to cGMP remains controversial. Here we show that STa, guanylin and cGMP each activate intestinal Cl- secretion, and that this is abolished by inhibitors of cAMP-dependent protein kinase (PKA), suggesting that PKA is a major mediator of this effect. These agents induce Cl- secretion only in cells expressing the wild-type CFTR, indicating that this molecule is the final common effector of the signaling pathway. The involvement of CFTR suggests a possible cystic fibrosis heterozygote advantage against STa-induced diarrhea. PMID:7510634

  20. 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%).

  1. 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.

  2. Recent advances and new perspectives in targeting CFTR for therapy of cystic fibrosis and enterotoxin-induced secretory diarrheas

    PubMed Central

    Zhang, Weiqiang; Fujii, Naoaki; Naren, Anjaparavanda P

    2012-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical surfaces of epithelial cells lining airway, gut and exocrine glands, where it is responsible for transepithelial salt and water transport. Several human diseases are associated with an altered channel function of CFTR. Cystic fibrosis (CF) is caused by the loss or dysfunction of CFTR-channel activity resulting from the mutations on the gene; whereas enterotoxin-induced secretory diarrheas are caused by the hyperactivation of CFTR channel function. CFTR is a validated target for drug development to treat these diseases. Significant progress has been made in developing CFTR modulator therapy by means of high-throughput screening followed by hit-to-lead optimization. Several oral administrated investigational drugs are currently being evaluated in clinical trials for CF. Also importantly, new ideas and methodologies are emerging. Targeting CFTR-containing macromolecular complexes is one such novel approach. PMID:22393940

  3. 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.

  4. 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.

  5. 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.

  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. Interaction of Escherichia coli heat-stable enterotoxin (STa) with its putative receptor on the intestinal tract of newborn kids.

    PubMed

    Al-Majali, Ahmad M; Ababneh, Mohammed M; Shorman, M; Saeed, A Mahdi

    2007-02-01

    The elaboration of heat stable enterotoxin (STa) is an important step in the pathogenesis of enterotoxigenic Escherichia coli (ETEC), which causes severe diarrhea in newborn animals. In this study, the distribution of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from the anterior jejunum, posterior jejunum, ileum and colon of newborn kids was investigated. The density of STa-receptors on enterocytes and BBMVs was higher in the posterior jejunum than that in other segments of the kids' intestines. Additionally, the affinity of the posterior jejunum STa-receptors was higher than the affinity of receptors present on the epithelium of other intestinal segments. Our findings suggest that the posterior jejunum is a major target for STa within the intestinal tract of newborn kids.

  8. 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.

  9. 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.

  10. Characterization of the interaction of staphylococcal enterotoxin B with CD1d expressed in human renal proximal tubule epithelial cells.

    PubMed

    Hammamieh, Rasha; Chakraborty, Nabarun; Lin, Yixin; Shupp, Jeffrey W; Miller, Stacy-Ann; Morris, Sam; Jett, Marti

    2015-02-04

    Participation of renal cells in the pathogenesis of staphylococcal enterotoxin B (SEB) is critical for late cleansing and sequestration of the antigens facilitated by CD1d mediated antigen sensing and recognition. This is a noted deviation from the typical antigen recognition process that recruits the major histocompatibility complex class II (MHC II) molecules. The immunological importance of CD1d is underscored by its influences on the performances of natural killer T-cells and thereby mediates the innate and adaptive immune systems. Using diffraction-based dotReady™ immunoassays, the present study showed that SEB directly and specifically conjugated to CD1d. The specificity of the conjugation between SEB and CD1d expressed on human renal proximal tubule epithelial cells (RPTEC) was further established by selective inhibition of CD1d prior to its exposure to SEB. We found that SEB induced the expression of CD1d on the cell surface prompting a rapid conjugation between them. The mRNA transcripts encoding CD1d remained elevated potentially after completing the antigen cleansing process. Molecular targets associated with the delayed pathogenic response have essential therapeutic values. Particularly in the event of bioterrorism, the caregivers are typically able to intervene much later than the toxic exposures. Given circumstances mandate a paradigm shift from the conventional therapeutic strategy that counts on targeting the host markers responding to the early assault of pathogens. We demonstrated the role of CD1d in the late stage of pathogen recognition and cleansing, and thereby underscored its clinical potential in treating bioweaponizable antigens, such as Staphylococcal enterotoxin B (SEB).

  11. Certified reference materials for testing of the presence/absence of Staphylococcus aureus enterotoxin A (SEA) in cheese.

    PubMed

    Zeleny, Reinhard; Nia, Yacine; Schimmel, Heinz; Mutel, Isabelle; Hennekinne, Jacques-Antoine; Emteborg, Håkan; Charoud-Got, Jean; Auvray, Frédéric

    2016-08-01

    Staphylococcal enterotoxins (SEs) account for a substantial number of food-poisoning outbreaks. European legislation (Commission Regulation 1441/2007) stipulates the reference procedure for SE analysis in milk and dairy products, which is based on extraction, dialysis concentration and immunochemical detection using one of two approved assays (VIDAS(®) SET2, Ridascreen(®) SET Total). However, certified reference materials (CRMs) are lacking to support laboratories in performing reliable detection of Staphylococcus aureus enterotoxin A (SEA) in relevant matrices at sub-nanogram per gram levels. The certification of a set of three reference materials (blank and two SEA-containing materials) for testing of the presence/absence of SEA in cheese is described. The reference procedure was applied in an intercomparison with 15 laboratories, and results were reported in a qualitative manner (presence or absence of SEA in the sample). No false-negative or false-positive results were obtained. The certified values were stated as diagnostic specificity (blank material) or diagnostic sensitivity (SEA-containing materials) and were 100 % in all cases. Stability studies demonstrated suitable material stability when stored cooled or frozen. An in-house study on the recovery of SEA in the cheese materials using a double-sandwich enzyme-linked immunosorbent assay (ELISA) revealed comparable recovery values of around 45 % at the two spiking levels and in both the SEA-containing CRMs as well as blank CRM freshly spiked prior to analysis. The values were also comparable over time and among different analysts. The materials provide valuable support to laboratories for method validation and method performance verification and will increase the reliability of measuring SEA in cheese.

  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. Application of r-PFE hyperimmune sera for concurrent detection of Bacillus anthracis, Yersinia pestis and staphylococcal enterotoxin B.

    PubMed

    Balakrishna, K; Tuteja, U; Murali, H S; Batra, H V

    2014-06-01

    To evaluate the potential of an intergeneric multidomain recombinant chimeric protein for the simultaneous detection of Bacillus anthracis, Yersinia pestis and Staphylococcal enterotoxin B. Truncated portions of protective antigen (pag) of B. anthracis, fraction 1 capsular antigen (F1) of Y. pestis and enterotoxin B (entB) of Staphylococcus aureus were PCR amplified and linked each other using ligation-dependent cloning. The fusion gene was codon-optimized for expression in Escherichia coli and encoded a 55 kDa recombinant PFE protein (rPFE). Hyperimmune antiserum raised against rPFE specifically reacted individually with the native PA of B. anthracis, F1 antigen of Y. pestis and SEB of S. aureus on Western blot analysis as well as in enzyme-linked immunosorbent assay (ELISA). For simultaneous detection of these three antigens from culture supernatants, common media consisting of BHI broth supplemented with 0·2% xylose were used. To assess the detection capability, a known number of these organisms (10(8) -10(2) CFU ml(-1)) were experimentally spiked on to the meat and blood samples, the polyclonal antibodies were again clearly able to identify all three target proteins up to a dilution of 10(5) CFU ml(-1). This recombinant chimeric protein-based immunodetection approach may eventually provide advantages over PCR formats during onsite investigations of biological emergencies or even during routine testing by laboratories. The trivalent recombinant PFE protein could be a novel intervention for possible diagnosis/detection of potential biological agents simultaneously in environmental and clinical samples to reduce the responding time and minimize the impact of the bioattack. © 2014 The Society for Applied Microbiology.

  14. Modulation of transforming growth factor‑β signaling transducers in colon adenocarcinoma cells induced by staphylococcal enterotoxin B.

    PubMed

    Akbari, Abolfazl; Mobini, Gholam Reza; Maghsoudi, Reza; Akhtari, Javad; Faghihloo, Ebrahim; Farahnejad, Zohreh

    2016-01-01

    Colorectal cancer (CRC) is a notable cause of cancer‑associated mortality worldwide, making it a pertinent topic for the study of cancer and its treatment. Staphylococcal enterotoxin B (SEB), an enterotoxin produced by Staphylococcus aureus, has been demonstrated to exert anticancer and antimetastatic effects due to its ability to modify cell immunity and cellular signaling pathways. In the current study, SEB was investigated, including whether it exerts its growth inhibitory effects on colon adenocarcinoma cells. This may occur through the manipulation of a key tumor growth factor, termed transforming growth factor‑β (TGF‑β), and its signaling pathway transducer, Smad2/3. The human colon adenocarcinoma HCT116 cell line was treated with different concentrations of SEB, and cell number was measured using MTT assay at different treatment times. Smad2/3 RNA expression level was analyzed in untreated or SEB‑treated cells using quantitative polymerase chain reaction, which indicated significant differences between cell viability and Smad2/3 expression levels. SEB effectively downregulated Smad2/3 expression in the HCT116 cells at concentrations of 1 and 2 µg/ml (P=0.0021 and P=0.0017, respectively). SEB concentrations that were effective at inhibiting Smad2/3 expression were correlated with those able to inhibit the proliferation of the cancer cells. SEB inhibited Smad2/3 expression at the mRNA level in a concentration‑ and time‑dependent manner. The present study thus proposed SEB as an agent able to significantly reduce Smad2/3 expression in colon cancer cells, provoking moderate TGF‑β growth signaling and the reduction of tumor cell proliferation.

  15. 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

  16. 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

  17. 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.

  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. Prevalence and Genetic Characteristics of Staphylococcus aureus and Staphylococcus argenteus Isolates Harboring Panton-Valentine Leukocidin, Enterotoxins, and TSST-1 Genes from Food Handlers in Myanmar.

    PubMed

    Aung, Meiji Soe; San, Thida; Aye, Mya Mya; Mya, San; Maw, Win Win; Zan, Khin Nyein; Htut, Wut Hmone Win; Kawaguchiya, Mitsuyo; Urushibara, Noriko; Kobayashi, Nobumichi

    2017-08-04

    Asymptomatic carriers of toxigenic Staphylococcus aureus are potential source of diseases, including food poisoning. Toxigenic potential and genetic traits of colonizing S. aureus were investigated for 563 healthy food handlers in Myanmar. Carriage of S. aureus was found in 110 individuals (19.5%), and a total of 144 S. aureus isolates were recovered from nasal cavities (110 isolates) and hands (34 isolates). Panton-Valentine leucocidin genes (pvl) were detected in 18 isolates (12.5%), among which 11 isolates were classified into coa-VIa, agr type III, and ST1930 (CC96) that had been also detected in pvl-positive clinical isolates in Myanmar. A pvl-positive, ST2250 nasal isolate was identified as S.