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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. PMID:26616654

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

    PubMed

    Lu, Xi; Fu, Enqing; Xie, Yonghong; Jin, Faguang

    2016-10-01

    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.

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

    PubMed

    Lu, Xi; Fu, Enqing; Xie, Yonghong; Jin, Faguang

    2016-10-01

    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

  4. 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

  5. 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

  6. Evaluation of a ganglioside immunosorbent assay for detection of Escherichia coli heat-labile enterotoxin.

    PubMed

    Bäck, E; Svennerholm, A M; Holmgren, J; Möllby, R

    1979-12-01

    The GM1 ganglioside enzyme-linked immunosorbent assay (GM1-ELISA), an immunological method for detection of Escherichia coli heat-labile enterotoxin (LT), was quantitatively and qualitatively compared with the conventional adrenal cell test for the identification of LT-producing strains. A micromodification model of the assay was developed. Enterotoxin preparations from 120 E. coli isolates from individuals with diarrhea, which had been previously shown to be enterotoxigenic by the adrenal cell test, and from 44 control strains of E. coli were compared in parallel by the two methods. Quantitatively the covariation of the enterotoxin titers was highly significant (RS = 0.98, P less than 0.001), the GM1-ELISA being somewhat more sensitive than the adrenal cell test. The methodological error was less than 5% in both tests. Qualitatively the overall agreement for positive and negative reactions for the two methods was 89%. The GM1-ELISA is practical for routine use in the diagnosis of enterotoxigenic E. coli, especially in laboratories without facilities for cell culture.

  7. Expression of functional pentameric heat-labile enterotoxin B subunit of Escherichia coli in Saccharomyces cerevisiae.

    PubMed

    Lim, Jung-Gu; Kim, Jung-Ae; Chung, Hea-Jong; Kim, Tae-Geum; Kim, Jung-Mi; Lee, Kyung-Ryul; Park, Seung-Moon; Yang, Moon-Sik; Kim, Dae-Hyuk

    2009-05-01

    Although the Escherichia coli heat-labile enterotoxin B subunit (LTB) has already been expressed in several different systems, including prokaryotic and eukaryotic organisms, studies regarding the synthesis of LTB into oligomeric structures of pentameric size in the budding yeast Saccharomyces cerevisiae have been limited. Therefore, this study used a functional signal peptide of the amylase 1A protein from rice to direct the yeast-expressed LTB towards the endoplasmic reticulum to oligomerize with the expected pentameric size. The expression and assembly of the recombinant LTB were confirmed in both the cell-free extract and culture media of the recombinant strain using a Western blot analysis. The binding of the LTB pentamers to intestinal epithelial cell membrane glycolipid receptors was further verified using a GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). On the basis of the GM1-ELISA results, pentameric LTB proteins comprised approximately 0.5-2.0% of the total soluble proteins, and the maximum quantity of secreted LTB was estimated to be 3 mg/l after a 3-day cultivation period. Consequently, the synthesis of LTB monomers and their assembly into biologically active oligomers in a recombinant S. cerevisiae strain demonstrated the feasibility of using a GRAS microorganism-based adjuvant, as well as the development of carriers against mucosal disease. PMID:19494699

  8. Immunological Study of the Heat-Labile Enterotoxins of Escherichia coli and Vibrio cholerae

    PubMed Central

    Gyles, Carlton L.

    1974-01-01

    Immunodiffusion experiments were conducted to associate a precipitin line with Escherichia coli heat-labile enterotoxin (LT). Wild strains of porcine and of human enteropathogenic E. coli as well as laboratory-derived enterotoxigenic variants of E. coli K-12 were used for LT antigen preparations. These were produced mainly by ultrafiltration and ammonium sulfate precipitation of broth culture supernatants. When antisera with anti-LT activity were reacted with antigen preparations from Ent+ and Ent− variants of E. coli K-12, a line “a” was given by Ent+ but not by Ent− preparations. Line “a” was removed by absorption of anti-LT serum with antigen preparation from an Ent+E. coli K-12, but was unaffected when the antigen preparation used to absorb the serum was from an Ent−E. coli K-12. A line identical to “a” was given by antigen preparations from wild strains of porcine enteropathogenic E. coli reacted with homologous or heterologous anti-LT sera. One human strain of enteropathogenic E. coli was shown to possess an antigen identical to that which gave rise to line “a.” To demonstrate this line it was necessary to use high concentrations of gammaglobulin and high concentrations of the crude antigen preparations. LT preparations reacted with anticholera toxin to give a line “c,” which showed a reaction of partial identity with line “b” produced by reaction of pure choleragenoid and anticholera toxin. Lines “a” and “c” gave reactions of identity. Images PMID:4206029

  9. Role of trypsin-like cleavage at arginine 192 in the enzymatic and cytotonic activities of Escherichia coli heat-labile enterotoxin.

    PubMed Central

    Grant, C C; Messer, R J; Cieplak, W

    1994-01-01

    Previous studies of cholera toxin and Escherichia coli heat-labile enterotoxin have suggested that proteolytic cleavage plays an important role in the expression of ADP-ribosyltransferase activity and toxicity. Specifically, several studies have implicated a trypsin-like cleavage at arginine 192, which lies within an exposed region subtended by a disulfide bond in the intact A subunit, in toxicity. To investigate the role of this modification in the enzymatic and cytotonic properties of heat-labile enterotoxin, the response of purified, recombinant A subunit to tryptic activation and the effect of substituting arginine 192 with glycine on the activities of the holotoxin were examined. The recombinant A subunit of heat-labile enterotoxin exhibited significant levels of ADP-ribosyltransferase activity that were only nominally increased (approximately twofold) by prior limited trypsinolysis. The enzymatic activity also did not appear to be affected by auto-ADP-ribosylation that occurs during the high-level synthesis of the recombinant A subunit in E. coli. A mutant form of the holotoxin containing the arginine 192-to-glycine substitution exhibited levels of cytotonic activity for CHO cells that were similar to that of the untreated, wild-type holotoxin but exhibited a marked delay in the ability to increase intracellular levels of cyclic AMP in Caco-2 cells. The results indicate that trypsin-like cleavage of the A subunit of E. coli heat-labile enterotoxin at arginine 192 is not requisite to the expression of enzymatic activity by the A subunit and further reveal that this modification, although it enhances the biological and enzymatic activities of the toxin, is not absolutely required for the enterotoxin to elicit cytotonic effects. Images PMID:7927684

  10. Intranuclear delivery of an antiviral peptide mediated by the B subunit of Escherichia coli heat-labile enterotoxin

    PubMed Central

    Loregian, Arianna; Papini, Emanuele; Satin, Barbara; Marsden, Howard S.; Hirst, Timothy R.; Palù, Giorgio

    1999-01-01

    We report an intracellular peptide delivery system capable of targeting specific cellular compartments. In the model system we constructed a chimeric protein consisting of the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) fused to a 27-mer peptide derived from the DNA polymerase of herpes simplex virus 1. Viral DNA synthesis takes places in the nucleus and requires the interaction with an accessory factor, UL42, encoded by the virus. The peptide, designated Pol, is able to dissociate this interaction. The chimeric protein, EtxB-Pol, retained the functional properties of both EtxB and peptide components and was shown to inhibit viral DNA polymerase activity in vitro via disruption of the polymerase-UL42 complex. When added to virally infected cells, EtxB-Pol had no effect on adenovirus replication but specifically interfered with herpes simplex virus 1 replication. Further studies showed that the antiviral peptide localized in the nucleus, whereas the EtxB component remained associated with vesicular compartments. The results indicate that the chimeric protein entered through endosomal acidic compartments and that the Pol peptide was cleaved from the chimeric protein before being translocated into the nucleus. The system we describe is suitable for delivery of peptides that specifically disrupt protein–protein interactions and may be developed to target specific cellular compartments. PMID:10220447

  11. 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.

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

    PubMed Central

    Greene, Christopher J.; Rong, Yinghui; Mandell, Lorrie M.; Connell, Terry D.

    2015-01-01

    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-IIbT13I, and LT-IIc, have never been compared side by side. We therefore conducted immunization studies in which LT-IIa, LT-IIb, LT-IIbT13I, 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. PMID:26491037

  13. Heat-labile enterotoxin-induced activation of NF-κB and MAPK pathways in intestinal epithelial cells impacts enterotoxigenic Escherichia coli (ETEC) adherence.

    PubMed

    Wang, Xiaogang; Gao, Xiaofei; Hardwidge, Philip R

    2012-08-01

    Enterotoxigenic Escherichia coli (ETEC) causes human morbidity and mortality in developing nations and is an emerging threat to food safety in developed nations. The ETEC heat-labile enterotoxin (LT) not only causes diarrheal disease by deregulating host adenylate cyclase, but also enhances ETEC adherence to intestinal epithelial cells. The mechanism governing this LT pro-adherence phenotype is unclear. Here we investigated intestinal epithelial cell signal transduction pathways activated by ETEC and quantified the relative importance of these host pathways to LT-induced ETEC adherence. We show that ETEC activates both NF-κB and mitogen-activated protein kinase signalling pathways through mechanisms that are primarily dependent upon LT. LT-induced NF-κB activation depends upon the cAMP-dependent activation of the Ras-like GTPase Rap1 but is independent of protein kinase A (PKA). By using inhibitors of these pathways, we demonstrate that inhibiting the p38 mitogen-activated protein kinase prevents LT from increasing ETEC adherence. By contrast, the LT pro-adherence phenotype appears unrelated to both LT-induced Rap1 activity and to subsequent NF-κB activation. We speculate that LT may alter host signal transduction to induce the presentation of ligands for ETEC adhesins in such a way that promotes ETEC adherence. Our findings provide insight into previously unexplored functions of LT and their relative importance to ETEC virulence. PMID:22452361

  14. Relationship between heat-labile enterotoxin secretion capacity and virulence in wild type porcine-origin enterotoxigenic Escherichia coli strains.

    PubMed

    Wijemanne, Prageeth; Xing, Jun; Berberov, Emil M; Marx, David B; Francis, David H; Moxley, Rodney A

    2015-01-01

    Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system (T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme-linked immunosorbent assay to measure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had the highest LT secretion level all porcine-origin WT strains tested (P<0.05). The level of LT secretion (concentration in supernatants at 6-h culture) explained 92% of the variation in time-to-a-moribund-condition (R2 = 0.92, P<0.0001) in gnotobiotic piglets inoculated with either strain 3030-2, or an ETEC strain of lesser virulence (2534-86), or a non-enterotoxigenic WT strain (G58-1). All 16 porcine ETEC strains were positive by PCR analysis for the T2SS genes, gspD and gspK, and bioinformatic analysis of 4 porcine-origin strains for which complete genomic sequences were available revealed a T2SS with a high degree of homology to that of H10407. Maximum Likelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18. Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml. This study demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and

  15. Antibodies to heat-labile Escherichia coli enterotoxins in human milk and sera. A study of Ethiopian and Swedish mothers and their children.

    PubMed

    Aust-Kettis, A; Gebre-Medhin, M; Habte, D; Khosla, N; Wadström, T

    1981-09-01

    Maternal serum and cord blood from 50 Ethiopian, 10 Costa Rican and 20 Swedish newly delivered mothers and their babies was examined for the presence of antibodies against heat labile (LT) enterotoxin from a human strain of E. coli. 96% of the Ethiopian, 80% of the Costa Rican and 30% of the Swedish mothers and infants had detectable antibody levels. The titres were significantly higher in the Ethiopian material. Furthermore, antibody titres to E. coli enterotoxin were determined in breast milk collected from Ethiopian mothers at 48 h and at 1 month after delivery. One third of these mothers had detectable levels of antibodies in samples from early lactation. Experiments performed with LT enterotoxin from another human and with LT from a porcine E. coli strain confirmed the results. Neutralization tests with cholera enterotoxin as antigen were all negative in sera and milk samples from all these groups. The material has been collected in three different geographical areas which are nonendemic for cholera. PMID:7032015

  16. 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

  17. Type II Heat-Labile Enterotoxins from 50 Diverse Escherichia coli Isolates Belong Almost Exclusively to the LT-IIc Family and May Be Prophage Encoded

    PubMed Central

    Jobling, Michael G.; Holmes, Randall K.

    2012-01-01

    Some enterotoxigenic Escherichia coli (ETEC) produce a type II heat-labile enterotoxin (LT-II) that activates adenylate cyclase in susceptible cells but is not neutralized by antisera against cholera toxin or type I heat-labile enterotoxin (LT-I). LT-I variants encoded by plasmids in ETEC from humans and pigs have amino acid sequences that are ≥95% identical. In contrast, LT-II toxins are chromosomally encoded and are much more diverse. Early studies characterized LT-IIa and LT-IIb variants, but a novel LT-IIc was reported recently. Here we characterized the LT-II encoding loci from 48 additional ETEC isolates. Two encoded LT-IIa, none encoded LT-IIb, and 46 encoded highly related variants of LT-IIc. Phylogenetic analysis indicated that the predicted LT-IIc toxins encoded by these loci could be assigned to 6 subgroups. The loci corresponding to individual toxins within each subgroup had DNA sequences that were more than 99% identical. The LT-IIc subgroups appear to have arisen by multiple recombinational events between progenitor loci encoding LT-IIc1- and LT-IIc3-like variants. All loci from representative isolates encoding the LT-IIa, LT-IIb, and each subgroup of LT-IIc enterotoxins are preceded by highly-related genes that are between 80 and 93% identical to predicted phage lysozyme genes. DNA sequences immediately following the B genes differ considerably between toxin subgroups, but all are most closely related to genomic sequences found in predicted prophages. Together these data suggest that the LT-II loci are inserted into lambdoid type prophages that may or may not be infectious. These findings raise the possibility that production of LT-II enterotoxins by ETEC may be determined by phage conversion and may be activated by induction of prophage, in a manner similar to control of production of Shiga-like toxins by converting phages in isolates of enterohemmorhagic E. coli. PMID:22242186

  18. Simultaneous Exposure to Escherichia coli Heat-Labile and Heat-Stable Enterotoxins Increases Fluid Secretion and Alters Cyclic Nucleotide and Cytokine Production by Intestinal Epithelial Cells

    PubMed Central

    Read, Lisa T.; Hahn, Rachel W.; Thompson, Carli C.; Bauer, David L.; Norton, Elizabeth B.

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC) is a significant cause of diarrheal disease and death, especially in children in developing countries. ETEC causes disease by colonizing the small intestine and producing heat-labile toxin (LT), heat-stable toxin (ST), or both LT and ST (LT+ST). The majority of ETEC strains produce both ST and LT. Despite the prevalence of LT+ST-producing organisms, few studies have examined the physiologic or immunologic consequences of simultaneous exposure to these two potent enterotoxins. In the current report, we demonstrate that when LT and ST are both present, they increase water movement into the intestinal lumen over and above the levels observed with either toxin alone. As expected, cultured intestinal epithelial cells increased their expression of intracellular cyclic GMP (cGMP) when treated with ST and their expression of intracellular cyclic AMP (cAMP) when treated with LT. When both toxins were present, cGMP levels but not cAMP levels were synergistically elevated compared with the levels of expression caused by the corresponding single-toxin treatment. Our data also demonstrate that the levels of inflammatory cytokines produced by intestinal epithelial cells in response to LT are significantly reduced in animals exposed to both enterotoxins. These findings suggest that there may be complex differences between the epithelial cell intoxication and, potentially, secretory outcomes induced by ETEC strains expressing LT+ST compared with strains that express LT or ST only. Our results also reveal a novel mechanism wherein ST production may reduce the hosts' ability to mount an effective innate or adaptive immune response to infecting organisms. PMID:25287923

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

    PubMed

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

    2010-05-11

    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 > or = 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.

  20. Subacute effects of maize-expressed vaccine protein, Escherichia coli heat-labile enterotoxin subunit B (LTB), on the Springtail, Folsomia candida , and the earthworm, Eisenia fetida.

    PubMed

    Kosaki, Hirofumi; Wolt, Jeffrey D; Wang, Kan; Coats, Joel R

    2008-12-10

    The ecotoxicological effects of transgenic maize-expressed vaccine protein, Escherichia coli heat-labile enterotoxin subunit B (LTB), on two soil invertebrates were studied under laboratory settings. After being reared for 28 days on LTB-maize-treated soils, no apparent mortality of the springtail, Folsomia candida , or the earthworm, Eisenia fetida , was observed at levels well above conservatively projected estimated environmental concentrations. Therefore, it is concluded that there would be no acutely toxic effect of LTB to these species. As for the subacute effect, no significant differences of F. candida mean reproduction and E. fetida mean growth were observed between LTB-maize-treated samples and non-GM-maize-treated controls. In addition, no LTB was detected in the E. fetida whole-body extraction assay, which indicates there was no tendency for bioaccumulation. On the basis of these observations, it is predicted that any adverse effects of LTB-maize on F. candida and E. fetida would be minimal, if any.

  1. Characterization of Receptor-Mediated Signal Transduction by Escherichia coli Type IIa Heat-Labile Enterotoxin in the Polarized Human Intestinal Cell Line T84

    PubMed Central

    Wimer-Mackin, Susan; Holmes, Randall K.; Wolf, Anne A.; Lencer, Wayne I.; Jobling, Michael G.

    2001-01-01

    Escherichia coli type IIa heat-labile enterotoxin (LTIIa) binds in vitro with highest affinity to ganglioside GD1b. It also binds in vitro with lower affinity to several other oligosialogangliosides and to ganglioside GM1, the functional receptor for cholera toxin (CT). In the present study, we characterized receptor-mediated signal transduction by LTIIa in the cultured T84 cell model of human intestinal epithelium. Wild-type LTIIa bound tightly to the apical surface of polarized T84 cell monolayers and elicited a Cl− secretory response. LTIIa activity, unlike CT activity, was not blocked by the B subunit of CT. Furthermore, an LTIIa variant with a T14I substitution in its B subunit, which binds in vitro to ganglioside GM1 but not to ganglioside GD1b, was unable to bind to intact T84 cells and did not elicit a Cl− secretory response. These findings show that ganglioside GM1 on T84 cells is not a functional receptor for LTIIa. The LTIIa receptor on T84 cells was inactivated by treatment with neuraminidase. Furthermore, LTIIa binding was blocked by tetanus toxin C fragment, which binds to gangliosides GD1b and GT1b. These findings support the hypothesis that ganglioside GD1b, or possibly a glycoconjugate with a GD1b-like oligosaccharide, is the functional receptor for LTIIa on T84 cells. The LTIIa-receptor complexes from T84 cells were associated with detergent-insoluble membrane microdomains (lipid rafts), extending the correlation between toxin binding to lipid rafts and toxin function that was previously established for CT. However, the extent of association with lipid rafts and the magnitude of the Cl− secretory response in T84 cells were less for LTIIa than for CT. These properties of LTIIa and the previous finding that enterotoxin LTIIb binds to T84 cells but does not associate with lipid rafts or elicit a Cl− secretory response may explain the low pathogenicity for humans of type II enterotoxin-producing isolates of E. coli. PMID:11705889

  2. Protection of piglets against enteric colibacillosis by intranasal immunization with K88ac (F4ac) fimbriae and heat labile enterotoxin of Escherichia coli.

    PubMed

    Lin, Jun; Mateo, Kristina S; Zhao, Mojun; Erickson, Alan K; Garcia, Nuria; He, Dong; Moxley, Rodney A; Francis, David H

    2013-03-23

    Enterotoxigenic Escherichia coli (ETEC) is an important diarrheal agent of young domestic animals. Currently, there are no commercially available non-living vaccines to protect weaned pigs from the disease and no major veterinary biologics company markets a postweaning ETEC vaccine of any kind. While efforts have been made to develop a non-living postweaning ETEC vaccine for pigs, studies have been limited to the assessment of immune responses to experimental immunogens. In the present study, we describe a reproducible gnotobiotic piglet model of post-weaning ETEC diarrhea and efficacy tests in that model of subunit vaccines consisting of K88 (F4) fimbriae and/or heat labile enterotoxin (LT) delivered by the intranasal route. We also report antibody responses to the vaccine antigens. Piglets vaccinated with both antigens mounted a substantial immune response with serum and cecal antibody titers to K88 antigen significantly greater than those of controls. Serum anti-LT antibody titers were also significantly greater than those of controls. Piglets vaccinated with both antigens remained healthy following challenge with ETEC. At least some pigs vaccinated with either antigen alone, and most of the control piglets developed dehydrating diarrhea and suffered significant weight loss. The results of this study suggest that an intranasal vaccine consisting of both antigens is highly protective against a vigorous experimental challenge of pigs with K88+ ETEC, while that against either antigen alone is not. The current study provides a system whereby various ETEC antigens and/or combinations of antigens can be tested in exploring strategies for the development of vaccines for ETEC. PMID:23089483

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

    PubMed Central

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

    2015-01-01

    DNA encoded adjuvants are well known for increasing the magnitude of cellular and/or humoral immune responses directed against vaccine antigens. DNA adjuvants can also tune immune responses directed against vaccine antigens to better protect against infection of the target organism. Two potent DNA adjuvants that have unique abilities to tune immune responses are the catalytic A1 domains of Cholera Toxin (CTA1) and Heat-Labile Enterotoxin (LTA1). Here, we have characterized the adjuvant activities of CTA1 and LTA1 using HIV and SIV genes as model antigens. Both of these adjuvants enhanced the magnitude of antigen-specific cellular immune responses on par with those induced by the well-characterized cytokine adjuvants IL-12 and GM-CSF. CTA1 and LTA1 preferentially enhanced cellular responses to the intracellular antigen SIVmac239-gag over those for the secreted HIVBaL-gp120 antigen. IL-12, GM-CSF and electroporation did the opposite suggesting differences in the mechanisms of actions of these diverse adjuvants. Combinations of CTA1 or LTA1 with IL-12 or GM-CSF generated additive and better balanced cellular responses to both of these antigens. Consistent with observations made with the holotoxin and the CTA1-DD adjuvant, CTA1 and LTA1 evoked mixed Th1/Th17 cellular immune responses. Together, these results show that CTA1 and LTA1 are potent DNA vaccine adjuvants that favor the intracellular antigen gag over the secreted antigen gp120 and evoke mixed Th1/Th17 responses against both of these antigens. The results also indicate that achieving a balanced immune response to multiple intracellular and extracellular antigens delivered via DNA vaccination may require combining adjuvants that have different and complementary mechanisms of action. PMID:26042527

  4. Comparative study on characterization of recombinant B subunit of E. coli heat-labile enterotoxin (rLTB) prepared from E. coli and P. patoris.

    PubMed

    Ma, Xingyuan; Yao, Bi; Zheng, Wenyun; Li, Linfeng

    2010-03-01

    Escherichia coli (E. coli) heat-labile enterotoxin B subunit (LTB) was regarded as one of the most powerful mucosal immunoadjuvants eliciting strong immunoresponse to coadministered antigens. In the research, the high-level secretory expression of functional LTB was achieved in P. pastoris through high-density fermentation in a 5-l fermentor. Meanwhile, the protein was expressed in E. coli by the way of inclusion body, although the gene was cloned from E. coli. Some positive yeast and E. coli transformants were obtained respectively by a series of screenings and identifications. Fusion proteins LTB-6x His could be secreted into the supernatant of the medium after the recombinant P. pastoris was induced by 0.5% (v/v) methanol at 30 degrees C, whereas E. coli transformants expressed target protein in inclusion body after being induced by 1 mM IPTG at 37 degrees C. The expression level increased dramatically to 250- 300 mg/l supernatant of fermentation in the former and 80-100 mg/l in the latter. The LTB-6x His were purified to 95% purity by affinity chromatography and characterized by SDS-PAGE and Western blot. Adjuvant activity of target protein was analyzed by binding ability with GM1 gangliosides. The MW of LTB-6x His expressed in P. pastoris was greater than that in E. coli, which was equal to the expected 11 kDa, possibly resulted from glycosylation by P. pastoris that would enhance the immunogenicity of co-administered antigens. These data demonstrated that P. pastoris producing heterologous LTB has significant advantages in higher expression level and in adjuvant activity compared with the homologous E. coli system. PMID:20372026

  5. Protective Mucosal Immunity to Ocular Herpes Simplex Virus Type 1 Infection in Mice by Using Escherichia coli Heat-Labile Enterotoxin B Subunit as an Adjuvant

    PubMed Central

    Richards, C. M.; Aman, A. T.; Hirst, T. R.; Hill, T. J.; Williams, N. A.

    2001-01-01

    The potential of nontoxic recombinant B subunits of cholera toxin (rCtxB) and its close relative Escherichia coli heat-labile enterotoxin (rEtxB) to act as mucosal adjuvants for intranasal immunization with herpes simplex virus type 1 (HSV-1) glycoproteins was assessed. Doses of 10 μg of rEtxB or above with 10 μg of HSV-1 glycoproteins elicited high serum and mucosal anti-HSV-1 titers comparable with that obtained using CtxB (10 μg) with a trace (0.5 μg) of whole toxin (Ctx-CtxB). By contrast, doses of rCtxB up to 100 μg elicited only meager anti-HSV-1 responses. As for Ctx-CtxB, rEtxB resulted in a Th2-biased immune response with high immunoglobulin G1 (IgG1)/IgG2a antibody ratios and production of interleukin 4 (IL-4) and IL-10 as well as gamma interferon by proliferating T cells. The protective efficacy of the immune response induced using rEtxB as an adjuvant was assessed following ocular challenge of immunized and mock-immunized mice. Epithelial disease was observed in both groups, but the immunized mice recovered by day 6 whereas mock-immunized mice developed more severe corneal disease leading to stromal keratitis. In addition, a significant reduction in the incidence of lid disease and zosteriform spread was observed in immunized animals and there was no encephalitis compared with 95% encephalitis in mock-immunized mice. The potential of such mucosal adjuvants for use in human vaccines against pathogens such as HSV-1 is discussed. PMID:11160664

  6. Attenuated Salmonella Gallinarum secreting an Escherichia coli heat-labile enterotoxin B subunit protein as an adjuvant for oral vaccination against fowl typhoid.

    PubMed

    Jeon, Byung Woo; Jawale, Chetan V; Kim, Seung Hwan; Lee, John Hwa

    2012-12-15

    In our previous study, we constructed a vaccine candidate (JOL916) for fowl typhoid (FT). A live adjuvant Salmonella Gallinarum (SG) strain was generated in the present study to facilitate efficacious oral vaccination with this vaccine. The Escherichia coli eltB gene secreting heat-labile enterotoxin B subunit (LTB) was cloned into an Asd(+) plasmid pJHL65. This was transformed into a Δlon ΔcpxR Δasd SG strain and the resulting strain was designated JOL1229. Secretion of LTB from JOL1229 was confirmed with an immunoblot assay. To determine the optimal dose of the strain, 50 six-week-old female chickens were divided into five groups (Groups A-E, n=10 per group) and orally inoculated with various doses of JOL1229 and JOL916. In Group B (consisting of four parts JOL916 and one part JOL1229), significant cell-mediated immune responses, plasma IgG levels and intestinal secretary IgA levels were induced after inoculation with both strains. On challenge with the wild-type strain, significant reductions in mortality were observed in the group. In addition, after inoculation the LTB strain was not recovered in feces samples, and resulted in no, or very mild, gross lesions in the liver and spleen. Both CD4(+) and CD8(+) T-cells were significantly increased in peripheral blood samples from the chickens immunized with the LTB strain. Expression of the interleukin-6 (IL-6) gene in splenocytes was induced in the chickens immunized with the LTB strain. These results suggest that oral immunization with the LTB-adjuvant strain, in particular with the four parts JOL916 and one part JOL1229 mixture, increased the immune response and provided efficient protection against FT in chickens.

  7. Salmonella enterica serovar enteritidis ghosts carrying the Escherichia coli heat-labile enterotoxin B subunit are capable of inducing enhanced protective immune responses.

    PubMed

    Jawale, Chetan V; Lee, John Hwa

    2014-06-01

    The Escherichia coli heat-labile enterotoxin B subunit (LTB) is a potent vaccine adjuvant. Salmonella enterica serovar Enteritidis ghosts carrying LTB (S. Enteritidis-LTB ghosts) were genetically constructed using a novel plasmid, pJHL187-LTB, designed for the coexpression of the LTB and E lysis proteins. S. Enteritidis-LTB ghosts were characterized using scanning electron microscopy to visualize their transmembrane tunnel structures. The expression of LTB in S. Enteritidis-LTB ghost preparations was confirmed by immunoblot and enzyme-linked immunosorbent assays. The parenteral adjuvant activity of LTB was demonstrated by immunizing chickens with either S. Enteritidis-LTB ghosts or S. Enteritidis ghosts. Chickens were intramuscularly primed at 5 weeks of age and subsequently boosted at 8 weeks of age. In total, 60 chickens were equally divided into three groups (n = 20 for each): group A, nonvaccinated control; group B, immunized with S. Enteritidis-LTB ghosts; and group C, immunized with S. Enteritidis ghosts. Compared with the nonimmunized chickens (group A), the immunized chickens (groups B and C) exhibited increased titers of plasma IgG and intestinal secretory IgA antibodies. The CD3(+) CD4(+) subpopulation of T cells was also significantly increased in both immunized groups. Among the immunized chickens, those in group B exhibited significantly increased titers of specific plasma IgG and intestinal secretory IgA (sIgA) antibodies compared with those in group C, indicating the immunomodulatory effects of the LTB adjuvant. Furthermore, both immunized groups exhibited decreased bacterial loads in their feces and internal organs. These results indicate that parenteral immunization with S. Enteritidis-LTB ghosts can stimulate superior induction of systemic and mucosal immune responses compared to immunization with S. Enteritidis ghosts alone, thus conferring efficient protection against salmonellosis. PMID:24671556

  8. 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

  9. Both enzymatic and non-enzymatic properties of heat-labile enterotoxin are responsible for LT-enhanced adherence of enterotoxigenic Escherichia coli to porcine IPEC-J2 cells.

    PubMed

    Fekete, Peter Z; Mateo, Kristina S; Zhang, Weiping; Moxley, Rodney A; Kaushik, Radhey S; Francis, David H

    2013-06-28

    Previous studies in piglets indicate that heat labile enterotoxin (LT) expression enhances intestinal colonization by K88 adhesin-producing enterotoxigenic Escherichia coli (ETEC) as wild-type ETEC adhered to intestinal epithelium in substantially greater numbers than did non-toxigenic constructs. Enzymatic activity of the toxin was also shown to contribute to the adhesion of ETEC and non-ETEC bacteria to epithelial cells in culture. To further characterize the contribution of LT to host cell adhesion, a nontoxigenic, K88-producing E. coli was transformed with either the gene encoding for LT holotoxin, a catalytically-attenuated form of the toxin [LT(R192G)], or LTB subunits, and resultant changes in bacterial adherence to IPEC-J2 porcine intestinal epithelial cells were measured. Strains expressing LT holotoxin or mutants were able to adhere in significantly higher numbers to IPEC-J2 cells than was an isogenic, toxin-negative construct. LT+ strains were also able to significantly block binding of a wild-type LT+ ETEC strain to IPEC-J2 cells. Adherence of isogenic strains to IPEC-J2 cells was unaltered by cycloheximide treatment, suggesting that LT enhances ETEC adherence to IPEC-J2 cells independent of host cell protein synthesis. However, pretreating IPEC-J2 cells with LT promoted adherence of negatively charged latex beads (a surrogate for bacteria which carry a negative change), which adherence was inhibited by cycloheximide, suggesting LT may induce a change in epithelial cell membrane potential. Overall, these data suggest that LT may enhance ETEC adherence by promoting an association between LTB and epithelial cells, and by altering the surface charge of the host plasma membrane to promote non-specific adherence.

  10. Both enzymatic and non-enzymatic properties of heat-labile enterotoxin are responsible for LT-enhanced adherence of enterotoxigenic Escherichia coli to porcine IPEC-J2 cells.

    PubMed

    Fekete, Peter Z; Mateo, Kristina S; Zhang, Weiping; Moxley, Rodney A; Kaushik, Radhey S; Francis, David H

    2013-06-28

    Previous studies in piglets indicate that heat labile enterotoxin (LT) expression enhances intestinal colonization by K88 adhesin-producing enterotoxigenic Escherichia coli (ETEC) as wild-type ETEC adhered to intestinal epithelium in substantially greater numbers than did non-toxigenic constructs. Enzymatic activity of the toxin was also shown to contribute to the adhesion of ETEC and non-ETEC bacteria to epithelial cells in culture. To further characterize the contribution of LT to host cell adhesion, a nontoxigenic, K88-producing E. coli was transformed with either the gene encoding for LT holotoxin, a catalytically-attenuated form of the toxin [LT(R192G)], or LTB subunits, and resultant changes in bacterial adherence to IPEC-J2 porcine intestinal epithelial cells were measured. Strains expressing LT holotoxin or mutants were able to adhere in significantly higher numbers to IPEC-J2 cells than was an isogenic, toxin-negative construct. LT+ strains were also able to significantly block binding of a wild-type LT+ ETEC strain to IPEC-J2 cells. Adherence of isogenic strains to IPEC-J2 cells was unaltered by cycloheximide treatment, suggesting that LT enhances ETEC adherence to IPEC-J2 cells independent of host cell protein synthesis. However, pretreating IPEC-J2 cells with LT promoted adherence of negatively charged latex beads (a surrogate for bacteria which carry a negative change), which adherence was inhibited by cycloheximide, suggesting LT may induce a change in epithelial cell membrane potential. Overall, these data suggest that LT may enhance ETEC adherence by promoting an association between LTB and epithelial cells, and by altering the surface charge of the host plasma membrane to promote non-specific adherence. PMID:23517763

  11. Protective efficacy of a Mycoplasma pneumoniae P1C DNA vaccine fused with the B subunit of Escherichia coli heat-labile enterotoxin.

    PubMed

    Zhu, Cuiming; Wang, Shiping; Hu, Shihai; Yu, Minjun; Zeng, Yanhua; You, Xiaoxing; Xiao, Jinhong; Wu, Yimou

    2012-06-01

    In the present study, we investigated the immunomodulatory responses of a DNA vaccine constructed by fusing Mycoplasma pneumoniae P1 protein carboxy terminal region (P1C) with the Escherichia coli heat-labile toxin B subunit (LTB). BALB/c mice were immunized by intranasal inoculation with control DNAs, the P1C DNA vaccine or the LTB-P1C fusion DNA vaccine. Levels of the anti-M. pneumoniae antibodies and levels of interferon-γ and IL-4 in mice were increased significantly upon inoculation of the LTB-P1C fusion DNA vaccine when compared with the inoculation with P1C DNA vaccine. The LTB-P1C fusion DNA vaccine efficiently enhanced the M. pneumoniae-specific IgA and IgG levels. The IgG2a/IgG1 ratio was significantly higher in bronchoalveolar lavages fluid and sera from mice fusion with LTB and P1C than mice receiving P1C alone. When the mice were challenged intranasally with 10(7) CFU M. pneumoniae strain (M129), the LTB-P1C fusion DNA vaccine conferred significantly better protection than P1C DNA vaccine (P < 0.05), as suggested by the results, such as less inflammation, lower histopathological score values, lower detectable number of M. pneumoniae strain, and lower mortality of challenging from 5 × 10(8) CFU M. pneumoniae. These results indicated that the LTB-P1C fusion DNA vaccine efficiently improved protective efficacy against M. pneumoniae infection and effectively attenuated development of M. pneumoniae in mice.

  12. The adjuvant effect of a non-toxic mutant of heat-labile enterotoxin of Escherichia coli for the induction of measles virus-specific CTL responses after intranasal co-immunization with a synthetic peptide.

    PubMed Central

    Partidos, C D; Pizza, M; Rappuoli, R; Steward, M W

    1996-01-01

    The intranasal route has been shown to be effective for immunization. However, immunization via this route may require the use of potent and safe adjuvant. The construction of non-toxic mutants of heat labile enterotoxin of Escherichia coli (LT), which is a potent mucosal adjuvant, is a major breakthrough for the development of mucosal vaccines. In this study we have assessed the ability of an LT mutant (LTK63) to act as an adjuvant following intranasal co-immunization with a peptide corresponding to a measles virus cytotoxic T lymphocyte (CTL) epitope. LTK63 was more effective at potentiating the in vivo induction of peptide-specific and measles virus-specific CTL responses than was administration of the peptide in saline. A concentration of 10 micrograms/dose of LTK63 was found to be the most effective in potentiating the in vivo priming of peptide-specific and measles virus-specific CTL responses. These findings highlight the potential of the non-toxic mutant of LT as a safe mucosal adjuvant for use in humans. PMID:9014810

  13. Construction of Bifidobacterium infantis as a live oral vaccine that expresses antigens of the major fimbrial subunit (CfaB) and the B subunit of heat-labile enterotoxin (LTB) from enterotoxigenic Escherichia coli.

    PubMed

    Ma, Yongping; Luo, Yaolin; Huang, Xueping; Song, Fangzhou; Liu, Geli

    2012-02-01

    We sought to develop Bifidobacterium infantis (BI) as a vehicle for the expression of heterologous antigens. Two proteins of enterotoxigenic Escherichia coli (ETEC) were expressed in BI: CfaB, a major fimbrial subunit protein, and LTB, the B subunit of heat-labile enterotoxin. The expression of CfaB and LTB in BI was verified by electrophoretic analysis. Sprague-Dawley rats were then subjected to intragastric immunization with BI-CfaB and BI-LTB systems both separately and together. ELISA was used to characterize the serum and mucosal immune responses against ETEC antigens. The immunized rats were intraperitoneally challenged with wild-type ETEC H10407 to study the immune response in vivo. The serum titres of IgG and faecal IgA antibodies in the BI-CfaB plus BI-LTB mixed vaccination group were significantly greater than those in the other two groups, which were immunized with a single vaccine (P<0.05). However, no significant difference was seen between the two groups that received a single immunization. These results suggest that expressing CfaB and LTB in BI provides a probiotic system with immunogenic properties. Furthermore, the expression of LTB in BI preserved its mucosal adjuvant effect. So this study confirms that BI can be used as a novel oral vaccine expression system for a heterologous antigen and BI-LTB can provide mucosal adjuvant properties. PMID:22053005

  14. 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.

  15. The Divergent CD8+ T Cell Adjuvant Properties of LT-IIb and LT-IIc, Two Type II Heat-Labile Enterotoxins, Are Conferred by Their Ganglioside-Binding B Subunits

    PubMed Central

    Hu, John C.; Greene, Christopher J.; King-Lyons, Natalie D.; Connell, Terry D.

    2015-01-01

    Poor immune responses elicited by vaccine antigens can be enhanced by the use of appropriate adjuvants. Type II heat-labile enterotoxins (HLT) produced by Escherichia coli are extremely potent adjuvants that augment both humoral and cellular immunity to co-administered antigens. Recent findings demonstrate that LT-IIb and LT-IIc, two type II HLT adjuvants, exhibit potent, yet distinguishable CD8+ T cell adjuvant properties. While LT-IIc elicits a robust and rapid response at one week after administration, LT-IIb engenders a more gradual and slower expansion of antigen-specific CD8+ T cells that correlates with improved immunity. The variations in immune effects elicited by the HLT adjuvants have been generally attributed to their highly divergent B subunits that mediate binding to various gangliosides on cell surfaces. Yet, HLT adjuvants with point mutations in the B subunit that significantly alter ganglioside binding retain similar adjuvant functions. Therefore, the contribution of the B subunits to adjuvanticity remains unclear. To investigate the influence of the B subunits on the enhancement of immune responses by LT-IIb and LT-IIc, chimeric HLT were engineered in which the B subunits of the two adjuvants were exchanged. Comparing the immune potentiating characteristics of both native and chimeric HLT adjuvants, it was found that not all the adjuvant characteristics of the HLT adjuvants were modulated by the respective B subunits. Specifically, the differences in the CD8+ T cell kinetics and protective responses elicited by LT-IIb and LT-IIc did indeed followed their respective B subunits. However, induction of IL-1 from macrophages and the capacity to intoxicate cells in a mouse Y1 adrenal cell bioassay did not correlate with the B subunits. Therefore, it is likely that additional factors other than the B subunits contribute to the effects elicited by the HLT adjuvants. PMID:26565800

  16. 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.

  17. 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

    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.

  18. 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.

  19. Immunization with a Double-Mutant (R192G/L211A) of the Heat-Labile Enterotoxin of Escherichia coli Offers Partial Protection against Campylobacter jejuni in an Adult Mouse Intestinal Colonization Model.

    PubMed

    Albert, M John; Haridas, Shilpa; Ebenezer, Mathew; Raghupathy, Raj; Khan, Islam

    2015-01-01

    We have previously shown that antibodies to cholera toxin (CT) reacted with the major outer membrane proteins (MOMPs) from Campylobacter jejuni strains on Western blot. Further, oral immunization with CT significantly protected against challenge with C. jejuni in an adult mouse colonization model of infection. CT and the heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli are structurally and functionally related. LT and its mutants including the double-mutant LT (R192G/L211A) (dmLT), are powerful mucosal adjuvants. Unlike LT which is reactogenic, dmLT has been shown to be safe for human use. In the current study, we determined whether rabbit anti-dmLT antibodies reacted with MOMPs from C. jejuni strains and whether immunization with dmLT would afford protection against C. jejuni. On Western blot, the MOMPs from C. jejuni 48 (Penner serotype O:19), C. jejuni 75 (O:3) and C. jejuni 111 (O:1,44) were probed with rabbit antibodies to dmLT or LT-E112K (a non-toxic LT mutant), which showed a lack of reaction. Adult BALB/c mice were orally immunized with dmLT and orally challenged with C. jejuni 48 or 111. Protection from colonization with the challenge bacteria was studied by enumerating Campylobacter colonies in feces daily for 9 days. Vaccination produced robust serum and stool antibody responses to dmLT and no antibody responses to C. jejuni MOMP. Vaccinated mice showed reduced colonization and excretion of both challenge strains compared to control mice. However, the differences were not statistically significant. The protective efficacy of the dmLT vaccine varied from 9.1% to 54.5%. The lack of cross-reaction between the MOMP and dmLT suggests that protection is not mediated by cross-reacting antibodies, but may be due to activation of innate immunity. As dmLT is safe for humans, it could be incorporated into a C. jejuni vaccine to enhance its efficacy. PMID:26540197

  20. 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

  1. 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, ...

  2. Heterogenic virulence in a diarrheagenic Escherichia coli: evidence for an EPEC expressing heat-labile toxin of ETEC.

    PubMed

    Dutta, Sanjucta; Pazhani, Gururaja P; Nataro, James P; Ramamurthy, Thandavarayan

    2015-01-01

    We have encountered an Escherichia coli strain isolated from a child with acute diarrhea. This strain harbored eae and elt genes encoding for E. coli attaching and effacing property and heat-labile enterotoxin of EPEC and ETEC, respectively. Due to the presence of these distinct virulence factors, we named this uncommon strain as EPEC/ETEC hybrid. The elt gene was identified in a conjugally transferable plasmid of the EPEC/ETEC hybrid. In addition, several virulence genes in the locus of enterocyte effacement have been identified, which confirms that the EPEC/ETEC has an EPEC genetic background. The hybrid nature of this strain was further confirmed by using tissue culture assays. In the multi locus sequence typing (MLST) analysis, the EPEC/ETEC belonged to the sequence type ST328 and was belonging to ST278 Cplx. Sequence analysis of the plasmid DNA revealed presence of six large contigs with several insertion sequences. A phage integrase gene and the prophages of gp48 and gp49 have been found in the upstream of eltAB. In the downstream of elt, an urovirulence loci adhesion encoding (pap) cluster containing papG, and papC were also identified. Similar to other reports, we have identified a heterogenic virulence in a diarrheagenic E. coli but with different combination of genes. PMID:25465159

  3. 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.

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

    PubMed Central

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

    2015-01-01

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

  5. Nanoparticulated heat-stable (STa) and heat-labile B subunit (LTB) recombinant toxin improves vaccine protection against enterotoxigenic Escherichia coli challenge in mouse.

    PubMed

    Deng, Guangcun; Zeng, Jin; Jian, Minjie; Liu, Wenmiao; Zhang, Zhong; Liu, Xiaoming; Wang, Yujiong

    2013-02-01

    Enterotoxigenic Escherichia coli (ETEC) remains a major cause of diarrheic disease in developing areas, for which there is no effective vaccine available. In this study, we genetically engineered a recombinant heat-stable enterotoxin (STa) coupled to the subunit B of heat-labile enterotoxin (LTB). This fusion protein, STa-LTB, possesses a single amino acid substitution at position 14 of STa. Our data demonstrates that the enterotoxicity of STa in STa-LTB was dramatically reduced. A gelatin nanovaccine candidate was prepared using the purified STa-LTB fusion protein characterized with an entrapment efficiency of 84.88 ± 6.37% and smooth spheres size ranges of 80-200 nm. Antigen-specific antibody responses against STa-LTB and STa in the sera and the intestinal mucus respectively were used to test the immunogenicity of the nanovaccine. This vaccine was further screened in mice by its ability to elicit neutralizing antibodies against STa and protect animals from the challenge with ETEC in mice. The STa-LTB nanoparticles delivered demonstrated a capacity to induce significantly higher and long-lasting antibody responses and increased immune protection against ETEC challenge relative to the control STa-LTB vaccine absorbed in conventional aluminum hydrate salt (p < 0.01). These results warrant the further studies of the development of a novel nanoparticulate vaccine as a broad-spectrum vaccine against ETEC infection.

  6. 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

  7. 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

  8. 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.

  9. Design and characterization of a chimeric multiepitope construct containing CfaB, heat-stable toxoid, CssA, CssB, and heat-labile toxin subunit B of enterotoxigenic Escherichia coli: a bioinformatic approach.

    PubMed

    Zeinalzadeh, Narges; Salmanian, Ali Hatef; Ahangari, Ghasem; Sadeghi, Mahdi; Amani, Jafar; Bathaie, S Zahra; Jafari, Mahyat

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains are the most common cause of bacterial diarrhea in children in developing countries and travelers to these areas. Enterotoxins and colonization factors (CFs) are two key virulence factors in ETEC pathogenesis, and the heterogeneity of the CFs is the bottleneck in reaching an effective vaccine. In this study, a candidate subunit vaccine, which is composed of CfaB, CssA and CssB, structural subunits of colonization factor antigen I and CS6 CFs, labile toxin subunit B, and the binding subunit of heat-labile and heat-stable toxoid, was designed to provide broad-spectrum protection against ETEC. The different features of chimeric gene, its mRNA stability, and chimeric protein properties were analyzed by using bioinformatic tools. The optimized chimeric gene was chemically synthesized and expressed successfully in a prokaryotic host. The purified protein was used for assessment of bioinformatic data by experimental methods.

  10. Design and characterization of a chimeric multiepitope construct containing CfaB, heat-stable toxoid, CssA, CssB, and heat-labile toxin subunit B of enterotoxigenic Escherichia coli: a bioinformatic approach.

    PubMed

    Zeinalzadeh, Narges; Salmanian, Ali Hatef; Ahangari, Ghasem; Sadeghi, Mahdi; Amani, Jafar; Bathaie, S Zahra; Jafari, Mahyat

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains are the most common cause of bacterial diarrhea in children in developing countries and travelers to these areas. Enterotoxins and colonization factors (CFs) are two key virulence factors in ETEC pathogenesis, and the heterogeneity of the CFs is the bottleneck in reaching an effective vaccine. In this study, a candidate subunit vaccine, which is composed of CfaB, CssA and CssB, structural subunits of colonization factor antigen I and CS6 CFs, labile toxin subunit B, and the binding subunit of heat-labile and heat-stable toxoid, was designed to provide broad-spectrum protection against ETEC. The different features of chimeric gene, its mRNA stability, and chimeric protein properties were analyzed by using bioinformatic tools. The optimized chimeric gene was chemically synthesized and expressed successfully in a prokaryotic host. The purified protein was used for assessment of bioinformatic data by experimental methods. PMID:24372617

  11. 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.

  12. Hereditary heat-labile hexosaminidase B: its implication for recognizing Tay-Sachs genotypes.

    PubMed

    Navon, R; Nutman, J; Kopel, R; Gaber, L; Gadoth, N; Goldman, B; Nitzan, M

    1981-11-01

    Two pairs of alleles, at the two loci of hexosaminidase (HEX), were found to segregate in an Arab inbred family: the normal and the mutant Tay-Sachs (TSD) alleles of HEX A, and the normal and a mutant allele of HEX B. Since the mutant HEX B is heat labile, no reliable identification of TSD genotypes can be obtained in its presence, as long as the proportions of HEX A and B are estimated by the routinely used heat-inactivation method. The genotypes may be correctly identified in such cases by separation of the two isoenzymes on ion-exchange chromatography, estimating their individual activities, and calculating the ratio between them. Of the nine genotype combinations possible with these two pairs of alleles, five have been identified in the reported family by this procedure. PMID:6459736

  13. Heat-labile enterotoxigenic Escherichia coli and intestinal protozoa in asymptomatic travellers.

    PubMed

    Echeverria, P; Cross, J H

    1977-12-01

    Thirty-two asymptomatic travellers who had recently journeyed in the Near, Middle, and Far East and had experienced a high incidence of diarrhoeal disease were screened for heat-labile enterotoxigenic Escherichia coli (ent+ E. coli) and other bacterial and parasitic pathogens. Six percent were colonized with ent+ E. coli and while other bacterial pathogens were not found, the intestinal protozoa Giardia lamblia (13%), Entamoeba histolytica (6%), Entamoeba coli (6%), Endolimax nana (6%), and Entamoeba hartmanni (3%) were detected in the stools. Ent+ E. coli, G. lamblia and E. histolytica should be considered in the differential diagnosis of gastrointestinal disease in travellers returning from the Orient. Furthermore, these travellers may be a potential source for the introduction of ent+ E. coli into communities where such organisms are relatively rare. PMID:351820

  14. Adhesin degradation accelerates delivery of heat-labile toxin by enterotoxigenic Escherichia coli.

    PubMed

    Roy, Koushik; Kansal, Rita; Bartels, Scott R; Hamilton, David J; Shaaban, Salwa; Fleckenstein, James M

    2011-08-26

    Many enteric pathogens, including enterotoxigenic Escherichia coli (ETEC), produce one or more serine proteases that are secreted via the autotransporter (or type V) bacterial secretion pathway. These molecules have collectively been referred to as SPATE proteins (serine protease autotransporter of the Enterobacteriaceae). EatA, an autotransporter previously identified in ETEC, possesses a functional serine protease motif within its secreted amino-terminal passenger domain. Although this protein is expressed by many ETEC strains and is highly immunogenic, its precise function is unknown. Here, we demonstrate that EatA degrades a recently characterized adhesin, EtpA, resulting in modulation of bacterial adhesion and accelerated delivery of the heat-labile toxin, a principal ETEC virulence determinant. Antibodies raised against the passenger domain of EatA impair ETEC delivery of labile toxin to epithelial cells suggesting that EatA may be an effective target for vaccine development. PMID:21757737

  15. Mutant Escherichia coli Heat-Labile Toxin B Subunit That Separates Toxoid-Mediated Signaling and Immunomodulatory Action from Trafficking and Delivery Functions

    PubMed Central

    Fraser, Sylvia A.; de Haan, Lolke; Hearn, Arron R.; Bone, Heather K.; Salmond, Robert J.; Rivett, A. Jennifer; Williams, Neil A.; Hirst, Timothy R.

    2003-01-01

    The homopentameric B-subunit components of Escherichia coli heat-labile enterotoxin (EtxB) and cholera toxin (CtxB) possess the capacity to enter mammalian cells and to activate cell-signaling events in leukocytes that modulate immune cell function. Both properties have been attributed to the ability of the B subunits to bind to GM1-ganglioside receptors, a ubiquitous glycosphingolipid found in the plasma membrane. Here we describe the properties of EtxB(H57S), a mutant B subunit with a His→Ser substitution at position 57. The mutant was found to be severely defective in inducing leukocyte signaling, as shown by failure to (i) trigger caspase 3-mediated CD8+-T-cell apoptosis, (ii) activate nuclear translocation of NF-κB in Jurkat T cells, (iii) induce a potent anti-B-subunit response in mice, or (iv) serve as a mucosal adjuvant. However, its GM1 binding, cellular uptake, and delivery functions remained intact. This was further validated by the finding that EtxB(H57S) was as effective as EtxB in delivering a conjugated model class I epitope into the major histocompatibility complex class I pathway of a dendritic cell line. These observations imply that GM1 binding alone is not sufficient to trigger the signaling events responsible for the potent immunomodulatory properties of EtxB. Moreover, they demonstrate that its signaling properties play no role in EtxB uptake and trafficking. Thus, EtxB(H57S) represents a novel tool for evaluating the complex cellular interactions and signaling events occurring after receptor interaction, as well as offering an alternative means of delivering attached peptides in the absence of the potent immunomodulatory signals induced by wild-type B subunits. PMID:12595472

  16. Heat-labile- and heat-stable-toxoid fusions (LTR₁₉₂G-STaP₁₃F) of human enterotoxigenic Escherichia coli elicit neutralizing antitoxin antibodies.

    PubMed

    Liu, Mei; Ruan, Xiaosai; Zhang, Chengxian; Lawson, Steve R; Knudsen, David E; Nataro, James P; Robertson, Donald C; Zhang, Weiping

    2011-10-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Adhesins and enterotoxins, including heat-labile (LT) and heat-stable (STa) toxins, are the key virulence factors. Antigenic adhesin and LT antigens have been used in developing vaccines against ETEC diarrhea. However, STa has not been included because of its poor immunogenicity and potent toxicity. Our recent study showed that porcine-type STa toxoids became immunogenic and elicited neutralizing anti-STa antibodies after being genetically fused to a full-length porcine-type LT toxoid, LT(R₁₉₂G) (W. Zhang et al., Infect. Immun. 78:316-325, 2010). In this study, we mutated human-type LT and STa genes, which are highly homologous to porcine-type toxin genes, for a full-length LT toxoid (LT(R₁₉₂)) and a full-length STa toxoid (STa(P₁₃F)) and genetically fused them to produce LT₁₉₂-STa₁₃ toxoid fusions. Mice immunized with LT₁₉₂-STa₁₃ fusion antigens developed anti-LT and anti-STa IgG (in serum and feces) and IgA antibodies (in feces). Moreover, secretory IgA antibodies from immunized mice were shown to neutralize STa and cholera toxins in T-84 cells. In addition, we fused the STa₁₃ toxoid at the N terminus and C terminus, between the A1 and A2 peptides, and between the A and B subunits of LT₁₉₂ to obtain different fusions in order to explore strategies for enhancing STa immunogenicity. This study demonstrated that human-type LT₁₉₂-STa₁₃ fusions induce neutralizing antitoxin antibodies and provided important information for developing toxoid vaccines against human ETEC diarrhea. PMID:21788385

  17. Evaluating the A-Subunit of the Heat-Labile Toxin (LT) As an Immunogen and a Protective Antigen Against Enterotoxigenic Escherichia coli (ETEC).

    PubMed

    Norton, Elizabeth B; Branco, Luis M; Clements, John D

    2015-01-01

    Diarrheal illness contributes to malnutrition, stunted growth, impaired cognitive development, and high morbidity rates in children worldwide. Enterotoxigenic Escherichia coli (ETEC) is a major contributor to this diarrheal disease burden. ETEC cause disease in the small intestine by means of colonization factors and by production of a heat-labile enterotoxin (LT) and/or a small non-immunogenic heat-stable enterotoxin (ST). Overall, the majority of ETEC produce both ST and LT. LT induces secretion via an enzymatically active A-subunit (LT-A) and a pentameric, cell-binding B-subunit (LT-B). The importance of anti-LT antibodies has been demonstrated in multiple clinical and epidemiological studies, and a number of potential ETEC vaccine candidates have included LT-B as an important immunogen. However, there is limited information about the potential contribution of LT-A to development of protective immunity. In the current study, we evaluate the immune response against the A-subunit of LT as well as the A-subunit's potential as a protective antigen when administered alone or in combination with the B-subunit of LT. We evaluated human sera from individuals challenged with a prototypic wild-type ETEC strain as well as sera from individuals living in an ETEC endemic area for the presence of anti-LT, anti-LT-A and anti-LT-B antibodies. In both cases, a significant number of individuals intentionally or endemically infected with ETEC developed antibodies against both LT subunits. In addition, animals immunized with the recombinant proteins developed robust antibody responses that were able to neutralize the enterotoxic and cytotoxic effects of native LT by blocking binding and entry into cells (anti-LT-B) or the intracellular enzymatic activity of the toxin (anti-LT-A). Moreover, antibodies to both LT subunits acted synergistically to neutralize the holotoxin when combined. Taken together, these data support the inclusion of both LT-A and LT-B in prospective vaccines

  18. Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa: purification and preliminary characterization.

    PubMed Central

    Berka, R M; Vasil, M L

    1982-01-01

    Phospholipase C (heat-labile hemolysin) was purified from Pseudomonas aeruginosa culture supernatants to near homogeneity by ammonium sulfate precipitation followed by a novel application of DEAE-Sephacel chromatography. Enzymatic activity remained associated with DEAE-Sephacel even in the presence of 1 M NaCl, but was eluted with a linear gradient of 0 to 5% tetradecyltrimethylammonium bromide. Elution from DEAE-Sephacel was also obtained with 2% lysophosphatidylcholine, and to a lesser extent with 2% phosphorylcholine, but not at all with choline. The enzyme was highly active toward phospholipids possessing substituted ammonium groups (e.g., phosphatidycholine, lysophosphatidylcholine, and sphingomyelin); however, it had little if any activity toward phospholipids lacking substituted ammonium groups (e.g., phosphatidylethanolamine, phosphatidylserine, and phosphaditylglycerol). Collectively, these data suggest that phospholipase C from P. aeruginosa exhibits high affinity for substituted ammonium groups, but requires an additional hydrophobic moiety for optimum binding. The specific activity of the purified enzyme preparation increased 1,900-fold compared with that of culture supernatants. The molecular weight of the phospholipase C was estimated to be 78,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephacryl S-200 column chromatography and was 76,000 by high-performance size exclusion chromatography. The isoelectric point was 5.5. Amino acid analysis showed that phospholipase C was rich in glycine, serine, threonine, aspartyl, glutamyl, and aromatic amino acids, but was cystine free. Images PMID:6811552

  19. Toxicity and immunogenicity of Enterotoxigenic Escherichia coli heat-labile and heat-stable toxoid fusion 3xSTa(A14Q)-LT(S63K/R192G/L211A) in a murine model.

    PubMed

    Zhang, Chengxian; Knudsen, David E; Liu, Mei; Robertson, Donald C; Zhang, Weiping

    2013-01-01

    Diarrhea is the second leading cause of death to young children. Enterotoxigenic Escherichia coli (ETEC) are the most common bacteria causing diarrhea. Adhesins and enterotoxins are the virulence determinants in ETEC diarrhea. Adhesins mediate bacterial attachment and colonization, and enterotoxins including heat-labile (LT) and heat-stable type Ib toxin (STa) disrupt fluid homeostasis in host cells that leads to fluid hyper-secretion and diarrhea. Thus, adhesins and enterotoxins have been primarily targeted in ETEC vaccine development. A recent study reported toxoid fusions with STa toxoid (STa(P13F)) fused at the N- or C-terminus, or inside the A subunit of LT(R192G) elicited neutralizing antitoxin antibodies, and suggested application of toxoid fusions in ETEC vaccine development (Liu et al., Infect. Immun. 79:4002-4009, 2011). In this study, we generated a different STa toxoid (STa(A14Q)) and a triple-mutant LT toxoid (LT(S63K/R192G/L211A), tmLT), constructed a toxoid fusion (3xSTa(A14Q)-tmLT) that carried 3 copies of STa(A14Q) for further facilitation of anti-STa immunogenicity, and assessed antigen safety and immunogenicity in a murine model to explore its potential for ETEC vaccine development. Mice immunized with this fusion antigen showed no adverse effects, and developed antitoxin antibodies particularly through the IP route. Anti-LT antibodies were detected and were shown neutralizing against CT in vitro. Anti-STa antibodies were also detected in the immunized mice, and serum from the IP immunized mice neutralized STa toxin in vitro. Data from this study indicated that toxoid fusion 3xSTa(A14Q)-tmLT is safe and can induce neutralizing antitoxin antibodies, and provided helpful information for vaccine development against ETEC diarrhea. PMID:24146989

  20. Toxicity and immunogenicity of Enterotoxigenic Escherichia coli heat-labile and heat-stable toxoid fusion 3xSTa(A14Q)-LT(S63K/R192G/L211A) in a murine model.

    PubMed

    Zhang, Chengxian; Knudsen, David E; Liu, Mei; Robertson, Donald C; Zhang, Weiping

    2013-01-01

    Diarrhea is the second leading cause of death to young children. Enterotoxigenic Escherichia coli (ETEC) are the most common bacteria causing diarrhea. Adhesins and enterotoxins are the virulence determinants in ETEC diarrhea. Adhesins mediate bacterial attachment and colonization, and enterotoxins including heat-labile (LT) and heat-stable type Ib toxin (STa) disrupt fluid homeostasis in host cells that leads to fluid hyper-secretion and diarrhea. Thus, adhesins and enterotoxins have been primarily targeted in ETEC vaccine development. A recent study reported toxoid fusions with STa toxoid (STa(P13F)) fused at the N- or C-terminus, or inside the A subunit of LT(R192G) elicited neutralizing antitoxin antibodies, and suggested application of toxoid fusions in ETEC vaccine development (Liu et al., Infect. Immun. 79:4002-4009, 2011). In this study, we generated a different STa toxoid (STa(A14Q)) and a triple-mutant LT toxoid (LT(S63K/R192G/L211A), tmLT), constructed a toxoid fusion (3xSTa(A14Q)-tmLT) that carried 3 copies of STa(A14Q) for further facilitation of anti-STa immunogenicity, and assessed antigen safety and immunogenicity in a murine model to explore its potential for ETEC vaccine development. Mice immunized with this fusion antigen showed no adverse effects, and developed antitoxin antibodies particularly through the IP route. Anti-LT antibodies were detected and were shown neutralizing against CT in vitro. Anti-STa antibodies were also detected in the immunized mice, and serum from the IP immunized mice neutralized STa toxin in vitro. Data from this study indicated that toxoid fusion 3xSTa(A14Q)-tmLT is safe and can induce neutralizing antitoxin antibodies, and provided helpful information for vaccine development against ETEC diarrhea.

  1. Staphylococcal Enterotoxins

    PubMed Central

    Pinchuk, Irina V.; Beswick, Ellen J.; Reyes, Victor E.

    2010-01-01

    Staphylococcus aureus (S. aureus) is a Gram positive bacterium that is carried by about one third of the general population and is responsible for common and serious diseases. These diseases include food poisoning and toxic shock syndrome, which are caused by exotoxins produced by S. aureus. Of the more than 20 Staphylococcal enterotoxins, SEA and SEB are the best characterized and are also regarded as superantigens because of their ability to bind to class II MHC molecules on antigen presenting cells and stimulate large populations of T cells that share variable regions on the β chain of the T cell receptor. The result of this massive T cell activation is a cytokine bolus leading to an acute toxic shock. These proteins are highly resistant to denaturation, which allows them to remain intact in contaminated food and trigger disease outbreaks. A recognized problem is the emergence of multi-drug resistant strains of S. aureus and these are a concern in the clinical setting as they are a common cause of antibiotic-associated diarrhea in hospitalized patients. In this review, we provide an overview of the current understanding of these proteins. PMID:22069679

  2. 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

  3. Parenteral Adjuvant Effects of an Enterotoxigenic Escherichia coli Natural Heat-Labile Toxin Variant.

    PubMed

    Braga, Catarina J M; Rodrigues, Juliana F; Medina-Armenteros, Yordanka; Farinha-Arcieri, Luís E; Ventura, Armando M; Boscardin, Silvia B; Sbrogio-Almeida, Maria E; Ferreira, Luís C S

    2014-01-01

    Native type I heat-labile toxins (LTs) produced by enterotoxigenic Escherichia coli (ETEC) strains exert strong adjuvant effects on both antibody and T cell responses to soluble and particulate antigens following co-administration via mucosal routes. However, inherent enterotoxicity and neurotoxicity (following intra-nasal delivery) had reduced the interest in the use of these toxins as mucosal adjuvants. LTs can also behave as powerful and safe adjuvants following delivery via parenteral routes, particularly for activation of cytotoxic lymphocytes. In the present study, we evaluated the adjuvant effects of a new natural LT polymorphic form (LT2), after delivery via intradermal (i.d.) and subcutaneous (s.c.) routes, with regard to both antibody and T cell responses. A recombinant HIV-1 p24 protein was employed as a model antigen for determination of antigen-specific immune responses while the reference LT (LT1), produced by the ETEC H10407 strain, and a non-toxigenic LT form (LTK63) were employed as previously characterized LT types. LT-treated mice submitted to a four dose-base immunization regimen elicited similar p24-specific serum IgG responses and CD4(+) T cell activation. Nonetheless, mice immunized with LT1 or LT2 induced higher numbers of antigen-specific CD8(+) T cells and in vivo cytotoxic responses compared to mice immunized with the non-toxic LT derivative. These effects were correlated with stronger activation of local dendritic cell populations. In addition, mice immunized with LT1 and LT2, but not with LTK63, via s.c. or i.d. routes developed local inflammatory reactions. Altogether, the present results confirmed that the two most prevalent natural polymorphic LT variants (LT1 or LT2) display similar and strong adjuvant effects for subunit vaccines administered via i.d. or s.c. routes.

  4. 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

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

    PubMed

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

    1989-06-01

    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.

  6. 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

  7. 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.

  8. Characterization of heat-labile toxin-subunit B from Escherichia coli by liquid chromatography-electrospray ionization-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    PubMed

    Sospedra, I; De Simone, C; Soriano, J M; Mañes, J; Ferranti, P; Ritieni, A

    2012-11-01

    The possibilities of characterizing the heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) by liquid chromatography electrospray mass spectrometry (LC/ESI-MS) and matrix-assisted laser desorption with time-of-flight mass spectrometry (MALDI-TOF-MS) were investigated. The B subunit from recombinant E. coli (expression in Pichia pastoris) can be detected by LC/ESI-MS expressed in P. pastoris and the charge envelope signals can be observed; LC/ESI-MS and MALDI-TOF-MS analysis allowed the acquisition of labile toxin subunit B (LTB) molecular weight and preliminary structural characterization of LTB toxin. MALDI-TOF analysis after reduction and alkylation of the protein evidenced the presence of one disulfide bond in the structure of the protein. Confirmatory analysis was carried out by detection of most of the tryptic fragments of the B subunit by MALDI-TOF-MS, obtaining total coverage of the protein sequence. Possible biovariations in the toxin can mostly be determined by sequencing, where an increase of molecular mass in the N-terminal side of the protein was identified. This modification may be due to an O-GlcNAc-1-phosphorylation. PMID:22921353

  9. Escherichia coli K88ac fimbriae expressing heat-labile and heat-stable (STa) toxin epitopes elicit antibodies that neutralize cholera toxin and STa toxin and inhibit adherence of K88ac fimbrial E. coli.

    PubMed

    Zhang, Chengxian; Zhang, Weiping

    2010-12-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Bacterial adhesins and heat-labile (LT) and heat-stable (ST) enterotoxins are the virulence determinants in ETEC diarrhea. It is believed that vaccines inducing anti-adhesin immunity to inhibit bacterial adherence and anti-toxin immunity to eliminate toxin activity would provide broad-spectrum protection against ETEC. In this study, an ETEC fimbrial adhesin was used as a platform to express LT and STa for adhesin-toxin fusion antigens to induce anti-toxin and anti-adhesin immunity. An epitope from the B subunit of LT toxin (LTP1, (8)LCSEYRNTQIYTIN(21)) and an STa toxoid epitope ((5)CCELCCNPQCAGCY(18)) were embedded in the FaeG major subunit of E. coli K88ac fimbriae. Constructed K88ac-toxin chimeric fimbriae were harvested and used for rabbit immunization. Immunized rabbits developed anti-K88ac, anti-LT, and anti-STa antibodies. Moreover, induced antibodies not only inhibited adherence of K88ac fimbrial E. coli to porcine small intestinal enterocytes but also neutralized cholera toxin and STa toxin. Data from this study demonstrated that K88ac fimbriae expressing LT and STa epitope antigens elicited neutralizing anti-toxin antibodies and anti-adhesin antibodies and suggested that E. coli fimbriae could serve as a platform for the development of broad-spectrum vaccines against ETEC. PMID:20980482

  10. Sequence Variability in Staphylococcal Enterotoxin Genes seb, sec, and sed

    PubMed Central

    Johler, Sophia; Sihto, Henna-Maria; Macori, Guerrino; Stephan, Roger

    2016-01-01

    Ingestion of staphylococcal enterotoxins preformed by Staphylococcus aureus in food leads to staphylococcal food poisoning, the most prevalent foodborne intoxication worldwide. There are five major staphylococcal enterotoxins: SEA, SEB, SEC, SED, and SEE. While variants of these toxins have been described and were linked to specific hosts or levels or enterotoxin production, data on sequence variation is still limited. In this study, we aim to extend the knowledge on promoter and gene variants of the major enterotoxins SEB, SEC, and SED. To this end, we determined seb, sec, and sed promoter and gene sequences of a well-characterized set of enterotoxigenic Staphylococcus aureus strains originating from foodborne outbreaks, human infections, human nasal colonization, rabbits, and cattle. New nucleotide sequence variants were detected for all three enterotoxins and a novel amino acid sequence variant of SED was detected in a strain associated with human nasal colonization. While the seb promoter and gene sequences exhibited a high degree of variability, the sec and sed promoter and gene were more conserved. Interestingly, a truncated variant of sed was detected in all tested sed harboring rabbit strains. The generated data represents a further step towards improved understanding of strain-specific differences in enterotoxin expression and host-specific variation in enterotoxin sequences. PMID:27258311

  11. Sequence Variability in Staphylococcal Enterotoxin Genes seb, sec, and sed.

    PubMed

    Johler, Sophia; Sihto, Henna-Maria; Macori, Guerrino; Stephan, Roger

    2016-01-01

    Ingestion of staphylococcal enterotoxins preformed by Staphylococcus aureus in food leads to staphylococcal food poisoning, the most prevalent foodborne intoxication worldwide. There are five major staphylococcal enterotoxins: SEA, SEB, SEC, SED, and SEE. While variants of these toxins have been described and were linked to specific hosts or levels or enterotoxin production, data on sequence variation is still limited. In this study, we aim to extend the knowledge on promoter and gene variants of the major enterotoxins SEB, SEC, and SED. To this end, we determined seb, sec, and sed promoter and gene sequences of a well-characterized set of enterotoxigenic Staphylococcus aureus strains originating from foodborne outbreaks, human infections, human nasal colonization, rabbits, and cattle. New nucleotide sequence variants were detected for all three enterotoxins and a novel amino acid sequence variant of SED was detected in a strain associated with human nasal colonization. While the seb promoter and gene sequences exhibited a high degree of variability, the sec and sed promoter and gene were more conserved. Interestingly, a truncated variant of sed was detected in all tested sed harboring rabbit strains. The generated data represents a further step towards improved understanding of strain-specific differences in enterotoxin expression and host-specific variation in enterotoxin sequences.

  12. 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.

  13. Search for heat-labile enterotoxigenic Escherichia coli in humans, livestock, food, and water in a community in the Philippines.

    PubMed

    Echeverria, P; Verhaert, L; Basaca-Sevilla, V; Banson, T; Cross, J; Orskov, F; Orskov, I

    1978-07-01

    Environmental sources of heat-labile enterotoxigenic Escherichia coli are unknown. The feces of 1,086 inhabitants (approximately 5%) of a small town in the Philippines, 28 pigs, and 10 water buffalo were cultured for enteric bacterial pathogens. Twenty-seven persons harbored pathogenic bacteria: five individuals had enterotoxigenic E. coli, 11 Salmonella species, nine Vibrio parahaemolyticus, one Shigella boydii, and one nonagglutinable Vibrio. Enterotoxigenic E. coli were isolated from two of 28 pigs and from one of 10 water buffalo. Cultures of 26 pieces of beef, 25 pieces of pork, and 52 leafy vegetables obtained from a community market failed to grow enterotoxigenic E. coli. None of 47 samples of contaminated surface water contained this pathogen. Serotypes of human and animal strains of enterotoxigenic E. coli were different, although E. coli O78:H12 isolated from a pig has previously been incriminated in human diarrheal disease. In this limited survey of a Philippine community, enterotoxigenic E. coli were isolated from humans and livestock. The possibility that enterotoxigenic E. coli infections are zoonotic warrants further investigation.

  14. Production of Enterotoxin A

    PubMed Central

    Kato, Eiichi; Khan, Mahmood; Kujovich, Lubinka; Bergdoll, Merlin S.

    1966-01-01

    A method for production of enterotoxin A in multiple liter lots is described. The medium contained 4% N-Z Amine NAK supplemented with 0.001% niacin and 0.00005% thiamine, and was adjusted to pH 6. The inoculated medium in lots of 400 to 600 ml, in 2-liter Erlenmeyer flasks, was incubated at 37 C for 24 hr on a gyrotory shaker at 280 rev/min. Production of 4 to 6 μg of enterotoxin A per ml occurred. PMID:16349703

  15. Allele variants of enterotoxigenic Escherichia coli heat-labile toxin are globally transmitted and associated with colonization factors.

    PubMed

    Joffré, Enrique; von Mentzer, Astrid; Abd El Ghany, Moataz; Oezguen, Numan; Savidge, Tor; Dougan, Gordon; Svennerholm, Ann-Mari; Sjöling, Åsa

    2015-01-01

    Enterotoxigenic Escherichia coli (ETEC) is a significant cause of morbidity and mortality in the developing world. ETEC-mediated diarrhea is orchestrated by heat-labile toxin (LT) and heat-stable toxins (STp and STh), acting in concert with a repertoire of more than 25 colonization factors (CFs). LT, the major virulence factor, induces fluid secretion after delivery of a monomeric ADP-ribosylase (LTA) and its pentameric carrier B subunit (LTB). A study of ETEC isolates from humans in Brazil reported the existence of natural LT variants. In the present study, analysis of predicted amino acid sequences showed that the LT amino acid polymorphisms are associated with a geographically and temporally diverse set of 192 clinical ETEC strains and identified 12 novel LT variants. Twenty distinct LT amino acid variants were observed in the globally distributed strains, and phylogenetic analysis showed these to be associated with different CF profiles. Notably, the most prevalent LT1 allele variants were correlated with major ETEC lineages expressing CS1 + CS3 or CS2 + CS3, and the most prevalent LT2 allele variants were correlated with major ETEC lineages expressing CS5 + CS6 or CFA/I. LTB allele variants generally exhibited more-stringent amino acid sequence conservation (2 substitutions identified) than LTA allele variants (22 substitutions identified). The functional impact of LT1 and LT2 polymorphisms on virulence was investigated by measuring total-toxin production, secretion, and stability using GM1-enzyme-linked immunosorbent assays (GM1-ELISA) and in silico protein modeling. Our data show that LT2 strains produce 5-fold more toxin than LT1 strains (P < 0.001), which may suggest greater virulence potential for this genetic variant. Our data suggest that functionally distinct LT-CF variants with increased fitness have persisted during the evolution of ETEC and have spread globally. PMID:25404692

  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. 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

  18. 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

  19. 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

  20. Bovine lactogenic immunity against cholera toxin-related enterotoxins and Vibrio cholerae outer membranes.

    PubMed Central

    Boesman-Finkelstein, M; Walton, N E; Finkelstein, R A

    1989-01-01

    The newly parturient cow secretes large quantities of immunoglobulin G1, a relatively protease- and heat-resistant immunoglobulin, in its colostrum and milk. This study establishes the feasibility of producing protective colostral immunoglobulins by immunizing pregnant cows with cholera toxin (CT), a CT-related enterotoxin from Escherichia coli, and Vibrio cholerae outer membranes (OMs). The OMs were prepared from bacteria grown under iron-replete or iron-deficient (to simulate the in vivo environment) conditions. Immunoglobulins were purified from the colostrum of newly parturient control and immunized cows. The bovine anti-CT and anti-H-LT (CT-related heat-labile enterotoxin produced by diarrheogenic E. coli strains of human origin) antibodies were quantitated by enzyme-linked immunosorbent assays and by neutralization of toxin activity in both Y-1 adrenal cell and infant rabbit assays. The bovine anti-OM antibodies from both high-iron-grown and low-iron-grown vibrios were assessed by bacterial agglutination and by Western blot (immunoblot) analysis of polyacrylamide gel electrophoresis of high-iron-grown and low-iron-grown OMs. To test their protective effect, immunoglobulin preparations were administered orally in infant feeding formula to 6-day-old rabbits. Anti-CT and anti-OM immunoglobulins elicited statistically significant protection against diarrhea in infant rabbits challenged intraintestinally with virulent cholera vibrios. Images PMID:2925248

  1. The LT1 and LT2 variants of the enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LT) are associated with major ETEC lineages.

    PubMed

    Joffré, Enrique; Sjöling, Åsa

    2016-01-01

    The heat-labile toxin (LT) is one of the major virulence factors of enterotoxigenic Escherichia coli (ETEC). We recently described that 20 polymorphic LT variants are present in ETEC strains isolated globally. Two of the variants, LT1 and LT2, are particularly common and we found that they were associated with clonal ETEC lineages that express the colonization factors (CFs), CFA/I, CS1+CS3, CS2+CS3, and CS5+CS6. ETEC expressing these CFs are frequently found among ETEC strains isolated from cases with diarrhea. ETEC expressing the colonization factors CS1+CS3, and CS2+CS3 are found in 2 discrete clonal lineages and express the LT1 variant and heat stable toxin (STh). Although they clearly are virulent they neither produce, nor secrete, high amounts of LT toxin. On the other hand ETEC strains expressing LT, STh, CFA/I and LT, STh, CS5+CS6, carry the LT2 variant and produce and secrete significantly more LT toxin. Despite differences in toxin production, LT1 and LT2 are found in ETEC lineages that have managed to spread globally confirming that these variants are important for ETEC virulence. PMID:26939855

  2. The LT1 and LT2 variants of the enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LT) are associated with major ETEC lineages

    PubMed Central

    Joffré, Enrique; Sjöling, Åsa

    2016-01-01

    ABSTRACT The heat-labile toxin (LT) is one of the major virulence factors of enterotoxigenic Escherichia coli (ETEC). We recently described that 20 polymorphic LT variants are present in ETEC strains isolated globally. Two of the variants, LT1 and LT2, are particularly common and we found that they were associated with clonal ETEC lineages that express the colonization factors (CFs), CFA/I, CS1+CS3, CS2+CS3, and CS5+CS6. ETEC expressing these CFs are frequently found among ETEC strains isolated from cases with diarrhea. ETEC expressing the colonization factors CS1+CS3, and CS2+CS3 are found in 2 discrete clonal lineages and express the LT1 variant and heat stable toxin (STh). Although they clearly are virulent they neither produce, nor secrete, high amounts of LT toxin. On the other hand ETEC strains expressing LT, STh, CFA/I and LT, STh, CS5+CS6, carry the LT2 variant and produce and secrete significantly more LT toxin. Despite differences in toxin production, LT1 and LT2 are found in ETEC lineages that have managed to spread globally confirming that these variants are important for ETEC virulence. PMID:26939855

  3. Cooperative role of antibodies against heat-labile toxin and the EtpA Adhesin in preventing toxin delivery and intestinal colonization by enterotoxigenic Escherichia coli.

    PubMed

    Roy, Koushik; Hamilton, David J; Fleckenstein, James M

    2012-10-01

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease in developing countries, where it is responsible for hundreds of thousands of deaths each year. Vaccine development for ETEC has been hindered by the heterogeneity of known molecular targets and the lack of broad-based sustained protection afforded by existing vaccine strategies. In an effort to explore the potential role of novel antigens in ETEC vaccines, we examined the ability of antibodies directed against the ETEC heat-labile toxin (LT) and the recently described EtpA adhesin to prevent intestinal colonization in vivo and toxin delivery to epithelial cells in vitro. We demonstrate that EtpA is required for the optimal delivery of LT and that antibodies against this adhesin play at least an additive role in preventing delivery of LT to target intestinal cells when combined with antibodies against either the A or B subunits of the toxin. Moreover, vaccination with a combination of LT and EtpA significantly impaired intestinal colonization. Together, these results suggest that the incorporation of recently identified molecules such as EtpA could be used to enhance current approaches to ETEC vaccine development. PMID:22875600

  4. 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. PMID:22541162

  5. 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

  6. 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

  7. A live attenuated Salmonella Enteritidis secreting detoxified heat labile toxin enhances mucosal immunity and confers protection against wild-type challenge in chickens.

    PubMed

    Lalsiamthara, Jonathan; Kamble, Nitin Machindra; Lee, John Hwa

    2016-01-01

    A live attenuated Salmonella Enteritidis (SE) capable of constitutively secreting detoxified double mutant Escherichia coli heat labile toxin (dmLT) was developed. The biologically adjuvanted strain was generated via transformation of a highly immunogenic SE JOL1087 with a plasmid encoding dmLT gene cassette; the resultant strain was designated JOL1641. A balanced-lethal host-vector system stably maintained the plasmid via auxotrophic host complementation with a plasmid encoded aspartate semialdehyde dehydrogenase (asd) gene. Characterization by western blot assay revealed the dmLT subunit proteins in culture supernatants of JOL1641. For the investigation of adjuvanticity and protective efficacy, chickens were immunized via oral or intramuscular routes with PBS, JOL1087 and JOL1641. Birds immunized with JOL1641 showed significant (P ≤ 0.05) increases in intestinal SIgA production at the 1(st) and 2(nd) weeks post-immunization via oral and intramuscular routes, respectively. Interestingly, while both strains showed significant splenic protection via intramuscular immunization, JOL1641 outperformed JOL1087 upon oral immunization. Oral immunization of birds with JOL1641 significantly reduced splenic bacterial counts. The reduction in bacterial counts may be correlated with an adjuvant effect of dmLT that increases SIgA secretion in the intestines of immunized birds. The inclusion of detoxified dmLT in the strain did not cause adverse reactions to birds, nor did it extend the period of bacterial fecal shedding. In conclusion, we report here that dmLT could be biologically incorporated in the secretion system of a live attenuated Salmonella-based vaccine, and that this construction is safe and could enhance mucosal immunity, and protect immunized birds against wild-type challenge. PMID:27262338

  8. 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

    2016-01-01

    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.

  9. 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

    2016-01-01

    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

  10. 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

  11. 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

  12. 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

  13. 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

  14. The staphylococcal enterotoxin (SE) family

    PubMed Central

    Krakauer, Teresa; Stiles, Bradley G

    2013-01-01

    Staphylococcus aureus plays an important role in numerous human cases of food poisoning, soft tissue, and bone infections, as well as potentially lethal toxic shock. This common bacterium synthesizes various virulence factors that include staphylococcal enterotoxins (SEs). These protein toxins bind directly to major histocompatibility complex class II on antigen-presenting cells and specific Vβ regions of T-cell receptors, resulting in potentially life-threatening stimulation of the immune system. Picomolar concentrations of SEs ultimately elicit proinflammatory cytokines that can induce fever, hypotension, multi-organ failure, and lethal shock. Various in vitro and in vivo models have provided important tools for studying the biological effects of, as well as potential vaccines/therapeutics against, the SEs. This review succinctly presents known physical and biological properties of the SEs, including various intervention strategies. In particular, SEB will often be portrayed as per biodefense concerns dating back to the 1960s. PMID:23959032

  15. 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. PMID:21929729

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

    PubMed

    Nassour, Hassan; Dubreuil, J Daniel

    2014-01-01

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

  17. Bacterial Toxins—Staphylococcal Enterotoxin B

    PubMed Central

    FRIES, BETTINA C.; VARSHNEY, AVANISH K.

    2015-01-01

    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. PMID:26184960

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

    PubMed Central

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

    1988-01-01

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

  19. Antigenicity and immunogenicity of fused B-subunit of heat labile toxin of Escherichia coli and colonization factor antigen I polyepitopes.

    PubMed

    Savar, Nastaran Sadat; Dashti, Amir; Darzi Eslam, Elham; Jahanian-Najafabadi, Ali; Jafari, Anis

    2014-11-01

    Linear B-cell epitopes ((93)AKEFEAAAL(101) and (66)PQLTDVLN(73)) of CfaB were genetically fused to ltb-(gly)5-cfaB(1-25). Sera of rabbits immunized with fusion proteins reacted strongly with solid-phase bound ETEC bacteria bearing CFA/I fimbriae. Sera failed to agglutinate or inhibit hemagglutination promoted by CFA/I-positive strain which may be due to solvent inaccessibility of epitope residues on intact fimbriae. PMID:25108290

  20. Specific inhibition of herpes virus replication by receptor-mediated entry of an antiviral peptide linked to Escherichia coli enterotoxin B subunit.

    PubMed Central

    Marcello, A; Loregian, A; Cross, A; Marsden, H; Hirst, T R; Palù, G

    1994-01-01

    Mimetic peptides capable of selectively disrupting protein-protein interactions represent potential therapeutic agents for inhibition of viral and cellular enzymes. This approach was first suggested by the observation that the peptide YAGAVVNDL, corresponding to the carboxyl-terminal 9 amino acids of the small subunit of ribonucleotide reductase of herpes simplex virus, specifically inhibited the viral enzyme in vitro. Evaluation and use of this peptide as a potential antiviral agent has, however, been thwarted by its failure to inhibit virus replication in vivo, presumably because the peptide is too large to enter eukaryotic cells unaided. Here, we show that the nontoxic B subunit of Escherichia coli heat-labile enterotoxin can be used as a recombinant carrier for the receptor-mediated delivery of YAGAVVNDL into virally infected cells. The resultant fusion protein specifically inhibited herpes simplex virus type 1 replication and ribonucleotide reductase activity in quiescent Vero cells. Preincubation of the fusion protein with soluble GM1 ganglioside abolished this antiviral effect, indicating that receptor-mediated binding to the target cell is necessary for its activity. This provides direct evidence of the usefulness of carrier-mediated delivery to evaluate the intracellular efficacy of a putative antiviral peptide. Images PMID:8090758

  1. 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

  2. Production of staphylococcal enterotoxin A in cream-filled cake.

    PubMed

    Anunciaçao, L L; Linardi, W R; do Carmo, L S; Bergdoll, M S

    1995-07-01

    Cakes were baked with normal ingredients and filled with cream, inoculated with different size enterotoxigenic-staphylococcal inocula. Samples of the cakes were incubated at room temperature and put in the refrigerator. Samples of cake and filling were taken at different times and analyzed for staphylococcal count and presence of enterotoxin. The smaller the inoculum, the longer the time required for sufficient growth (10(6)) to occur for production of detectable enterotoxin. Enterotoxin added to the cake dough before baking (210 degrees C, 45 min) did not survive the baking. The presence of enterotoxin in the contaminated cream filling indicated this as the cause of staphylococcal food poisoning from cream-filled cakes. Refrigeration of the cakes prevented the growth of the staphylococci.

  3. Molecular Screening of Staphylococcal Enterotoxin B Gene in Clinical Isolates

    PubMed Central

    Ataee, Ramezan Ali; Karami, Ali; Izadi, Morteza; Aghania, Aref; Ataee, Mohammad Hossein

    2011-01-01

    Objective: The role of staphylococcal enterotoxin B (SEB) in food poisoning is well known, however its role in other diseases remains to be explored. The aim of this study is the molecular screening and characterization of the SEB gene in clinically isolated strains. Materials and Methods: In this experimentally study, 300 Staphylococcus aureus (S. aureus) strains isolated from clinical samples were assayed. The isolated strains were confirmed by conventional bacteriological methods. Polymerase chain reaction (PCR) was used to determine the enterotoxin B (ent B) gene. Assessment of toxin production in all strains that contained the ent B gene was then performed. Finally, using specific antibody against SEB, a Western-blot was applied to confirm detection of enterotoxin B production. Results: Results indicated that only 5% of the 300 clinically isolated S. aureus contained the ent B gene. All strains which contained the ent B gene produced a proteinous enterotoxin B. The results of sequence determination of the PCR product were compared with the gene bank database and 98% similarity was achieved. The results of the Western-blot confirmed that enterotoxin B was produced in strains that contained the ent B gene. Conclusion: The results of this study indicate that 5% of clinically isolated S. aureus strains produce enterotoxin B. Considering that the enterotoxin B is an important superantigen, it is possible that a delay in diagnosis and lack of early proper treatment can cause an incidence of late complications, particularly in staphylococcal chronic infections. For this reason, it is suggested that in addition to detecting bacteria, an enterotoxin B detection test should be performed to control its toxigenicity. PMID:23508641

  4. Growth and enterotoxin production of Staphylococcus aureus in shrimp.

    PubMed Central

    Beckers, H. J.; Van Leusden, F. M.; Tips, P. D.

    1985-01-01

    Strains of Staphylococcus aureus isolated from shrimp were examined for phage pattern and enterotoxin production; 63% of the strains isolated from North Sea shrimp were typable with the International and additional set of phages, as were 38% of the strains isolated from South-East Asian shrimp. Staphylococcal enterotoxin(s) (SE) were produced by 48% and 35% of strains isolated from North Sea and South-East Asian shrimp respectively. Growth and enterotoxin production by S. aureus in shrimp was examined in storage experiments at 22 degrees C. S. aureus increased by 1-2 log units in 24 h when the organism was only a minor part of the total microflora of shrimp. When S. aureus was an equivalent part of the total flora its numbers increased by 3-4 log units in 24 h. Enterotoxins A and B became detectable when the number of S. aureus exceeded 10(7) per g in aseptically peeled shrimp. Results indicate that S. aureus is able to produce enterotoxin in shrimp, but its production depends upon a number of factors, including the relationship between S. aureus and competitive micro-organisms. It is concluded that the presence of S. aureus on commercially produced shrimp represents a potential hazard to health. PMID:4093610

  5. Enterotoxin gene profiles among Staphylococcus aureus isolated from raw milk

    PubMed Central

    Nazari, R; Godarzi, H; Rahimi Baghi, F; Moeinrad, M

    2014-01-01

    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 nine Staphylococcal enterotoxins (SEs) and enterotoxin gene profiles in S. aureus isolates derived from raw bovine milk. A total of 52 S. aureus isolates were obtained from 246 milk samples of 246 dairy cows from eight different farms in Qom, Iran. On the basis of cultural and biochemical properties as well as by amplification of the 23S rRNA specific to S. aureus, all isolates could be identified as S. aureus. Of the 52 isolates studied, 80.7% were positive for one or more genes encoding the enterotoxins, and 12 different genotypes were identified. The gene encoding for enterotoxin A (Sea) was the most frequent (16 isolates, 30.7%), followed by Seb (14 isolates, 26.9%) and Sed (8 isolates, 15.37%). Among the genes encoding the other enterotoxins, Seg and Seh were the most frequently observed (8 isolates each, 15.38%), followed by Sej (6 isolates, 11.5%) and Sei (1 isolates, 3.84%). With the recent identification of new SEs, the frequency of enterotoxigenic strains has increased, suggesting that the pathogenic potential of Staphylococci may be higher than previously thought. These results of enterotoxin genes positivity of milk-derived Staphylococci constitute a potential risk for consumers’ health. PMID:27175141

  6. 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. PMID:27054573

  7. 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

  8. 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.

  9. 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

  10. Tool for Quantification of Staphylococcal Enterotoxin Gene Expression in Cheese▿

    PubMed Central

    Duquenne, Manon; Fleurot, Isabelle; Aigle, Marina; Darrigo, Claire; Borezée-Durant, Elise; Derzelle, Sylviane; Bouix, Marielle; Deperrois-Lafarge, Véronique; Delacroix-Buchet, Agnès

    2010-01-01

    Cheese is a complex and dynamic microbial ecosystem characterized by the presence of a large variety of bacteria, yeasts, and molds. Some microorganisms, including species of lactobacilli or lactococci, are known to contribute to the organoleptic quality of cheeses, whereas the presence of other microorganisms may lead to spoilage or constitute a health risk. Staphylococcus aureus is recognized worldwide as an important food-borne pathogen, owing to the production of enterotoxins in food matrices. In order to study enterotoxin gene expression during cheese manufacture, we developed an efficient procedure to recover total RNA from cheese and applied a robust strategy to study gene expression by reverse transcription-quantitative PCR (RT-qPCR). This method yielded pure preparations of undegraded RNA suitable for RT-qPCR. To normalize RT-qPCR data, expression of 10 potential reference genes was investigated during S. aureus growth in milk and in cheese. The three most stably expressed reference genes during cheese manufacture were ftsZ, pta, and gyrB, and these were used as internal controls for RT-qPCR of the genes sea and sed, encoding staphylococcal enterotoxins A and D, respectively. Expression of these staphylococcal enterotoxin genes was monitored during the first 72 h of the cheese-making process, and mRNA data were correlated with enterotoxin production. PMID:20061456

  11. Tool for quantification of staphylococcal enterotoxin gene expression in cheese.

    PubMed

    Duquenne, Manon; Fleurot, Isabelle; Aigle, Marina; Darrigo, Claire; Borezée-Durant, Elise; Derzelle, Sylviane; Bouix, Marielle; Deperrois-Lafarge, Véronique; Delacroix-Buchet, Agnès

    2010-03-01

    Cheese is a complex and dynamic microbial ecosystem characterized by the presence of a large variety of bacteria, yeasts, and molds. Some microorganisms, including species of lactobacilli or lactococci, are known to contribute to the organoleptic quality of cheeses, whereas the presence of other microorganisms may lead to spoilage or constitute a health risk. Staphylococcus aureus is recognized worldwide as an important food-borne pathogen, owing to the production of enterotoxins in food matrices. In order to study enterotoxin gene expression during cheese manufacture, we developed an efficient procedure to recover total RNA from cheese and applied a robust strategy to study gene expression by reverse transcription-quantitative PCR (RT-qPCR). This method yielded pure preparations of undegraded RNA suitable for RT-qPCR. To normalize RT-qPCR data, expression of 10 potential reference genes was investigated during S. aureus growth in milk and in cheese. The three most stably expressed reference genes during cheese manufacture were ftsZ, pta, and gyrB, and these were used as internal controls for RT-qPCR of the genes sea and sed, encoding staphylococcal enterotoxins A and D, respectively. Expression of these staphylococcal enterotoxin genes was monitored during the first 72 h of the cheese-making process, and mRNA data were correlated with enterotoxin production.

  12. Chromosomal locus for staphylococcal enterotoxin B.

    PubMed Central

    Shafer, W M; Iandolo, J J

    1978-01-01

    The genetic locus of staphylococcal enterotoxin B (SEB) was investigated in the Staphylococcus aureus food-poisoning isolates, strains S6 and 277. Direct neutral sucrose gradient centrifugation analysis of sodium dodecyl sulfate-sodium chloride-mediated cleared lysates demonstrated that strain S6 contained a single 37S plasmid. Transductional analysis revealed that the 37S plasmid in S6 encoded for cadmium resistance (Cad) but not SEB. Additionally, elimination of cadmium resistance in S6 provided a plasmid-negative derivative that produced SEB at the same level as the parent. Examination of strain 277 showed two plasmids, a 37S species encoding for penicillin resistance (Penr) and a 21S species containing the gene(s) responsible for tetracycline resistance (Tetr). Elimination of the 37S, penr plasmid in 277 had no effect on SEB production, whereas introduction of the 21S tetr plasmid via transformation into strain 8325 (SEB--) did not confer enterotoxigenesis upon the transformants. The data obtained in this investigation suggest that the SEB gene(s) in these food-poisoning isolates of S. aureus is chromosomal. Images PMID:669796

  13. Production of enterotoxin A by supposedly nonenterotoxigenic Staphylococcus aureus strains.

    PubMed Central

    Gomez-Lucía, E; Goyache, J; Orden, J A; Blanco, J L; Ruiz-Santa-Quiteria, J A; Domínguez, L; Suárez, G

    1989-01-01

    The production of staphylococcal enterotoxins A (SEA) and B (SEB) was studied by inoculating six well-defined staphylococcal collection strains into cow's, goat's, or sheep's milk (individually or as a 50% mixture of cow's + goat's or cow's + sheep's), into brain heart infusion, and into a medium generally used to enhance the synthesis of enterotoxins (3+3 medium). Four of the strains used are considered to be SEB producers, another is considered an SEA producer, and the remaining strain is nonenterotoxigenic but produces large quantities of staphylococcal protein A. Staphylococcal protein A masked the results in most cases. Only one strain secreted exclusively SEB, while the other three SEB producers synthesized SEA in different amounts. We conclude that enterotoxin production depends on the natural substrate and may differ from the results obtained when the strain is grown on cellophane over agar to determine its toxigenicity. PMID:2764561

  14. Inverse relationship between heat stable enterotoxin-b induced fluid accumulation and adherence of F4ac-positive enterotoxigenic Escherichia coli in ligated jejunal loops of F4ab/ac fimbria receptor-positive swine.

    PubMed

    Erume, Joseph; Wijemanne, Prageeth; Berberov, Emil M; Kachman, Stephen D; Oestmann, Daniel J; Francis, David H; Moxley, Rodney A

    2013-01-25

    Heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) increases bacterial adherence to porcine enterocytes in vitro and enhances small intestinal colonization in swine. Heat-stable enterotoxin-b (STb) is not known to affect colonization; however, through an induction of net fluid accumulation it might reduce bacterial adherence. The relationship between fluid accumulation and bacterial adherence in jejunal loops inoculated with ETEC strains that produce LT, STb, both, or neither toxin was studied. Ligated jejunal loops were constructed in weaned Yorkshire pigs in two independent experiments (Exp. 1, n=5, 8-week-old; Exp. 2, n=6, 6-8-week-old). Each pig was inoculated with six F4ac(+)E. coli strains: (1) LT(+), STb(+) parent (WAM2317); (2) STb(-) (ΔestB) mutant (MUN297); (3) MUN297 complemented with STb (MUN298); (4) LT(-) STb(-) (ΔeltAB ΔestB) mutant (MUN300); (5) MUN300 complemented with LT (MUN301); and (6) 1836-2 (non-enterotoxigenic, wild-type). Pigs were confirmed to be K88 (F4)ab/ac receptor-positive in Exp. 2 by testing for intestinal mucin-type glycoproteins and inferred to be receptor-positive in both Exp. 1 and 2 based on histopathologic evidence of bacterial adherence. Strains that produced STb induced marked fluid accumulation with the response (ml/cm) to WAM2317 and MUN298 significantly greater than that to the other strains (P<0.0001). Conversely, bacterial adherence scores based on immunohistochemistry and CFU/g of washed mucosa were both lowest in the strains that expressed STb and highest in those that did not. For the two experiments combined, the Pearson correlation coefficient (R) between fluid volume (ml/cm) and log CFU per gram was -0.57021 (P<0.0001); R(2)=0.3521 (n=197). These results support the hypothesis that enterotoxin-induced fluid accumulation flushes progeny organisms into the lumen of the bowel, thereby increasing the likelihood of fecal shedding and transmission of the pathogen to new hosts. PMID

  15. 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

  16. Contamination of beef products with staphylococcal classical enterotoxins in Egypt and Saudi Arabia

    PubMed Central

    Shawish, Reyad R.; Al-Humam, Naser A.

    2016-01-01

    Food-borne pathogens are of high concern for public health and food safety. Staphylococcus aureus food poisoning is one of the most economically devastating types of food poisoning globally. The purpose of this study was to detect staphylococcal classical enterotoxins (SEs) in processed beef from Kingdom of Saudi Arabia (KSA) and Egypt. In the present investigation a total of 250 random processed meat samples (50 each of minced meat, beef burger, beef sausage, beef kofta and beef luncheon) were collected from different super markets in the study area. Using conventional cultural methods, samples were cultured for isolation and identification of S. aureus. Multiplex PCR was used to detect SEs of the classical type SEA, SEB, SEC and SED from isolates. The percentage presence of S. aureus in minced meat, beef burger, beef sausage, beef kofta and beef luncheon was 38%, 22%, 30%, 32% and 12%, respectively. Multiplex PCR indicated that all examined samples contain different types of classical staphylococcal enterotoxins and only minced meat samples contained all four types of toxins. Multiplex PCR is efficient in detection of SEs from food and may be used in tracing of toxins to promote food hygiene. Implications of contamination of processed meat to food hygiene in the study area are highlighted. PMID:27088066

  17. Contamination of beef products with staphylococcal classical enterotoxins in Egypt and Saudi Arabia.

    PubMed

    Shawish, Reyad R; Al-Humam, Naser A

    2016-01-01

    Food-borne pathogens are of high concern for public health and food safety. Staphylococcus aureus food poisoning is one of the most economically devastating types of food poisoning globally. The purpose of this study was to detect staphylococcal classical enterotoxins (SEs) in processed beef from Kingdom of Saudi Arabia (KSA) and Egypt. In the present investigation a total of 250 random processed meat samples (50 each of minced meat, beef burger, beef sausage, beef kofta and beef luncheon) were collected from different super markets in the study area. Using conventional cultural methods, samples were cultured for isolation and identification of S. aureus. Multiplex PCR was used to detect SEs of the classical type SEA, SEB, SEC and SED from isolates. The percentage presence of S. aureus in minced meat, beef burger, beef sausage, beef kofta and beef luncheon was 38%, 22%, 30%, 32% and 12%, respectively. Multiplex PCR indicated that all examined samples contain different types of classical staphylococcal enterotoxins and only minced meat samples contained all four types of toxins. Multiplex PCR is efficient in detection of SEs from food and may be used in tracing of toxins to promote food hygiene. Implications of contamination of processed meat to food hygiene in the study area are highlighted. PMID:27088066

  18. Growth and enterotoxin A production by Staphylococcus aureus S6 in Manchego type cheese.

    PubMed

    Gomez-Lucia, E; Blanco, J L; Goyache, J; de la Fuente, R; Vazquez, J A; Ferri, E F; Suarez, G

    1986-12-01

    Milk (from cow, goat and sheep) was inoculated with Staphylococcus aureus strain S6, which is generally considered to be a strong enterotoxin B producer and a weak enterotoxin A producer. It was then used to make Manchego type cheese as prepared industrially. Two concentrations of starter culture (1% and 0.1%) were tested. Staphylococcal growth was good in both but better in the more dilute culture. Staphylococcal enterotoxin B was not detected at any stage of the ripening process of any cheese tested. However enterotoxin A was detected in both starter concentrations, reaching as high as 769 ng/100 g of cheese in the 0.1% starter batches. PMID:3558164

  19. 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.

  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. Prevalence of enterotoxigenic Escherichia coli in some processed raw food from animal origin.

    PubMed

    Reis, M H; Vasconcelos, J C; Trabulsi, L R

    1980-01-01

    Eighteen of 1,200 colonies of Escherichia coli isolated from "keebe," hamburger, or sausage produced heat-labile enterotoxin. None of them produced heat-stable enterotoxin. The characteristics of 9 of the 18 strains are presented. PMID:6986850

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. 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

  5. Production of Enterotoxin by Yersinia bercovieri, a Recently Identified Yersinia enterocolitica-Like Species

    PubMed Central

    Sulakvelidze, Alexander; Kreger, Arnold; Joseph, Aaron; Robins-Browne, Roy M.; Fasano, Alessio; Wauters, Georges; Harnett, Norma; DeTolla, Louis; Morris, J. Glenn

    1999-01-01

    Yersinia bercovieri, a recently identified Y. enterocolitica-like species, produces a heat-stable enterotoxin (designated YbST) which has biologic activity in infant mice and increases short circuit current in Ussing chambers. Although YbST has some properties in common with the heat-stable enterotoxins of Y. enterocolitica (YST I and YST II), it appears to be a novel toxin because (i) it was not neutralized by anti-YST I antiserum, (ii) YbST-neutralizing antiserum did not neutralize YST I, and (iii) Y. bercovieri strains did not hybridize with genetic probes for yst I, yst II, and other known enterotoxins. PMID:9916117

  6. 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. PMID:27074376

  7. 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.

  8. Enterotoxin B is the predominant toxin involved in staphylococcal scarlet fever in Taiwan.

    PubMed

    Wang, Chih-Chien; Lo, Wen-Tsung; Hsu, Chen-Fang; Chu, Mong-Ling

    2004-05-15

    This study retrospectively reviewed all pediatric cases of staphylococcal scarlet fever (SSF) that occurred during a 10-year period in a 1400-bed tertiary medical center in northern Taiwan. All 20 cases of SSF occurred in previously healthy individuals. Skin and soft-tissue infections predominated among children from whom Staphylococcus aureus was isolated. Polymerase chain reaction testing was used to detect known staphylococcal toxin genes, and of the isolates studied, most (18 [90%] of 20) contained only the staphylococcal enterotoxin B. One of the other strains was positive for staphylococcal enterotoxin A only, and the last strain was positive for both staphylococcal enterotoxin G and staphylococcal enterotoxin I. Pulsed-field gel electrophoresis identified a small cluster of isolates (6 [30%] of 20) that were genetically related, but these strains came from epidemiologically unrelated patients during a 3-year period.

  9. Staphylococcal Enterotoxin C in Synovial Fluid of Patients With Rheumatoid Arthritis

    PubMed Central

    Ataee, Ramezan Ali; Ataee, Mohammad Hosein; Alishiri, Gholam Hosein; Esmaeili, Davoud

    2014-01-01

    Background: In the previous studies using the commercial ELISA kit, the existence of staphylococcal superantigens has been reported in synovial fluid of patients with rheumatoid arthritis (RA). Objectives: This study aimed to design molecular methods to detect staphylococcal enterotoxin C in synovial fluid of patients with rheumatoid arthritis. Materials and Methods: In this experimental study, Staphylococcus aureus strain producing enterotoxin C was used as the reference strain. The polymerase chain reaction (PCR) was set up by design a specific pair of primers. Besides bacterial culture, 50 synovial fluid samples of patients with rheumatoid arthritis were subjected to DNA extraction, and then PCR amplification was carried out according to the protocol. All samples were examined by ELISA method for enterotoxin C. The data were descriptively analyzed. Results: The results of bacterial culture were negative for all samples. The results showed that 66% (33 cases) of samples contained entC gene and only 46% (23 cases) have also enterotoxin C. The interesting finding was that the results of ELISA and PCR were the same and have shown only 22 positive cases (44%samples) for staphylococcal enterotoxin C. Conclusions: Based on the findings of this study, S. aureus enterotoxin C (SEC) has been detected in synovial fluid of patients with rheumatoid arthritis by PCR and ELISA methods. These valuable findings may describe the exact etiology of the RA and as well as change the methods of its diagnosis and treatment. This is the first research, which has shown the staphylococcal entC gene in synovial fluid of RA patients. However, S. aureus strains can produce more than 20 types of enterotoxins. Therefore, its involvement on rheumatoid arthritis pathogenesis makes an important challenge in the future. In this regard, further investigation on the other enterotoxins is necessary. PMID:25558381

  10. Growth of Staphylococcus aureus and synthesis of enterotoxin during ripening of experimental Manchego-type cheese.

    PubMed

    Gómez-Lucía, E; Goyache, J; Orden, J A; Domenech, A; Javier Hernandez, F; Ruiz-Santa Quiteria, J A; Lopez, B; Blanco, J L; Suárez, G

    1992-01-01

    To study the possible presence of staphylococcal enterotoxins in Manchego-type cheese, milk was inoculated with the enterotoxigenic Staphylococcus aureus collection strains FRI-100, S6, FRI-137, and FRI-472 to a final concentration of 10,000 to 25,000 cfu/ml. Cheese was prepared following the industrial specifications and ripened for 60 d. Batches were prepared with 1 and .1% lactic acid culture and labeled with the abbreviated name of the strain and the concentration of lactic acid culture. Mean staphylococcal counts in .1% lactic bacteria cheeses were usually more than 1 log higher than the corresponding 1% ones. Staphylococcal counts declined markedly after d 35 to 42, and, by the end of ripening, they had disappeared from some cheeses. Enterotoxins were present in five of the cheeses, three prepared with .1% and two with 1% lactic bacteria. Enterotoxins detected were A and D, the enterotoxins most commonly associated with human intoxication. The maximum level of enterotoxin A detected in cheese with strain FRI-100 and with the .1% culture was 222 ng/100 g of cheese; in cheese FRI-100 with 1%, 111 ng/100 g; in cheese S6 with .1%, 769 ng/100 g; and in cheese S6 with 1%, 33 ng/100 g. Maximum level of enterotoxin D detected in cheese FRI-472 with .1% was 38 ng/100 g. PMID:1541730

  11. 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

  12. Enterotoxin production by strains of Staphylococcus aureus isolated from clinical and non-clinical specimens with special reference to enterotoxin F and toxic shock syndrome.

    PubMed Central

    de Nooij, M. P.; van Leeuwen, W. J.; Notermans, S.

    1982-01-01

    Enterotoxin production by strains of Staphylococcus aureus isolated from clinical specimens of human and animal origin and from healthy human carriers was investigated. All nine patients admitted to hospital with symptoms of toxic shock syndrome (TSS) yielded enterotoxin-producing strains of S. aureus. Eight of these produced staphylococcal enterotoxin F (SEF). A significantly smaller proportion of strains (42% of 50 strains tested) isolated from other clinical specimens of hospitalized patients produced SEF. Production of SEF by strains isolated from clinical specimens of animal origin (48 strains) was not observed. Twenty-nine per cent of 24 S. aureus strains isolated from noses of hospital staff produced SEF. This result was not significantly different from that obtained from strains isolated from clinical specimens other than TSS. A similar percentage of strains isolated from healthy human carriers outside hospital produced SEF (25% of 24 strains tested). The results indicated that enterotoxin production, especially that of SEF, is associated with S. aureus isolated from patients suspected of TSS. There was no indication of an association between S. aureus isolated from other staphylococcal infections and SEF production. All strains were phage typed and 79% of the strains belonging to the international phage-group I produced SEF. All strains lysed by phage 187 were found to produce SEF. PMID:6218196

  13. A tripartite fusion, FaeG-FedF-LT(192)A2: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

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

    2011-10-01

    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 (LT(192)) A2 and B subunits for a tripartite adhesin-adhesin-toxoid fusion (FaeG-FedF-LT(192)A2: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-LT(192)A2: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-LT(192)A2: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

  14. Further evidence for staphylococcal food poisoning outbreaks caused by egc-encoded enterotoxins.

    PubMed

    Johler, Sophia; Giannini, Petra; Jermini, Marco; Hummerjohann, Jörg; Baumgartner, Andreas; Stephan, Roger

    2015-03-20

    Staphylococcal food poisoning represents the most prevalent foodborne intoxication worldwide. It is caused by oral intake of enterotoxins preformed by Staphylococcus aureus in food. The relevance of newly described enterotoxins in outbreaks of staphylococcal food poisoning is controversially discussed. Although the staphylococcal enterotoxins SEG, SEI, SEM, SEN, and SEO elicit emesis in a monkey feeding assay, there has been no conclusive proof of their emetic activity in humans. In this study, we provide further evidence suggesting that one of these enterotoxins or a combination of SEG, SEI, SEM, SEN, and SEO cause staphylococcal food poisoning. We investigated two outbreaks registered with the Swiss Federal Office of Public Health, in which only Staphylococcus aureus strains harboring the egc cluster, including seg, sei, sem, sen, and seo linked to typical signs of staphylococcal food poisoning were isolated. The outbreaks were caused by consumption of raw goat cheese and semi-hard goat cheese, and were linked to strains assigned to CC45 (agr type I) and CC9 (agr type II), respectively. These outbreaks provide further evidence that newly-described staphylococcal enterotoxins are likely to cause staphylococcal food poisoning in humans.

  15. Virulence Factors of Staphylococcus aureus Isolated from Korean Pork bulgogi: Enterotoxin Production and Antimicrobial Resistance.

    PubMed

    Jung, Byeong Su; Lee, Yong Ju; Lee, Na-Kyoung; Kim, Hyoun Wook; Oh, Mi-Hwa; Paik, Hyun-Dong

    2015-01-01

    The aim of this study was to investigate the antimicrobial resistance profiles of and the enterotoxin gene distribution in 4 strains of Staphylococcus aureus (S10-2, S10-3, S12-2, and S13-2) isolated from 90 bulgogi samples. The S. aureus enterotoxin H gene (seh) was found in all the strains, while the S. aureus enterotoxin A gene (sea) was found only in 3 of the 4 strains. The S10-2 strain expressed a combination of enterotoxin genes - seg, seh, sei, sej, selm, and seln. The strains S10-2 and S13-2 were resistant to ampicillin and penicillin G, and all the isolated strains were resistant to tetracycline. The S10-2 strain was the only mecA-positive strain; it was also resistant to β-lactam antibiotics. Thus, genes encoding enterotoxin as well as those conferring antibiotic resistance were identified in the S. aureus strains isolated from pork bulgogi. These results represents the potential occurrence of MRSA in pork bulgogi, and the need for a monitoring system for pork bulgogi in order to prevent an outbreak of staphylococcal food poisoning.

  16. 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

  17. 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

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

    PubMed

    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

  19. Staphylococcal enterotoxin and its rapid identification in foods by enzyme-linked immunosorbent assay-based methodology.

    PubMed

    Bennett, Reginald W

    2005-06-01

    The problem of Staphylococcus aureus and other species as contaminants in the food supply remains significant on a global level. Time and temperature abuse of a food product contaminated with enterotoxigenic staphylococci can result in formation of enterotoxin, which can produce foodborne illness when the product is ingested. Between 100 and 200 ng of enterotoxin can cause symptoms consistent with staphylococcal intoxication. Although humans are the primary reservoirs of contamination, animals, air, dust, and food contact surfaces can serve as vehicles in the transfer of this pathogen to the food supply. Foods may become contaminated during production or processing and in homes or food establishments, where the organism can proliferate to high concentrations and subsequently produce enterotoxin. The staphylococcal enterotoxins are highly heat stable and can remain biologically active after exposure to retort temperatures. Prior to the development of serological methods for the identification of enterotoxin, monkeys (gastric intubation) and later kittens (intravenous injection) were used in assays for toxin detection. When enterotoxins were identified as mature proteins that were antigenic, serological assays were developed for use in the laboratory analysis of foods suspected of containing preformed enterotoxin. More recently developed methods are tracer-labeled immunoassays. Of these methods, the enzyme-linked immunosorbent assays are highly specific, highly sensitive, and rapid for the detection of enterotoxin in foods.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  1. Detection of Staphylococcus Enterotoxin B at Picogram Levels Using Piezoelectric-Excited Millimeter-Sized Cantilever Sensors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report a highly sensitive and rapid method for thhe detection of Staphylocoiccus aureus enterotoxin B (SEB) at picogram levels using a piezolelectric-excited millimeter cantilever (PEMC) sensor. Affinity purified polyclonal antibody to staphylococcal enterotoxin B (anti-SEB) was immobilized on th...

  2. Inhibition of neuromuscular transmission in isolated mouse phrenic nerve-diaphragm by the enterotoxin of Clostridium perfringens type A.

    PubMed

    Sugimoto, N; Miyamoto, A; Horiguchi, Y; Okabe, T; Matsuda, M

    1992-08-01

    The enterotoxin of Clostridium perfringens type A, a channel forming protein toxin, inhibited neuromuscular transmission under conditions of low calcium. Twitch tension of isolated phrenic nerve-diaphragm preparations elicited by electrical stimulations to the phrenic nerve was recorded isometrically, and the preparations were exposed to the purified enterotoxin. In Krebs solution containing 0.5 mM calcium, the enterotoxin (20 micrograms/ml) reduced within 10 min the amplitude of the twitch tension to 34 +/- 7% (mean +/- S.D., n = 11) of that recorded before the treatment. The effects of the enterotoxin on the twitch tension were irreversible and proceeded independently of stimulation. The reduction of the twitch tension by the enterotoxin was apparent in Krebs solution containing less than 0.6 mM calcium and the degree of reduction was inversely related to the concentration of calcium. The reduction of the twitch tension by the enterotoxin was also dependent on temperature and concentration of the toxin. At temperatures below 20 degrees C, no obvious reduction of twitch tension was observed with 20 micrograms/ml of the enterotoxin. Enterotoxin at a concentration of 0.4 micrograms/ml caused 16 +/- 2% (mean +/- S.D., n = 4) reduction of twitch tension, and the degree of the reduction in twitch tension increased with toxin concentration, reaching a plateau of 65 +/- 4% (mean +/- S.D., n = 7) at 6.5 micrograms/ml of the enterotoxin. The effects of the enterotoxin were antagonized by 2 microM physostigmine. Unlike curare, pretreatment of the preparation with enterotoxin did not antagonize the neuromuscular block by decamethonium. Neither the tension of muscular twitch elicited by direct electrical stimulation to the muscle nor the resting membrane potentials of muscle fibers recorded intracellularly were affected by the enterotoxin. The enterotoxin (2.2 micrograms/ml) reduced the frequency, but not mean amplitude or amplitude distribution, of miniature end

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

    PubMed Central

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

    2010-01-01

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

  4. 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.

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

    SciTech Connect

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

    1988-11-01

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

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

    PubMed

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

    2015-11-01

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

  7. Late Multiple Organ Surge in Interferon-Regulated Target Genes Characterizes Staphylococcal Enterotoxin B Lethality

    PubMed Central

    Ferreyra, Gabriela A.; Elinoff, Jason M.; Demirkale, Cumhur Y.; Starost, Matthew F.; Buckley, Marilyn; Munson, Peter J.; Krakauer, Teresa; Danner, Robert L.

    2014-01-01

    Background Bacterial superantigens are virulence factors that cause toxic shock syndrome. Here, the genome-wide, temporal response of mice to lethal intranasal staphylococcal enterotoxin B (SEB) challenge was investigated in six tissues. Results The earliest responses and largest number of affected genes occurred in peripheral blood mononuclear cells (PBMC), spleen, and lung tissues with the highest content of both T-cells and monocyte/macrophages, the direct cellular targets of SEB. In contrast, the response of liver, kidney, and heart was delayed and involved fewer genes, but revealed a dominant genetic program that was seen in all 6 tissues. Many of the 85 uniquely annotated transcripts participating in this shared genomic response have not been previously linked to SEB. Nine of the 85 genes were subsequently confirmed by RT-PCR in every tissue/organ at 24 h. These 85 transcripts, up-regulated in all tissues, annotated to the interferon (IFN)/antiviral-response and included genes belonging to the DNA/RNA sensing system, DNA damage repair, the immunoproteasome, and the ER/metabolic stress-response and apoptosis pathways. Overall, this shared program was identified as a type I and II interferon (IFN)-response and the promoters of these genes were highly enriched for IFN regulatory matrices. Several genes whose secreted products induce the IFN pathway were up-regulated at early time points in PBMCs, spleen, and/or lung. Furthermore, IFN regulatory factors including Irf1, Irf7 and Irf8, and Zbp1, a DNA sensor/transcription factor that can directly elicit an IFN innate immune response, participated in this host-wide SEB signature. Conclusion Global gene-expression changes across multiple organs implicated a host-wide IFN-response in SEB-induced death. Therapies aimed at IFN-associated innate immunity may improve outcome in toxic shock syndromes. PMID:24551153

  8. Novel Tissue Level Effects of the Staphylococcus aureus Enterotoxin Gene Cluster Are Essential for Infective Endocarditis

    PubMed Central

    Stach, Christopher S.; Vu, Bao G.; Merriman, Joseph A.; Herrera, Alfa; Cahill, Michael P.; Schlievert, Patrick M.; Salgado-Pabón, Wilmara

    2016-01-01

    Background Superantigens are indispensable virulence factors for Staphylococcus aureus in disease causation. Superantigens stimulate massive immune cell activation, leading to toxic shock syndrome (TSS) and contributing to other illnesses. However, superantigens differ in their capacities to induce body-wide effects. For many, their production, at least as tested in vitro, is not high enough to reach the circulation, or the proteins are not efficient in crossing epithelial and endothelial barriers, thus remaining within tissues or localized on mucosal surfaces where they exert only local effects. In this study, we address the role of TSS toxin-1 (TSST-1) and most importantly the enterotoxin gene cluster (egc) in infective endocarditis and sepsis, gaining insights into the body-wide versus local effects of superantigens. Methods We examined S. aureus TSST-1 gene (tstH) and egc deletion strains in the rabbit model of infective endocarditis and sepsis. Importantly, we also assessed the ability of commercial human intravenous immunoglobulin (IVIG) plus vancomycin to alter the course of infective endocarditis and sepsis. Results TSST-1 contributed to infective endocarditis vegetations and lethal sepsis, while superantigens of the egc, a cluster with uncharacterized functions in S. aureus infections, promoted vegetation formation in infective endocarditis. IVIG plus vancomycin prevented lethality and stroke development in infective endocarditis and sepsis. Conclusions Our studies support the local tissue effects of egc superantigens for establishment and progression of infective endocarditis providing evidence for their role in life-threatening illnesses. In contrast, TSST-1 contributes to both infective endocarditis and lethal sepsis. IVIG may be a useful adjunct therapy for infective endocarditis and sepsis. PMID:27124393

  9. 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. PMID:25572397

  10. Staphylococcal enterotoxin B influences the DNA methylation pattern in nasal polyp tissue: a preliminary study

    PubMed Central

    2013-01-01

    Staphylococcal enterotoxins may influence the pro-inflammatory pattern of chronic sinus diseases via epigenetic events. This work intended to investigate the potential of staphylococcal enterotoxin B (SEB) to induce changes in the DNA methylation pattern. Nasal polyp tissue explants were cultured in the presence and absence of SEB; genomic DNA was then isolated and used for whole genome methylation analysis. Results showed that SEB stimulation altered the methylation pattern of gene regions when compared with non stimulated tissue. Data enrichment analysis highlighted two genes: the IKBKB and STAT-5B, both playing a crucial role in T- cell maturation/activation and immune response. PMID:24341752

  11. Polymerase chain reaction detection of enterotoxins genes in coagulase-negative staphylococci isolated from Brazilian Minas cheese.

    PubMed

    Rall, Vera Lúcia Mores; Sforcin, José Maurício; de Deus, Maria Fernanda Ramos; de Sousa, Daniel Casaes; Camargo, Carlos Henrique; Godinho, Natália Cristina; Galindo, Luciane Almeida; Soares, Taíssa Cook Siqueira; Araújo, João Pessoa

    2010-09-01

    For a long time, Staphylococcus aureus has been always thought to be the only pathogenic species among Staphylococcus, while coagulase-negative staphylococci (CNS) were classified as contaminant agents. However, molecular techniques have shown that these microorganisms also possess enterotoxin-encoding genes. The aim of this study was to analyze the frequency of genes for staphylococcal enterotoxins SEA, SEB, SEC, and SED in CNS strains isolated from Minas soft cheese and to assess the in vitro production of toxins. CNS were found in 65 (72.2%) samples of cheese: 23 were Staphylococcus saprophyticus, 16 Staphylococcus warneri, 10 Staphylococcus epidermidis, 9 Staphylococcus xylosus, 3 Staphylococcus haemolyticus, 2 Staphylococcus schleiferi subsp. schleiferi, and 1 each Staphylococcus capitis subsp. urealyticus and Staphylococcus caprae. Seventeen (26.2%) CNS strains had genes for enterotoxins, and sea was more frequently found (18.5%), followed by sec in three and seb in two strains, whereas the sed gene was not found. S. saprophyticus showed enterotoxin genes in 6 of 23 isolates, but only sea was observed. On the other hand, five strains of S. warneri showed the sea, seb, or sec gene. In spite of the presence of these enterotoxin genes, these strains did not produce enterotoxins in vitro. It is essential to understand the real role of CNS in food, and based on the presence of enterotoxin genes, CNS should not be ignored in epidemiological investigations of foodborne outbreaks.

  12. 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 Central

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

    2015-01-01

    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. PMID:26341202

  13. 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.

  14. Incidence and characterization of diarrheal enterotoxins of fecal Bacillus cereus isolates associated with diarrhea.

    PubMed

    Al-Khatib, Mariam Saleh; Khyami-Horani, Hala; Badran, Eman; Shehabi, Asem A

    2007-12-01

    A total of 490 stool specimens were collected from patients with diarrhea and healthy controls without diarrhea to investigate the incidence of Bacillus cereus and its enterotoxins. B. cereus was found more significant in stools of persons with diarrhea than without diarrhea (9.5% versus 1.8%, P < 0.05), and was also detected more frequent but not significant in individuals aged > or =1 year and in adults than in children aged <1 year (11% and 8% versus 7.8%, P > 0.05). The hemolytic enterotoxin HBL genes of B. cereus isolates (hblA, hblC, hblD) were detected in 58%, 58%, and 68%, respectively, whereas the nonhemolytic enterotoxin NHE genes (nheA, nheB, nheC) were detected more frequent in 71.%, 84%, and 90% of the isolates, respectively. This study suggests that B. cereus isolates harboring 1 or more enterotoxin gene(s) can be a potential cause of diarrhea in Jordanian population.

  15. 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. PMID:19722395

  16. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  18. 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. PMID:180885

  19. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. 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

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

    PubMed

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

    2015-09-14

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

  4. 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...

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

    PubMed

    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.

  6. 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.

  7. 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....

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. Staphylococcal Enterotoxin B Causes Proliferation of Sensory C-Fibers and Subsequent Enhancement of Neurogenic Inflammation in Rat Skin

    PubMed Central

    Ohshima, Mihoko; Miyake, Mio; Takeda, Masanori; Kamijima, Michihiro

    2011-01-01

    Background. Staphylococcal enterotoxin B (SEB) may be associated with the exacerbation of atopic dermatitis. We investigated whether SEB causes proliferation of sensory C-fibers and subsequent enhancement of plasma leakage induced by sensorineural stimulation in rat skin. Methods. SEB was applied intracutaneously to the abdomen of preweaning and adult rats. Evans blue dye leakage into the skin induced by topical 10% formalin was measured as an index of neurogenic skin vascular permeability. Local expression of substance P, tachykinin NK1 receptors, and nerve growth factor was assessed immunohistochemically. In addition, we assessed the effects of topical tacrolimus on these skin responses induced by SEB. Results. Increased neurogenic skin plasma leakage was seen 7 days after SEB treatment in 2 different age groups. Innervation of substance P-immunoreactive nerves and expression of tachykinin NK1 receptors and nerve growth factor were also promoted by SEB, peaking at 7 days, 7 days, and 56 h after SEB treatment, respectively. Tacrolimus markedly inhibited these skin changes. Conclusions. SEB increased the innervation of sensory C-fibers and tachykinin NK1 receptors in rat skin, probably because of upregulated production of neurotrophins, including nerve growth factor, leading to enhancement of neurogenic skin inflammation. T cell activation induced by SEB may initiate these changes. PMID:21252260

  14. Inhibition of enterotoxin from Escherichia coli and Vibrio cholerae by gangliosides from human milk.

    PubMed Central

    Otnaess, A B; Laegreid, A; Ertresvåg, K

    1983-01-01

    Inhibitory activity of enterotoxin from Escherichia coli and Vibrio cholerae was associated with the ganglioside fraction of human milk. Both the milk fat and skim milk contained gangliosides that inhibited the toxins. The most purified milk fraction contained three glycolipid components, of which two migrated close to ganglioside GM1 on thin-layer chromatography plates. A component with a slightly different mobility from GM1 appeared to be associated with the inhibitory activity. Milk ganglioside fraction, derived from 2 ml of human milk, contained 1 to 4 micrograms of lipid-bound sialic acid and completely inhibited 0.1 micrograms of cholera toxin in rabbit intestinal loop experiments. It is suggested that human milk gangliosides, although present only in trace amounts, may be important in protecting infants against enterotoxin-induced diarrhea. PMID:6341242

  15. 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

  16. 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.

  17. 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.

  18. An Enterotoxin-Bearing Pathogenicity Island in Staphylococcus epidermidis▿†

    PubMed Central

    Madhusoodanan, Jyoti; Seo, Keun Seok; Remortel, Brian; Park, Joo Youn; Hwang, Sun Young; Fox, Lawrence K.; Park, Yong Ho; Deobald, Claudia F.; Wang, Dan; Liu, Song; Daugherty, Sean C.; Gill, Ann Lindley; Bohach, Gregory A.; Gill, Steven R.

    2011-01-01

    Cocolonization of human mucosal surfaces causes frequent encounters between various staphylococcal species, creating opportunities for the horizontal acquisition of mobile genetic elements. The majority of Staphylococcus aureus toxins and virulence factors are encoded on S. aureus pathogenicity islands (SaPIs). Horizontal movement of SaPIs between S. aureus strains plays a role in the evolution of virulent clinical isolates. Although there have been reports of the production of toxic shock syndrome toxin 1 (TSST-1), enterotoxin, and other superantigens by coagulase-negative staphylococci, no associated pathogenicity islands have been found in the genome of Staphylococcus epidermidis, a generally less virulent relative of S. aureus. We show here the first evidence of a composite S. epidermidis pathogenicity island (SePI), the product of multiple insertions in the genome of a clinical isolate. The taxonomic placement of S. epidermidis strain FRI909 was confirmed by a number of biochemical tests and multilocus sequence typing. The genome sequence of this strain was analyzed for other unique gene clusters and their locations. This pathogenicity island encodes and expresses staphylococcal enterotoxin C3 (SEC3) and staphylococcal enterotoxin-like toxin L (SElL), as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and immunoblotting. We present here an initial characterization of this novel pathogenicity island, and we establish that it is stable, expresses enterotoxins, and is not obviously transmissible by phage transduction. We also describe the genome sequence, excision, replication, and packaging of a novel bacteriophage in S. epidermidis FRI909, as well as attempts to mobilize the SePI element by this phage. PMID:21317317

  19. 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.

  20. Staphylococcus aureus agr Genotypes with Enterotoxin Production Capabilities Can Resist Neutrophil Bactericidal Activity

    PubMed Central

    Mullarky, I. K.; Su, C.; Frieze, N.; Park, Y. H.; Sordillo, L. M.

    2001-01-01

    Staphylococcus aureus pathogenicity is mainly due to the production of a number of secreted and cell surface-associated proteins under the regulation of the agr gene. A region of the agr gene was used to subgroup S. aureus strains according to restriction fragment length polymorphisms. Additionally, strains were subtyped according to the coagulase gene in order to strengthen discriminatory power. Virulence capabilities of agr genotype subgroups were evaluated using an in vitro neutrophil bactericidal assay, which showed that prevalent genotypes were significantly better at evading this primary host defense. Multiplex PCR was then used to detect enterotoxin genes among the genotype subgroups in order to determine possible virulence candidates that enable strains to combat neutrophil killing. The prevalent genotype strains were found to possess higher production capabilities for enterotoxin A than did low-prevalence strains. The significance of enterotoxin A production capabilities in affecting pathogenicity of S. aureus strains was evaluated and found to have a profound effect on neutrophil killing abilities. The use of a large epidemiological database as a tool for subgrouping strains with varying degrees of pathogenicity has allowed the identification of relevant and previously undefined virulence factors that affect a pathogen's capability to overcome host immune defenses. PMID:11119487

  1. Structural basis for disruption of claudin assembly in tight junctions by an enterotoxin

    PubMed Central

    Shinoda, Takehiro; Shinya, Naoko; Ito, Kaori; Ohsawa, Noboru; Terada, Takaho; Hirata, Kunio; Kawano, Yoshiaki; Yamamoto, Masaki; Kimura-Someya, Tomomi; Yokoyama, Shigeyuki; Shirouzu, Mikako

    2016-01-01

    The food-poisoning bacterium Clostridium perfringens produces an enterotoxin (~35 kDa) that specifically targets human claudin-4, among the 26 human claudin proteins, and causes diarrhea by fluid accumulation in the intestinal cavity. The C-terminal domain of the Clostridium perfringens enterotoxin (C-CPE, ~15 kDa) binds tightly to claudin-4, and disrupts the intestinal tight junction barriers. In this study, we determined the 3.5-Å resolution crystal structure of the cell-free synthesized human claudin-4•C-CPE complex, which is significantly different from the structure of the off-target complex of an engineered C-CPE with mouse claudin-19. The claudin-4•C-CPE complex structure demonstrated the mechanism underlying claudin assembly disruption. A comparison of the present C-CPE-bound structure of claudin-4 with the enterotoxin-free claudin-15 structure revealed sophisticated C-CPE-induced conformation changes of the extracellular segments, induced on the foundation of the rigid four-transmembrane-helix bundle structure. These conformation changes provide a mechanistic model for the disruption of the lateral assembly of claudin molecules. Furthermore, the present novel structural mechanism for selecting a specific member of the claudin family can be used as the foundation to develop novel medically important technologies to selectively regulate the tight junctions formed by claudin family members in different organs. PMID:27647526

  2. Structural basis for disruption of claudin assembly in tight junctions by an enterotoxin.

    PubMed

    Shinoda, Takehiro; Shinya, Naoko; Ito, Kaori; Ohsawa, Noboru; Terada, Takaho; Hirata, Kunio; Kawano, Yoshiaki; Yamamoto, Masaki; Kimura-Someya, Tomomi; Yokoyama, Shigeyuki; Shirouzu, Mikako

    2016-01-01

    The food-poisoning bacterium Clostridium perfringens produces an enterotoxin (~35 kDa) that specifically targets human claudin-4, among the 26 human claudin proteins, and causes diarrhea by fluid accumulation in the intestinal cavity. The C-terminal domain of the Clostridium perfringens enterotoxin (C-CPE, ~15 kDa) binds tightly to claudin-4, and disrupts the intestinal tight junction barriers. In this study, we determined the 3.5-Å resolution crystal structure of the cell-free synthesized human claudin-4•C-CPE complex, which is significantly different from the structure of the off-target complex of an engineered C-CPE with mouse claudin-19. The claudin-4•C-CPE complex structure demonstrated the mechanism underlying claudin assembly disruption. A comparison of the present C-CPE-bound structure of claudin-4 with the enterotoxin-free claudin-15 structure revealed sophisticated C-CPE-induced conformation changes of the extracellular segments, induced on the foundation of the rigid four-transmembrane-helix bundle structure. These conformation changes provide a mechanistic model for the disruption of the lateral assembly of claudin molecules. Furthermore, the present novel structural mechanism for selecting a specific member of the claudin family can be used as the foundation to develop novel medically important technologies to selectively regulate the tight junctions formed by claudin family members in different organs. PMID:27647526

  3. 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.

  4. 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.

  5. 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.

  6. Association between the enterotoxin production and presence of Coa, Nuc genes among Staphylococcus aureus isolated from various sources, in Shiraz.

    PubMed

    Moghassem Hamidi, R; Hosseinzadeh, S; Shekarforoush, S S; Poormontaseri, M; Derakhshandeh, A

    2015-01-01

    The present study was aimed to identify the frequency of coagulase (Coa) and thermonuclease (Nuc) genes and Staphylococcal enterotoxin A (Sea) production among Staphylococcus aureus isolated from various sources in Shiraz. Moreover, the correlation between the Sea gene and coagulase and thermonuclease enzymes is also considered. A total of 100 S. aureus were isolated from various sources including 40 humans, 30 animals and 30 food samples by the routine biochemical tests. The frequency of Coa, Nuc and Sea genes was evaluated by PCR assay. Correlation among those genes was finally evaluated by statistical analysis. The PCR results showed that the prevalence of Coa, Nuc and Sea genes was 91%, 100% and 14%, respectively. The evaluation of the enterotoxin production indicated that 78.6% of the Sea gene was expressed. The presence of enterotoxin A was not necessarily correlated to the production of toxin. As a final conclusion to detect the enterotoxigenic strains, both genotypic and phenotypic methods are highly recommended.

  7. Association between the enterotoxin production and presence of Coa, Nuc genes among Staphylococcus aureus isolated from various sources, in Shiraz

    PubMed Central

    Moghassem Hamidi, R; Hosseinzadeh, S; Shekarforoush, S. S.; Poormontaseri, M; Derakhshandeh, A

    2015-01-01

    The present study was aimed to identify the frequency of coagulase (Coa) and thermonuclease (Nuc) genes and Staphylococcal enterotoxin A (Sea) production among Staphylococcus aureus isolated from various sources in Shiraz. Moreover, the correlation between the Sea gene and coagulase and thermonuclease enzymes is also considered. A total of 100 S. aureus were isolated from various sources including 40 humans, 30 animals and 30 food samples by the routine biochemical tests. The frequency of Coa, Nuc and Sea genes was evaluated by PCR assay. Correlation among those genes was finally evaluated by statistical analysis. The PCR results showed that the prevalence of Coa, Nuc and Sea genes was 91%, 100% and 14%, respectively. The evaluation of the enterotoxin production indicated that 78.6% of the Sea gene was expressed. The presence of enterotoxin A was not necessarily correlated to the production of toxin. As a final conclusion to detect the enterotoxigenic strains, both genotypic and phenotypic methods are highly recommended. PMID:27175208

  8. 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

  9. 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

    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. PlcR binding sites are

  10. 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

    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. PlcR binding sites are

  11. Isolation of a Highly Thermal Stable Lama Single Domain Antibody Specific for Staphylococcus aureus Enterotoxin B

    PubMed Central

    2011-01-01

    Background Camelids and sharks possess a unique subclass of antibodies comprised of only heavy chains. The antigen binding fragments of these unique antibodies can be cloned and expressed as single domain antibodies (sdAbs). The ability of these small antigen-binding molecules to refold after heating to achieve their original structure, as well as their diminutive size, makes them attractive candidates for diagnostic assays. Results Here we describe the isolation of an sdAb against Staphyloccocus aureus enterotoxin B (SEB). The clone, A3, was found to have high affinity (Kd = 75 pM) and good specificity for SEB, showing no cross reactivity to related molecules such as Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin D (SED), and Shiga toxin. Most remarkably, this anti-SEB sdAb had an extremely high Tm of 85°C and an ability to refold after heating to 95°C. The sharp Tm determined by circular dichroism, was found to contrast with the gradual decrease observed in intrinsic fluorescence. We demonstrated the utility of this sdAb as a capture and detector molecule in Luminex based assays providing limits of detection (LODs) of at least 64 pg/mL. Conclusion The anti-SEB sdAb A3 was found to have a high affinity and an extraordinarily high Tm and could still refold to recover activity after heat denaturation. This combination of heat resilience and strong, specific binding make this sdAb a good candidate for use in antibody-based toxin detection technologies. PMID:21933444

  12. Heat-Stable Enterotoxin of Enterotoxigenic Escherichia coli as a Vaccine Target ▿

    PubMed Central

    Taxt, Arne; Aasland, Rein; Sommerfelt, Halvor; Nataro, James; Puntervoll, Pål

    2010-01-01

    Enterotoxigenic Escherichia coli (ETEC) is responsible for 280 million to 400 million episodes of diarrhea and about 380,000 deaths annually. Epidemiological data suggest that ETEC strains which secrete heat-stable toxin (ST), alone or in combination with heat-labile toxin (LT), induce the most severe disease among children in developing countries. This makes ST an attractive target for inclusion in an ETEC vaccine. ST is released upon colonization of the small intestine and activates the guanylate cyclase C receptor, causing profuse diarrhea. To generate a successful toxoid, ST must be made immunogenic and nontoxic. Due to its small size, ST is nonimmunogenic in its natural form but becomes immunogenic when coupled to an appropriate large-molecular-weight carrier. This has been successfully achieved with several carriers, using either chemical conjugation or recombinant fusion techniques. Coupling of ST to a carrier may reduce toxicity, but further reduction by mutagenesis is desired to obtain a safe vaccine. More than 30 ST mutants with effects on toxicity have been reported. Some of these mutants, however, have lost the ability to elicit neutralizing immune responses to the native toxin. Due to the small size of ST, separating toxicity from antigenicity is a particular challenge that must be met. Another obstacle to vaccine development is possible cross-reactivity between anti-ST antibodies and the endogenous ligands guanylin and uroguanylin, caused by structural similarity to ST. Here we review the molecular and biological properties of ST and discuss strategies for developing an ETEC vaccine that incorporates immunogenic and nontoxic derivatives of the ST toxin. PMID:20231404

  13. 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

  14. A simple and rapid fluorescence-based immunoassay for the detection of staphylococcal enterotoxin B.

    PubMed

    Khan, Akbar S; Cao, Cheng J; Thompson, Roy G; Valdes, James J

    2003-01-01

    The bioterrorism threat is perceived to be a real challenge to our nation's security. This threat has necessitated the design of better and faster assays for the detection of biothreat agents including staphylococcal enterotoxin B (SEB), a causative agent of food poisoning. This study describes a simple, fast and highly sensitive fluorescence-based immunoassay, in which the antibody is fluorescently-labeled for use in this assay. Use of labeled antibodies resulted in very low level of detection of SEB, 100 pg/well. This method is four times faster than classical and conventional enzyme-linked immunosorbent assay (ELISA). PMID:12788034

  15. 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

  16. The enterotoxin of Clostridium perfringens type A binds to the presynaptic nerve endings in neuromuscular junctions of mouse phrenic nerve-diaphragm.

    PubMed

    Senda, T; Sugimoto, N; Horiguchi, Y; Matsuda, M

    1995-04-01

    The enterotoxin of Clostridium perfringens type A, a channel-pore forming protein toxin, inhibited neuromuscular transmission in isolated mouse phrenic nerve-diaphragm preparation at low concentrations of calcium. We investigated immunohistochemically the localization of the binding sites of the enterotoxin in the preparation under the conditions in which the enterotoxin reduced maximally the amplitudes of the twitch tension elicited by electrical stimulations to the phrenic nerve. Under the conditions, double immunohistochemical staining of the preparation with (1) rabbit anti-enterotoxin IgG-rhodamine-labeled goat anti-rabbit IgG and (2) mouse anti-synaptophysin (one of the synaptic vesicle-specific membrane proteins)-fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG showed that the enterotoxin binds specifically to most of the sites which were stained with anti-synaptophysin exactly in the same configurations having the shapes of the nerve endings in the endplates. The thin section electron micrographs of the enterotoxin-intoxicated preparation showed no alterations in the ultrastructure of the neuromuscular junction and the nerve endings filled with numerous synaptic vesicles. The present results, together with our previous electrophysiological findings, indicate that the enterotoxin binds specifically to the presynaptic nerve endings and inhibits neurotransmitter release at the neuromuscular junction.

  17. Staphylococcal Enterotoxin O Exhibits Cell Cycle Modulating Activity

    PubMed Central

    Hodille, Elisabeth; Alekseeva, Ludmila; Berkova, Nadia; Serrier, Asma; Badiou, Cedric; Gilquin, Benoit; Brun, Virginie; Vandenesch, François; Terman, David S.; Lina, Gerard

    2016-01-01

    Maintenance of an intact epithelial barrier constitutes a pivotal defense mechanism against infections. Staphylococcus aureus is a versatile pathogen that produces multiple factors including exotoxins that promote tissue alterations. The aim of the present study is to investigate the cytopathic effect of staphylococcal exotoxins SEA, SEG, SEI, SElM, SElN and SElO on the cell cycle of various human cell lines. Among all tested exotoxins only SEIO inhibited the proliferation of a broad panel of human tumor cell lines in vitro. Evaluation of a LDH release and a DNA fragmentation of host cells exposed to SEIO revealed that the toxin does not induce necrosis or apoptosis. Analysis of the DNA content of tumor cells synchronized by serum starvation after exposure to SEIO showed G0/G1 cell cycle delay. The cell cycle modulating feature of SEIO was confirmed by the flow cytometry analysis of synchronized cells exposed to supernatants of isogenic S. aureus strains wherein only supernatant of the SElO producing strain induced G0/G1 phase delay. The results of yeast-two-hybrid analysis indicated that SEIO’s potential partner is cullin-3, involved in the transition from G1 to S phase. In conclusion, we provide evidence that SEIO inhibits cell proliferation without inducing cell death, by delaying host cell entry into the G0/G1 phase of the cell cycle. We speculate that this unique cell cycle modulating feature allows SEIO producing bacteria to gain advantage by arresting the cell cycle of target cells as part of a broader invasive strategy. PMID:27148168

  18. Antibiotic resistance and enterotoxin genes in Staphylococcus sp. isolates from polluted water in Southern Brazil.

    PubMed

    Basso, Ana P; Martins, Paula D; Nachtigall, Gisele; Van Der Sand, Sueli; De Moura, Tiane M; Frazzon, Ana Paula G

    2014-12-01

    The aim of this study was to evaluate the species distribution, antibiotic-resistance profile and presence of enterotoxin (SE) genes in staphylococci isolated from the Dilúvio stream in South Brazil. Eighty-eight staphylococci were identified, 93.18% were identified as coagulase-negative (CNS) and 6.82% coagulase-positive (CPS). Fourteen Staphylococcus species were detected and the most frequently were Staphylococcus cohnii (30.48%) and S. haemolyticus (21.95%). Resistance to erythromycin was verified in 37.50% of the strains, followed by 27.27% to penicillin, 12.50% to clindamycin, 6.81% to trimethoprim-sulfamethoxazole, 5.68% to chloramphenicol and 2.27% to norfloxacin. None of the investigated strains showed gentamicin and ciprofloxacin resistance. The strains were tested for the presence of sea, seb, sec, sed and see genes by PCR and only CNS strains (43.18%) showed positive results to one or more SE genes. The scientific importance of our results is due to the lack of data about these topics in polluted waters in Brazil. In conclusion, polluted waters from the Dilúvio stream may constitute a reservoir for disseminating antibiotic-resistance and enterotoxin into the community. In addition, the detection of staphylococci in the polluted waters of the Dilúvio stream indicated a situation of environmental contamination and poor sanitation conditions.

  19. Antibiotic resistance and enterotoxin genes in Staphylococcus sp. isolates from polluted water in Southern Brazil.

    PubMed

    Basso, Ana P; Martins, Paula D; Nachtigall, Gisele; Sand, Sueli VAN DER; Moura, Tiane M DE; Frazzon, Ana Paula G

    2014-10-01

    The aim of this study was to evaluate the species distribution, antibiotic-resistance profile and presence of enterotoxin (SE) genes in staphylococci isolated from the Dilúvio stream in South Brazil. Eighty-eight staphylococci were identified, 93.18% were identified as coagulase-negative (CNS) and 6.82% coagulase-positive (CPS). Fourteen Staphylococcus species were detected and the most frequently were Staphylococcus cohnii (30.48%) and S. haemolyticus (21.95%). Resistance to erythromycin was verified in 37.50% of the strains, followed by 27.27% to penicillin, 12.50% to clindamycin, 6.81% to trimethoprim-sulfamethoxazole, 5.68% to chloramphenicol and 2.27% to norfloxacin. None of the investigated strains showed gentamicin and ciprofloxacin resistance. The strains were tested for the presence of sea, seb, sec, sed and see genes by PCR and only CNS strains (43.18%) showed positive results to one or more SE genes. The scientific importance of our results is due to the lack of data about these topics in polluted waters in Brazil. In conclusion, polluted waters from the Dilúvio stream may constitute a reservoir for disseminating antibiotic-resistance and enterotoxin into the community. In addition, the detection of staphylococci in the polluted waters of the Dilúvio stream indicated a situation of environmental contamination and poor sanitation conditions.

  20. Detection of Staphylococcus enterotoxin B using fluorescent immunoliposomes as label for immunochromatographic testing.

    PubMed

    Khreich, Nathalie; Lamourette, Patricia; Boutal, Hervé; Devilliers, Karine; Créminon, Christophe; Volland, Hervé

    2008-06-15

    Staphylococcus enterotoxin B (SEB) is one of several toxins produced by the gram positive bacterium Staphylococcus aureus. SEB is a major cause of food poisoning and represents a significant biological threat with regard to bioterrorism. A rapid, sensitive, and specific method is required to monitor food and water in cases of both natural and intentional contamination by this toxin. This report presents an improved immunochromatographic test (ICT) using immunoliposomes as label for the detection of SEB. For the first time in an ICT, the signal generated by the sulforhodamine B encapsulated into immunoliposomes was measured by fluorescence, allowing a 15-fold increase in sensitivity compared with that for visual detection of colored labels. The ICT was completed within 30 min, providing a limit of detection close to 20 pg/ml in buffer and showing no cross-reactivity with the other major toxin of the bacterium, Staphylococcus enterotoxin A. This sensitivity was retained when analyzing SEB spiked in various alimentary matrices, mimicking contaminated foods. Due to the use of fluorescent immunoliposomes as label, the present assay offers the inherent simplicity and speed of a dipstick assay while providing detection of low levels of SEB in real samples. PMID:18384739

  1. The toxicity of staphylococcal enterotoxin B in mice is mediated by T cells

    PubMed Central

    1990-01-01

    Staphylococcal enterotoxin B (SEB) has been shown in the past to be a potent T cell stimulant in mouse or man. The toxin acts as a superantigen that is, it binds to class II MHC proteins and, as such a complex, stimulates T cells bearing particular V beta s as part of their receptors. The toxin also has several pathological effects, causing, in mice, rapid weight loss, thymus atrophy, immunosuppression, and, at high doses, death. The data in this paper show that at least one of these effects, weight loss, is T cell mediated. Staphylococcal enterotoxin-mediated weight loss is MHC dependent, and is almost absent in animals expressing MHC class II molecules, which, complexed with SEB, are poor T cell stimulants. Also, mice that lack T cell function, genetically or because of cyclosporin A treatment, lose no or less weight than controls in response to SEB. Finally, animals bred such that they express few T cells bearing V beta s with which SEB can interact lose much less weight in response to the toxin than littermate controls that have higher numbers of reactive T cells. It is therefore suggested that the pathological effects of the staphylococcal, T cell- stimulating toxins in mouse and man may be partially or wholly the consequence of massive T cell stimulation. PMID:2303780

  2. 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.

  3. 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.

  4. [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.

  5. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. Association and dissociation of Escherichia coli heat-stable enterotoxin from rat brush border membrane receptors

    SciTech Connect

    Cohen, M.B.; Thompson, M.R.; Overmann, G.J.; Giannella, R.A.

    1987-02-01

    Escherichia coli heat-stable enterotoxin (ST) binds to receptors on rat intestinal cells and brush border membranes (BBM). We devised experiments to examine the reversibility of ST binding. We found that both /sup 125/I-labeled ST and native ST were spontaneously dissociable from the BBM receptor. Radiolabeled ST bound to BBM was also dissociated by the addition of avid goat anti-ST antiserum. Furthermore, using a computer program for analysis of ligand binding, we calculated an apparent Ka of 10(8) liters/mol from competitive inhibition and saturation-binding data. This is significantly lower than the value previously reported by others. Our findings, of a lower Ka and a reversible ST-binding process, suggest that a therapeutic strategy of removing bound ST from its receptor or competing with the enterocyte receptor for unbound ST might be successful in terminating ST-induced secretion.

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

    PubMed

    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

  9. Genetic control of rat T-cell response to Staphylococcus aureus enterotoxins (SE).

    PubMed Central

    Fu, Y; Villas, P A; Blankenhorn, E P

    1991-01-01

    Rat T cells, like those of mouse and human origin, respond strongly to superantigens (SAg) derived from Staphylococcus aureus enterotoxins A and B (SEA, SEB). Lewis and ACI are high responders, whereas Brown Norway (BN) is a low responder. Congenic and back-cross rat studies indicate that the degree of responsiveness is controlled by at least one non-MHC gene. The action of these genes may reside in the antigen-presenting cells (APC), since both Sephadex G10 non-adherent BN spleen cells and purified BN T cells in the presence of Lewis APC can respond well to SE. Responses to concanavalin A (Con A) and SEA generally segregate together in back-cross rats. Surprisingly, the degree of responsiveness to Con A and SEA is not correlated with the susceptibility to experimental allergic encephalomyelitis (EAE) either in independently derived inbred rat strains or in (Lewis x BN) x BN back-cross rats. PMID:1769696

  10. Survival of Staphylococcus aureus and enterotoxin A in glassy and rubbery states of gelatin.

    PubMed

    Bolton, K J; Dodd, C E; Gould, G W; Waites, W M

    1996-08-01

    About 1% of Staphylococcus aureus cells survived the production of gelatin sheets containing nutrient broth. Those cells which survived showed no evidence of injury. Growth occurred in rubbery state gelatin with a(w) values of 0.98 and 0.93 ; viability decreased during storage at a(w) values of 0.89, 0.62 and 0.36 but there was little loss of viability in gelatin at an aw of 0.25 over 27 d storage at 26 degrees C. Assays for enterotoxin A detected no synthesis of new toxin but no loss in pre-formed toxin. The results suggest that high levels of Staph. aureus and its toxins should be excluded from glassy and rubbery state food products in order to prevent illness. PMID:8760328

  11. Staphylococcus aureus, thermostable nuclease and staphylococcal enterotoxins in raw ewes' milk Manchego cheese.

    PubMed

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

    1988-07-01

    Growth and survival of two enterotoxigenic strains of Staphylococcus aureus were studied during manufacture and ripening of eight batches of raw ewes' milk Manchego cheese. Only 2-3 generations of Staph. aureus occurred in the vat and during pressing. The death rate of Staph. aureus (mean decrease in log cfu/g/week of ripening) from day 1 to day 60 was 0.421 in cheese made with 1% Streptococcus lactis starter and 0.404 in cheese made without starter. Thermostable nuclease was produced in the vat by growing Staph. aureus cells; it was inactivated by rennet during the first 24 h and synthesized again by surviving cells of Staph. aureus from day 1 to day 60. Staphylococcal enterotoxins A, B, C and D were not detected in any batches of cheese, even though Staph. aureus counts exceeded 10(7) cfu/g. PMID:3209513

  12. 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

  13. 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.

  14. Enterotoxin-producing bacteria and parasites in stools of Ethiopian children with diarrhoeal disease.

    PubMed Central

    Wadström, T; Aust-Kettis, A; Habte, D; Holmgren, J; Meeuwisse, G; Möllby, R; Söderlind, O

    1976-01-01

    Enterotoxinogenic bacteria were isolated from 131 (37%) of 354 Ethiopian infants and children with acute gastrointestinal symptoms. Only one of these isolates belonged to the classical enteropathogenic serotypes of Esch. coli. Two colonies from each patient were isolated and tested for production of enterotoxin by the rabbit ileal loop test, the rabbit skin test, and an adrenal cell assay. However, only 38% of the isolated enterotoxinogenic strains were Esch. coli; the others belonged to Klebsiella, Enterobacter, Proteus, Citrobacter, Serratia, and Aeromonas. In 18 patients both isolates were toxinogenic and belonged to different species. The incidence of intestinal parasites was 35% with no apparent correlation to the occurrence of toxinogenic bacteria in the stools. PMID:1008593

  15. Enterotoxin-producing bacteria and parasites in stools of Ethiopian children with diarrhoeal disease.

    PubMed

    Wadström, T; Aust-Kettis, A; Habte, D; Holmgren, J; Meeuwisse, G; Möllby, R; Söderlind, O

    1976-11-01

    Enterotoxinogenic bacteria were isolated from 131 (37%) of 354 Ethiopian infants and children with acute gastrointestinal symptoms. Only one of these isolates belonged to the classical enteropathogenic serotypes of Esch. coli. Two colonies from each patient were isolated and tested for production of enterotoxin by the rabbit ileal loop test, the rabbit skin test, and an adrenal cell assay. However, only 38% of the isolated enterotoxinogenic strains were Esch. coli; the others belonged to Klebsiella, Enterobacter, Proteus, Citrobacter, Serratia, and Aeromonas. In 18 patients both isolates were toxinogenic and belonged to different species. The incidence of intestinal parasites was 35% with no apparent correlation to the occurrence of toxinogenic bacteria in the stools. PMID:1008593

  16. 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)

  17. 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.

  18. 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. PMID:26584048

  19. 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

  20. Effects of Acute and Repeated Administration of Staphylococcal Enterotoxin A On Morris Water Maze Learning, Corticosterone and Hippocampal IL-1β and TNFα

    PubMed Central

    Woodruff, Randall T.; Schorpp, Kristen M.; Lawrenczyk, Agniesczka J.; Chakraborty, Trisha; Kusnecov, Alexander W.

    2014-01-01

    Staphylococcal enterotoxin A (SEA) is a bacterial superantigen that induces pronounced T cell expansion and cytokine production. In addition, SEA activates the HPA axis and forebrain regions relevant to cognitive functions. Since learning-related cognitive changes have not been assessed in response to SEA, spatial learning in the morris water maze (MWM) was determined in male C57BL/6J mice subjected to acute or repeated injections of 5 μg SEA or Saline. Injections were given 2 hrs prior to 4–5 days of hidden platform sessions. Animals were then rested for 1 month and given retraining without further injections. In addition, splenic IL-1β, IL-2 and TNFα, plasma corticosterone, and hippocampal IL-1β and TNFα were measured after the regimen of treatment used in the behavioral experiments. The results showed no learning impairment following acute or repeated SEA challenge. Moreover, when retested one month later, and without further injections, the SEA group showed more rapid relearning of the MWM. This suggested that coincidental superantigenic T cell activation and training served to promote long-term improvement in recovery of learning. Furthermore, repeated SEA challenge continued to drive increases in plasma corticosterone, but with a compensatory reduction in hippocampal IL-1β. However, while hippocampal TNFα was reduced after acute and repeated SEA treatment, this was not statistically significant. In view of the importance of modest glucocorticoid elevations and hippocampal IL-1β in promoting contextual learning, the data point to the hypothesis that SEA promotes long-term plasticity by restraining disruptive increases in hippocampal IL-1β, and possibly TNFα, during learning. PMID:20946950

  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. PMID:25172706

  2. Phenotypic and genotypic (pulsed-field gel electrophoresis) characteristics of enterotoxin-A-producing Staphylococcus aureus strains.

    PubMed

    Gouloumès, C; Bes, M; Renaud, F; Lina, B; Reverdy, M E; Brun, Y; Fleurette, J

    1996-05-01

    The phenotypic (antibiotype, serotype, phagetype) and genotypic (SmaI restriction patterns using pulsed-field gel electrophoresis) characters of 162 Staphylococcus aureus epidemiologically unrelated strains were studied. Eighty-two of the isolates produced enterotoxin-A (SEA+), while 80 produced none (SEA-). None of the phenotypic characters observed were characteristic of SEA+ strains. On the other hand, the electrophoretic profiles revealed a non-random distribution of the SEA+ strains (p < 0.01 in groups PI and PIII, and p < 0.03 in group PII). It can therefore reasonably be assumed that the enterotoxin-A-producing strains did not constitute a single clone, but rather, seemed to belong to strains derived from at least three clones with distinct genetic organization. PMID:8763613

  3. Survival and germination of Bacillus cereus spores without outgrowth or enterotoxin production during in vitro simulation of gastrointestinal transit.

    PubMed

    Ceuppens, Siele; Uyttendaele, Mieke; Drieskens, Katrien; Heyndrickx, Marc; Rajkovic, Andreja; Boon, Nico; Van de Wiele, Tom

    2012-11-01

    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.

  4. The effects of cholera enterotoxin on intestinal tissue water as measured by nuclear magnetic resonance (NMR) spectroscopy.

    PubMed

    Udall, J N; Alvarez, L A; Nichols, B L; Hazlewood, C F

    1975-01-01

    Cholera enterotoxin has been postulated to change the configuration of the intracellular protein-water system, altering the permeability of the cell to water. Using nuclear magnetic resonance (NMR) spectroscopy, this protein-water relationship can be examined. Small intestinal loops in the rat were injected with 0.5 ml of either Schwarz/Mann cholera enterotoxin (40 mug/cc saline solution) or normal saline. Full thickness segments of intestine from each loop were taken and percentage water (using a gravimetric procedure which includes a correction for fat) and NMR relaxation times were determined. The mean value +/- S.D. for tissue water was 79.49 +/- 2.65% in the controls and 84.52 +/- 2.01% in the cholera specimens (p less than .001). T1 (spin-lattice) relaxation times were 521.22 +/- 69.5 msec in the controls and 667.96 +/- 119.25 msec in cholera tissue (p less than .001). T2 (spin-spin) relaxation times were 62.34 +/- 9.59 msec in controls and 80.35 +/- 21.46 msec in cholera tissue (p less than .02). These findings are consistent with the theory that cholera enterotoxin acts to alter intracellular protein-water relationship.

  5. Genotype analysis of enterotoxin H-positive Staphylococcus aureus strains isolated from food samples in the Czech Republic.

    PubMed

    Růzicková, Vladislava; Karpísková, Renata; Pantůcek, Roman; Pospísilová, Markéta; Cerníková, Pavla; Doskar, Jirí

    2008-01-15

    Twenty-eight enterotoxin H-positive Staphylococcus aureus strains isolated from food samples collected in eleven districts of the Czech Republic between 2000 and 2005 were genotypically characterized by pulsed-field gel electrophoresis (PFGE) profiling, spa gene polymorphism analysis, enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) fingerprinting and prophage carriage detection. These strains accounted for about 21% of the food-derived, staphylococcal enterotoxin (SE)-positive isolates. One strain, detected in feta cheese, was implicated in a case of enterotoxinosis. Sixteen of the twenty-eight isolates carried the seh gene alone. The remaining twelve strains harbored the seh gene in combination with other enterotoxin genes, most often the seg and sei genes, followed by the sea, seb, sec and sed genes. Comparison of various genomic profiles resulted in the determination of twenty genotypes designated G-1 to G-20. Two new, to date not defined, spa types (t2000 and t2002) were identified in one strain isolated from raw meat and two strains obtained from prepacked pizza. Evidence has been given that the seh-positive S. aureus isolates from foodstuffs did not originate from a single source or a common ancestor.

  6. 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

  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.

  8. Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy.

    PubMed Central

    Beuchat, L R; Clavero, M R; Jaquette, C B

    1997-01-01

    The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 micrograms of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 microgram/ml, nisin was lethal to B. cereus, the effect being more pronounced in BHI broth than in beef gravy. The inhibitory effect of nisin (1 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C. Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees C, respectively, for 14 days. Growth of vegetative cells and spores of B. cereus after an initial period of inhibition is attributed to loss of activity of nisin. One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15 degrees C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C. Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 microgram of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B. cereus in beef gravy at 8 degrees C, a

  9. Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy.

    PubMed

    Beuchat, L R; Clavero, M R; Jaquette, C B

    1997-05-01

    The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 micrograms of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 microgram/ml, nisin was lethal to B. cereus, the effect being more pronounced in BHI broth than in beef gravy. The inhibitory effect of nisin (1 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C. Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees C, respectively, for 14 days. Growth of vegetative cells and spores of B. cereus after an initial period of inhibition is attributed to loss of activity of nisin. One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15 degrees C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C. Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 microgram of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B. cereus in beef gravy at 8 degrees C, a

  10. Assessment of high and low enterotoxin A producing Staphylococcus aureus strains on pork sausage.

    PubMed

    Zeaki, Nikoleta; Cao, Rong; Skandamis, Panagiotis N; Rådström, Peter; Schelin, Jenny

    2014-07-16

    Three Staphylococcus aureus strains representing different alleles of the Siphoviridae prophage-encoded enterotoxin A (SEA) gene, including two high-SEA-producing strains and one low-SEA-producing strain were studied to investigate sea expression and SEA formation on a frankfurter type of sausage. The effect of lactic acid, an antimicrobial compound used as a preservative in food, was also investigated on the same product. All three strains were grown on pork sausages at 15°C for 14days in the presence or absence of lactic acid (1 or 2% v/v). Growth, sea mRNA expression and SEA formation were regularly monitored and compared between non-treated and treated sausages. For all experiments performed, the extracellular SEA formation significantly differed between the high- and low-SEA-producing strains, although growth and viability were overall the same. For the low producer (Sa51), the accumulated amount of extracellular SEA formed after 14days was close to the detection limit (less than 1ng/g) in all conditions; while Sa21 and Sa17, the two high-producing strains, formed 250±25.37ng/g and 750±82.65ng/g in non-treated sausage and 150±75.75ng/g and 300±83.89ng/g when treated with 1% lactic acid, respectively, after 14days. Sausages treated with 2% lactic acid followed the same pattern as above, but with an extended lag phase to 4days and reduced levels of enterotoxin formed for all strains. The difference in the level of SEA between the two high-producing strains is most likely due to the different clonal lineages of the sea-encoded Siphoviridae phages where induction of the prophage potentially could be the reason for higher production of SEA in one of the lines. Furthermore, a prolonged expression of sea gene in the two high-producing strains was observed during the entire incubation period, while the sea expression was under the detection limit in the low-producing strain. This study indicates that the high-SEA-producing strains, especially the strains with the

  11. Enterotoxin-producing Staphylococcus aureus genotype B as a major contaminant in Swiss raw milk cheese.

    PubMed

    Hummerjohann, J; Naskova, J; Baumgartner, A; Graber, H U

    2014-03-01

    The objective of this study was to characterize Staphylococcus aureus isolates from Swiss raw milk cheeses that had been found to be contaminated with coagulase-positive staphylococci and to estimate the frequency of the various genotypes, in particular the mastitis-associated Staph. aureus genotype B (GTB). The isolates were also tested for staphylococcal enterotoxin (SE) genes and other virulence factors. From 623 coagulase-positive staphylococci isolated from 78 contaminated raw milk cheeses, 609 were found to be Staphylococcus aureus. Genotyping of all Staph. aureus isolates was performed by PCR amplification of the 16S-23S rRNA intergenic spacer region, as this method was used previously to differentiate between mastitis subtypes associated with their clinical outcome. In total, 20 different genotypes were obtained and the 5 most frequently occurring genotypes were distributed in 6.4% or more of the samples. The enterotoxin-producing Staph. aureus GTB, known for its high contagiousness and increased pathogenicity in Swiss mastitis herds, was found to be the most abundant subtype at the sample level (71.8%) as well as among the isolates (62.0%). A subset of 107 isolates of the different genotypes were analyzed for the presence of SE genes and revealed 9 different SE gene patterns, with sed being most frequently detected and 26% being PCR-negative for SE genes. Almost all isolates of the major contaminant GTB contained the SE gene pattern sed, sej, ser, with half of them additionally carrying sea. Production of SE in vitro was consistent with the SE genes detected in most of the cases; however, some isolated GTB did not produce SEA. Staphylococcus aureus Protein A (spa) typing revealed 30 different subtypes and most GTB isolates belonged to the bovine spa type t2953; GTB/t2953 was linked among other subtypes to SE production in cheese and staphylococcal intoxication cases. Furthermore, 1 of the 623 isolates was a methicillin-resistant Staph. aureus, which was an

  12. Comparative observations of fever and associated clinical hematological and blood biochemical changes after intravenous administration of staphylococcal enterotoxins B and F (toxic shock syndrome toxin-1) in goats.

    PubMed Central

    Van Miert, A S; Van Duin, C T; Schotman, A J

    1984-01-01

    The present investigation was undertaken to examine the characteristics of purified toxic shock syndrome toxin-1 (staphylococcal enterotoxin F) given intravenously to dwarf goats (dose, 0.02 to 20 micrograms kg-1). Rectal temperature, heart rate, rumen motility, plasma zinc and iron concentrations, and certain other blood biochemical and hematological values were studied and compared with the changes seen after intravenous administration of staphylococcal enterotoxin B (dose, 0.02 to 0.5 micrograms kg-1). Similar changes such as fever, tachycardia, inhibition of rumen contractions, drop in plasma zinc and iron concentrations, lymphopenia, and a decrease in serum alkaline phosphatase activity were observed. In contrast to the effects of toxic shock syndrome toxin-1, staphylococcal enterotoxin B induced colic, watery diarrhea with pseudomembranes, hemoconcentration, and a more pronounced increase in blood urea nitrogen. The results obtained demonstrate that (i) in the goat staphylococcal enterotoxin B is much more potent than toxic shock syndrome toxin-1 and (ii) the goat is a useful model to study the gastro-intestinal effects caused by staphylococcal enterotoxin B. The present finding that no clear relationship could be found between the temperature response and the alterations in zinc and iron levels in plasma support the theory that the febrile reactions and the changes in plasma trace metals are mediated by different polypeptides released by activated macrophages. PMID:6500695

  13. [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.

  14. Plant-Derived Polyphenols Interact with Staphylococcal Enterotoxin A and Inhibit Toxin Activity

    PubMed Central

    Shimamura, Yuko; Aoki, Natsumi; Sugiyama, Yuka; Tanaka, Takashi; Murata, Masatsune; Masuda, Shuichi

    2016-01-01

    This study was performed to investigate the inhibitory effects of 16 different plant-derived polyphenols on the toxicity of staphylococcal enterotoxin A (SEA). Plant-derived polyphenols were incubated with the cultured Staphylococcus aureus C-29 to investigate the effects of these samples on SEA produced from C-29 using Western blot analysis. Twelve polyphenols (0.1–0.5 mg/mL) inhibited the interaction between the anti-SEA antibody and SEA. We examined whether the polyphenols could directly interact with SEA after incubation of these test samples with SEA. As a result, 8 polyphenols (0.25 mg/mL) significantly decreased SEA protein levels. In addition, the polyphenols that interacted with SEA inactivated the toxin activity of splenocyte proliferation induced by SEA. Polyphenols that exerted inhibitory effects on SEA toxic activity had a tendency to interact with SEA. In particular, polyphenol compounds with 1 or 2 hexahydroxydiphenoyl groups and/or a galloyl group, such as eugeniin, castalagin, punicalagin, pedunculagin, corilagin and geraniin, strongly interacted with SEA and inhibited toxin activity at a low concentration. These polyphenols may be used to prevent S. aureus infection and staphylococcal food poisoning. PMID:27272505

  15. Structural features of Escherichia coli heat-stable enterotoxin that activates membrane-associated guanylyl cyclase.

    PubMed

    Sato, T; Shimonishi, Y

    2004-03-01

    Heat-stable enterotoxin (ST), a small peptide of 18 or 19 amino acid residues produced by enterotoxigenic Escherichia coli, is the cause of acute diarrhea in infants and travelers in developing countries. ST triggers a biological response by binding to a membrane-associated guanylyl cyclase C (GC-C) which is located on intestinal epithelial cell membranes. This binding causes an increase in the concentration of cGMP as a second messenger in cells and activates protein kinase A and cystic fibrosis transmembrane conductance regulator. Here we describe the crystal structure of an ST at 0.89 A resolution. The molecule has a ring-shaped molecular architecture consisting of six peptide molecules with external and internal diameters of approximately 35 and 7 A, respectively and a thickness of approximately 11 A. The conserved residues at the central portion of ST are distributed on the outer surface of the ring-shaped peptide hexamer, suggesting that the hexamer may be implicated in the association with GC-C through these invariant residues. PMID:15049831

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

    PubMed

    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; Waldman, Scott A

    2016-10-01

    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

  17. Purification and partial characterization of a putative precursor to staphylococcal enterotoxin B.

    PubMed Central

    Tweten, R K; Iandolo, J J

    1981-01-01

    A putative precursor to staphylococcal enterotoxin B (SEB) has been identified as a component of purified membranes from Staphylococcus aureus S6. Agarose gel immunodiffusion analysis of the solubilized membranes demonstrated an immunoreactive protein that formed complete lines of identity with purified extracellular SEB. This putative precursor (pSEB) also had a different electrophoretic mobility from that of extracellular SEB when analyzed by immunoelectrophoresis. When membrane proteins from S6 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose sheets and probed with I-125 labeled, affinity-purified anti-SEB, the pSEB band was identified. The pSEB was approximately 3,500 daltons larger than extracellular SEB. This component was purified by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two-dimensional peptide maps of the putative SEB precursor revealed that most of the tryptic peptides were identical to those of mature extracellular SEB. When purified membranes of other SEB+ (DU4916 and 10-275) and SEB- (RN450, RN451, S6R, and FR1100) S. aureus strains were analyzed by the nitrocellulose blot procedure, only the SEB+ strains contained this putative SEB precursor on their membranes. Images PMID:7333675

  18. Inhibition of the superantigenic activities of Staphylococcal enterotoxin A by an aptamer antagonist.

    PubMed

    Wang, Kaiyu; Wu, Dong; Chen, Zhuang; Zhang, Xianhui; Yang, Xiangyue; Yang, Chaoyong James; Lan, Xiaopeng

    2016-09-01

    Staphylococcal enterotoxin A (SEA) is an important component of Staphylococcus aureus pathogenesis. SEA induces T lymphocytes activation and proliferation, resulting in the release of a large number of inflammatory cytokines. Blocking the toxic cascade triggered by SEA may be an effective strategy for the treatment of SEA-induced diseases. Through a systematic evolution of ligands by exponential enrichment process, we obtained an aptamer (S3) that could bind SEA with both high affinity and specificity, with a Kd value 36.93 ± 7.29 nM (n = 3). This aptamer antagonist effectively inhibited SEA-mediated human peripheral blood mononuclear cells proliferation and inflammatory cytokines (IFN-γ, TNF-α, IL-2 and IL-6) secretion. Moreover, PEGylated S3 significantly reduced mortality in murine lethal toxic shock models established by lipopolysaccharide-potentiated SEA. Therefore, this novel aptamer antagonist has the potential to become a new strategy for treating S. aureus infections and SEA-induced diseases. PMID:27179422

  19. 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

  20. 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.

  1. From Escherichia coli heat-stable enterotoxin to mammalian endogenous guanylin hormones

    PubMed Central

    Lima, A.A.M.; Fonteles, M.C.

    2014-01-01

    The isolation of heat-stable enterotoxin (STa) from Escherichia coli and cholera toxin from Vibrio cholerae has increased our knowledge of specific mechanisms of action that could be used as pharmacological tools to understand the guanylyl cyclase-C and the adenylyl cyclase enzymatic systems. These discoveries have also been instrumental in increasing our understanding of the basic mechanisms that control the electrolyte and water balance in the gut, kidney, and urinary tracts under normal conditions and in disease. Herein, we review the evolution of genes of the guanylin family and STa genes from bacteria to fish and mammals. We also describe new developments and perspectives regarding these novel bacterial compounds and peptide hormones that act in electrolyte and water balance. The available data point toward new therapeutic perspectives for pathological features such as functional gastrointestinal disorders associated with constipation, colorectal cancer, cystic fibrosis, asthma, hypertension, gastrointestinal barrier function damage associated with enteropathy, enteric infection, malnutrition, satiety, food preferences, obesity, metabolic syndrome, and effects on behavior and brain disorders such as attention deficit, hyperactivity disorder, and schizophrenia. PMID:24652326

  2. 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. PMID:24016577

  3. Evaluation of Potential Effects of NaCl and Sorbic Acid on Staphylococcal Enterotoxin A Formation

    PubMed Central

    Zeaki, Nikoleta; Rådström, Peter; Schelin, Jenny

    2015-01-01

    The prophage-encoded staphylococcal enterotoxin A (SEA) is recognized as the main cause of staphylococcal food poisoning (SFP), a common foodborne intoxication disease, caused by Staphylococcus aureus. Studies on the production of SEA suggest that activation of the SOS response and subsequent prophage induction affect the regulation of the sea gene and the SEA produced, increasing the risk for SFP. The present study aims to evaluate the effect of NaCl and sorbic acid, in concentrations relevant to food production, on SOS response activation, prophage induction and SEA production. The impact of stress was initially evaluated on steady state cells for a homogenous cell response. NaCl 2% was found to activate the SOS response, i.e., recA expression, and trigger prophage induction, in a similar way as the phage-inducer mitomycin C. In contrast, sorbic acid decreased the pH of the culture to a level where prophage induction was probably suppressed, even when combined with NaCl stress. The impact of previous physiological state of the bacteria was also addressed on cells pre-exposed to NaCl, and was found to potentially affect cell response upon exposure to further stress. The results obtained highlight the possible SFP-related risks arising from the use of preservatives during food processing.

  4. Plant-Derived Polyphenols Interact with Staphylococcal Enterotoxin A and Inhibit Toxin Activity.

    PubMed

    Shimamura, Yuko; Aoki, Natsumi; Sugiyama, Yuka; Tanaka, Takashi; Murata, Masatsune; Masuda, Shuichi

    2016-01-01

    This study was performed to investigate the inhibitory effects of 16 different plant-derived polyphenols on the toxicity of staphylococcal enterotoxin A (SEA). Plant-derived polyphenols were incubated with the cultured Staphylococcus aureus C-29 to investigate the effects of these samples on SEA produced from C-29 using Western blot analysis. Twelve polyphenols (0.1-0.5 mg/mL) inhibited the interaction between the anti-SEA antibody and SEA. We examined whether the polyphenols could directly interact with SEA after incubation of these test samples with SEA. As a result, 8 polyphenols (0.25 mg/mL) significantly decreased SEA protein levels. In addition, the polyphenols that interacted with SEA inactivated the toxin activity of splenocyte proliferation induced by SEA. Polyphenols that exerted inhibitory effects on SEA toxic activity had a tendency to interact with SEA. In particular, polyphenol compounds with 1 or 2 hexahydroxydiphenoyl groups and/or a galloyl group, such as eugeniin, castalagin, punicalagin, pedunculagin, corilagin and geraniin, strongly interacted with SEA and inhibited toxin activity at a low concentration. These polyphenols may be used to prevent S. aureus infection and staphylococcal food poisoning. PMID:27272505

  5. 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

  6. Investigation of synthetic Escherichia coli heat-stable enterotoxin as an immunogen for swine and cattle.

    PubMed Central

    Frantz, J C; Bhatnagar, P K; Brown, A L; Garrett, L K; Hughes, J L

    1987-01-01

    In its native form Escherichia coli heat-stable enterotoxin (STa) is nonantigenic; however, neutralizing antibodies are elicited in animals vaccinated with toxin-carrier conjugates. To study the immunogenicity of STa, peptides STa1-18 and STa5-18 were synthesized, characterized, and conjugated to carrier proteins. Pregnant gilts and heifers were hyperimmunized with the respective conjugates. Following parturition neonates were challenged with virulent E. coli (K99+ STa+). Peptides coupled to ovalbumin and emulsified with Freund adjuvant elicited antibodies that neutralized toxin-induced fluid accumulation in suckling mice. Peptides coupled to particulate carriers, with or without muramyl dipeptide adjuvant, failed to induce a measurable response. Peak antibody levels in sera were observed following three doses of conjugate and persisted for several weeks. The serological response in cattle was superior to that observed in swine; however, antibody levels in porcine colostrum were higher than those observed in cattle. Clinical observations of neonates from vaccinated dams indicated that passively obtained antibody provided partial protection from disease, but not as complete as that demonstrable with whole cell bacterins that induce antibody to pili. However, the data suggest the potential for utility of synthetically prepared antigens. PMID:3552985

  7. [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. PMID:10932731

  8. 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

    2016-04-01

    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

  9. Functional piglet model for the clinical syndrome and postmortem findings induced by staphylococcal enterotoxin B.

    PubMed

    van Gessel, Yvonne A; Mani, Sachin; Bi, Shuguang; Hammamieh, Rasha; Shupp, Jeffrey W; Das, Rina; Coleman, Gary D; Jett, Marti

    2004-11-01

    Staphylococcal enterotoxin (SE) B causes serious gastrointestinal illness, and intoxication with this exotoxin can lead to lethal toxic shock syndrome. In order to overcome significant shortcomings of current rodent and nonhuman primate models, we developed a piglet model of lethal SEB intoxication. Fourteen-day-old Yorkshire piglets were given intravenous SEB, observed clinically, and sacrificed at 4, 6, 24, 48, 72, or 96 hrs posttreatment. Clinical signs were biphasic with pyrexia, vomiting, and diarrhea within 4 hrs, followed by terminal hypotension and shock by 96 hrs. Mild lymphoid lesions were identified as early as 24 hrs, with severe lymphadenopathy, splenomegaly, and prominent Peyer's patches found by 72 hrs. Widespread edema-most prominent in the mesentery, between loops of spiral colon, and in retroperitoneal connective tissue-was found in animals at 72 hrs. Additional histologic changes included perivascular aggregates of large lymphocytes variably present in the lung and brain, circulating lymphoblasts, and lymphocytic portal hepatitis. Preliminary molecular investigation using gene array has uncovered several gene profile changes that may have implications in the pathophysiology leading to irreversible shock. Five genes were selected for further study, and all showed increased mRNA levels subsequent to SEB exposure. The use of this piglet model will continue to elucidate the pathogenesis of SEB intoxication and facilitate the testing of new therapeutic regimens that may better correlate with human lesions.

  10. 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.

  11. NSP4 enterotoxin of rotavirus induces paracellular leakage in polarized epithelial cells.

    PubMed

    Tafazoli, F; Zeng, C Q; Estes, M K; Magnusson, K E; Svensson, L

    2001-02-01

    The nonstructural NSP4 protein of rotavirus has been described as the first viral enterotoxin. In this study we have examined the effect of NSP4 on polarized epithelial cells (MDCK-1) grown on permeable filters. Apical but not basolateral administration of NSP4 was found to cause a reduction in the transepithelial electrical resistance, redistribution of filamentous actin, and an increase in paracellular passage of fluorescein isothiocyanate-dextran. Significant effects on transepithelial electrical resistance were noted after a 20- to 30-h incubation with 1 nmol of NSP4. Most surprisingly, the epithelium recovered its original integrity and electrical resistance upon removal of NSP4. Preincubation of nonconfluent MDCK-1 cells with NSP4 prevented not only development of a permeability barrier but also lateral targeting of the tight-junction-associated Zonula Occludens-1 (ZO-1) protein. Taken together, these data indicate new and specific effects of NSP4 on tight-junction biogenesis and show a novel effect of NSP4 on polarized epithelia. PMID:11152526

  12. Molecular analysis of non structural rotavirus group A enterotoxin gene of bovine origin from India.

    PubMed

    Malik, Yashpal Singh; Kumar, Naveen; Sharma, Kuldeep; Ghosh, Souvik; Bányai, Krisztián; Balasubramanian, Ganesh; Kobayashi, Nobumichi; Matthijnssens, Jelle

    2014-07-01

    The rotavirus enterotoxin NSP4 (nonstructural protein 4), plays a pivotal role in viral morphogenesis as well as pathogenesis. In this study, the NSP4 gene of rotavirus group A (RVA) isolates of bovine origin isolated in several states of India from 2008 to 2011 were characterized. The complete open reading frame of 23 RVA strains were sequenced and analyzed phylogenetically. Genotype E1 was detected for the first time in bovines from India, in addition to the more common bovine genotype E2. Sequence similarity analysis of the E1 sequences showed a close genetic relatedness to human strains. Six of the bovine E2 genotypes strains clustered near bovine and unusual human strains (possible human animal reassortant) from Thailand, while the remaining E2 sequences clustered with Indian bovine strains. Analysis pointed out one positively selected site (154aa), believe to be part of an antigenic region and 123 negatively selected sites. Unexpectedly, a pentameric NSP4 structure of the coiled coil domain in the E1 carrying strains and a monomeric NSP4 in RVA strain P14 (E2) was predicted based on homology modeling, potentially affecting the biological properties of NSP4. The close relationship between bovine and human rotavirus strains further highlights the complex interaction among rotaviruses of different species. PMID:24747605

  13. Transcytosis, Antitumor Activity and Toxicity of Staphylococcal Enterotoxin C2 as an Oral Administration Protein Drug

    PubMed Central

    Zhao, Wenbin; Li, Yangyang; Liu, Wenhui; Ding, Ding; Xu, Yingchun; Pan, Liqiang; Chen, Shuqing

    2016-01-01

    Staphylococcal enterotoxin C2 (SEC2) is a classical superantigen (SAg), which can tremendously activate T lymphocytes at very low dosage, thus exerting its powerful antitumor activity. As an intravenous protein drug and a bacterial toxin, SEC2 has some limitations including poor patient compliance and toxic side effects. In this research, we devoted our attention to studying the antitumor activity and toxicity of SEC2 as a potential oral administration protein drug. We proved that His-tagged SEC2 (SEC2-His) could undergo facilitated transcytosis on human colon adenocarcinoma (Caco-2) cells and SEC2-His was detected in the blood of rats after oral administration. Furthermore, oral SEC2-His caused massive cytokine release and immune cell enrichment around tumor tissue, leading to inhibition of tumor growth in vivo. Meanwhile, although SEC2-His was dosed up to 32 mg/kg in mice, no significant toxicity was observed. These data showed that SEC2 can cross the intestinal epithelium in an immunologically integral form, maintaining antitumor activity but with reduced systemic toxicity. Therefore, these results may have implications for developing SEC2 as an oral administration protein drug. PMID:27322320

  14. Cure of Trypanosoma musculi infection by heat-labile activity in immune plasma.

    PubMed

    Wechsler, D S; Kongshavn, P A

    1984-06-01

    Passive transfer of plasma from a mouse cured of parasitemia to a Trypanosoma musculi-infected host rapidly eliminates parasitemia; this curative activity, presumably mediated by an immunoglobulin, is sensitive to heat treatment (56 degrees C, 30 min). In addition, pretreatment with immune plasma, even after heat treatment, prevents the development of a patent parasitemia in a naive host (protective activity).

  15. 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.

  16. 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

  17. 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. PMID:24341350

  18. 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.

  19. 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.

  20. 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.

  1. The Redox Regulator Fnr Is Required for Fermentative Growth and Enterotoxin Synthesis in Bacillus cereus F4430/73▿

    PubMed Central

    Zigha, Assia; Rosenfeld, Eric; Schmitt, Philippe; Duport, Catherine

    2007-01-01

    Glucose-grown cells of Bacillus cereus respond to anaerobiosis and low extracellular oxidoreduction potentials (ORP), notably by enhancing enterotoxin production. This response involves the ResDE two-component system. We searched the B. cereus genome for other redox response regulators potentially involved in this adaptive process, and we identified one gene encoding a protein predicted to have an amino acid sequence 58% identical (80% similar) to that of the Bacillus subtilis Fnr redox regulator. The fnr gene of the food-borne pathogen B. cereus F4430/73 has been cloned and partially characterized. We showed that fnr was up-regulated during anaerobic fermentation, especially when fermentation occurred at low ORP (under highly reducing conditions). The expression of fnr was down-regulated in the presence of O2 and nitrate which, unlike fumarate, stimulated the respiratory pathways. The inactivation of B. cereus fnr abolished fermentative growth but only moderately affected aerobic and anaerobic nitrate respiratory growth. Analyses of glucose by-products and the transcription profiles of key catabolic genes confirmed the strong regulatory impact of Fnr on B. cereus fermentative pathways. More importantly, the fnr mutation strongly decreased the expression of PlcR-dependent hbl and nhe genes, leading to the absence of hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) secretion by the mutant. These data indicate that fnr is essential for both fermentation and toxinogenesis. The results also suggest that both Fnr and the ResDE two-component system belong to a redox regulatory pathway that functions at least partially independently of the pleiotropic virulence gene regulator PlcR to regulate enterotoxin gene expression. PMID:17259311

  2. 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.

  3. 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

  4. Immunogenicity and protective efficacy of a recombinant adenoviral based vaccine expressing heat-stable enterotoxin (STa) and K99 adhesion antigen of enterotoxigenic Escherichia coli in mice.

    PubMed

    Deng, Guangcun; Li, Wu; Wu, Xiaoling; Bao, Shaowen; Zeng, Jin; Zhao, Ning; Luo, Meihui; Liu, Xiaoming; Wang, Yujiong

    2015-12-01

    The diarrheal disease of domestic animals or in humans caused by enterotoxigenic Escherichia coli (ETEC) infections remains a major issue for public health in developing countries. Unfortunately, there is no effective vaccine available for preventing from an ETEC infection. Therefore, the development of a safe and effective vaccine against ETEC is urgently needed. In the present study, A recombinant adenoviral vector Ad5-STa-K99 that capable of expressing a fusion protein of heat-stable enterotoxin (STa) and K99 adhesion antigen of ETEC was generated and its immunogenicity was evaluated in a murine model. The intestinal mucosal secretory IgA(sIgA), serum anti-STa-K99 antibody responses, antigen-specific CD4(+) and CD8(+) T cells frequencies, as well as T-cell proliferation of mice immunized with the viral vector were determined as immunological indexes. The results demonstrated that Ad5-STa-K99 was able to enhance humoral responses with a dramatically augmented antigen-specific serum IgG antibody, and an elevated production of intestinal sIgA in immunized mice, suggesting the elicitation of both of humoral and mucosal immune responses. In addition, this adenoviral vector could significantly promote splenic T cell proliferation and increase the frequencies of CD4(+) and CD8(+) T cell populations in mice, indicative of a capacity to activate T cell responses. More importantly, vaccination of the Ad5-STa-K99 showed a potential to evoke a protective effect from ETEC challenge in mice. These data indicate that the Ad5-STa-K99 is a highly immunogenic vector able to induce a broad range of antigen-specific immune responses in vivo, and evoke a protective immune response against ETEC infections, implying that it may be a novel vaccine candidate warranted for further investigation.

  5. A tripartite cocktail of chimeric monoclonal antibodies passively protects mice against ricin, staphylococcal enterotoxin B and Clostridium perfringens epsilon toxin.

    PubMed

    Sully, Erin K; Whaley, Kevin; Bohorova, Natasha; Bohorov, Ognian; Goodman, Charles; Kim, Do; Pauly, Michael; Velasco, Jesus; Holtsberg, Frederick W; Stavale, Eric; Aman, M Javad; Tangudu, Chandra; Uzal, Francisco A; Mantis, Nicholas J; Zeitlin, Larry

    2014-12-15

    Due to the fast-acting nature of ricin, staphylococcal enterotoxin B (SEB), and Clostridium perfringens epsilon toxin (ETX), it is necessary that therapeutic interventions following a bioterrorism incident by one of these toxins occur as soon as possible after intoxication. Moreover, because the clinical manifestations of intoxication by these agents are likely to be indistinguishable from each other, especially following aerosol exposure, we have developed a cocktail of chimeric monoclonal antibodies that is capable of neutralizing all three toxins. The efficacy of this cocktail was demonstrated in mouse models of lethal dose toxin challenge. PMID:25260254

  6. A Tripartite Cocktail of Chimeric Monoclonal Antibodies Passively Protects Mice against Ricin, Staphylococcal Enterotoxin B and Clostridium perfringens Epsilon Toxin

    PubMed Central

    Sully, Erin K.; Whaley, Kevin; Bohorova, Natasha; Bohorov, Ognian; Goodman, Charles; Kim, Do; Pauly, Michael; Velasco, Jesus; Holtsberg, Frederick W.; Stavale, Eric; Aman, M. Javad; Tangudu, Chandra; Uzal, Francisco A.; Mantis, Nicholas J.; Zeitlin, Larry

    2014-01-01

    Due to the fast-acting nature of ricin, staphylococcal enterotoxin (SEB), and Clostridium perfringens epsilon toxin (ETX), it is necessary that therapeutic interventions following a bioterrorism incident by one of these toxins occur as soon as possible after intoxication. Moreover, because the clinical manifestations of intoxication by these toxins are likely to be indistinguishable from each other, especially following aerosol exposure, we have developed a cocktail of chimeric monoclonal antibodies that is capable of neutralizing all three toxins. The efficacy of this cocktail was demonstrated in mouse models of lethal dose toxin challenge. PMID:25260254

  7. Use of erythrocytes sensitized with purified enterotoxin from Vibrio cholerae for the assay of antibody and antibody-forming cells.

    PubMed

    Kateley, J R; Friedman, H

    1975-02-01

    A method was developed for the sensitization of ovine erythrocytes with a purified enterotoxin from Vibrio cholerae. Sensitized cells were used for the titration of serum antibody by passive hemagglutination and in a hemolytic plaque assay for both IgM and IgG antibody-secreting cells. Inhibition experiments with various antigens of V. cholerae indicated that the toxin, whether unheated or heat-inactivated, significantly reduced the expected antitoxic plaque-forming cell response, whereas a lipopolysaccharide-rich extract from homologous vibiros was not inhibitory.

  8. DIARRHEA OUTBREAK IN PERNAMBUCO, BRAZIL, ASSOCIATED WITH A HEAT-STABLE CYTOTOXIC ENTEROTOXIN PRODUCED BY Aeromonas caviae.

    PubMed

    Lopes, Ana Carolina Amaral; Martins, Luciano Moura; Gatti, Maria Silvia Viccari; Falavina Dos Reis, Cristhiane Moura; Hofer, Ernesto; Yano, Tomomasa

    2015-01-01

    In the present study enterotoxic and cytotoxic activities of twenty Aeromonas caviae strains were examined. They originated from fecal specimens of patients with acute diarrhea during an outbreak in Brazil in 2004. Culture supernatants of fourteen strains (70%) caused fluid accumulation in rabbit ileal intestinal loops and in suckling mice assays, and also showed a cytotoxic activity in Vero and Caco-2 cells. The enterotoxic and cytotoxic factors were heat-stable after culture supernatants treatment at 100 ºC. The results revealed that A. caviae strains produce a putative diarrheagenic virulence factor, a heat-stable cytotoxic enterotoxin that could be linked to the diarrhea outbreak that took place in Brazil.

  9. A tripartite cocktail of chimeric monoclonal antibodies passively protects mice against ricin, staphylococcal enterotoxin B and Clostridium perfringens epsilon toxin.

    PubMed

    Sully, Erin K; Whaley, Kevin; Bohorova, Natasha; Bohorov, Ognian; Goodman, Charles; Kim, Do; Pauly, Michael; Velasco, Jesus; Holtsberg, Frederick W; Stavale, Eric; Aman, M Javad; Tangudu, Chandra; Uzal, Francisco A; Mantis, Nicholas J; Zeitlin, Larry

    2014-12-15

    Due to the fast-acting nature of ricin, staphylococcal enterotoxin B (SEB), and Clostridium perfringens epsilon toxin (ETX), it is necessary that therapeutic interventions following a bioterrorism incident by one of these toxins occur as soon as possible after intoxication. Moreover, because the clinical manifestations of intoxication by these agents are likely to be indistinguishable from each other, especially following aerosol exposure, we have developed a cocktail of chimeric monoclonal antibodies that is capable of neutralizing all three toxins. The efficacy of this cocktail was demonstrated in mouse models of lethal dose toxin challenge.

  10. 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

  11. Association of Specific Immunoglobulin E to Staphylococcal Enterotoxin with Airway Hyperresponsiveness in Asthma Patients

    PubMed Central

    Kim, Seong Han; Yang, Seo Yeon; You, Jihong; Lee, Sang Bae; You, Jin; Chang, Yoon Soo; Kim, Hyung Jung; Ahn, Chul Min; Byun, Min Kwang; Park, Jung-Won

    2016-01-01

    Background Specific immunoglobulin E (IgE) sensitization to staphylococcal enterotoxin (SE) has been recently considered to be related to allergic disease, including asthma. Despite studies on specific IgE (sIgE) to SE and its relationship to asthma diagnosis and severity, the association of sIgE to SE with airway hyperresponsiveness (AHR) remains unclear. Methods We enrolled 81 asthma patients admitted to the Severance Hospital in Korea from March 1, 2013, to February 28, 2015 and retrospectively reviewed the electronic medical records of the enrolled subjects. The serum levels of sIgE to SE (A/B) of all subjects was measured using the ImmunoCAP 250 (Phadia) system with SE-sIgE positive defined as >0.10 kU/mL. Results The SE-sIgE level was not significantly correlated with asthma severity (forced expiratory volume in 1 second [FEV1], FEV1/forced vital capacity, sputum eosinophils, and serum eosinophils), whereas the SE-sIgE level in patients with positive AHR (mean±standard error of the mean, 0.606±0.273 kU/mL) was significantly higher than that in patients with negative AHR (0.062±0.015 kU/mL, p=0.034). In regression analysis, SE sensitization (sIgE to SE ≥0.010 kU/mL) was a significant risk factor for AHR, after adjustment for age, sex, FEV1, and sputum eosinophils (odds ratio, 7.090; 95% confidence interval, 1.180–42.600; p=0.032). Prevalence of SE sensitization was higher in patients with allergic rhinitis and non-atopic asthma patients, as compared to patients without allergic rhinitis and atopic asthma patients, respectively, but without statistical significance. Conclusion SE sensitization is significantly associated with AHR. PMID:27790282

  12. Construction and characterization of VL-VH tail-parallel genetically engineered antibodies against staphylococcal enterotoxins.

    PubMed

    He, Xianzhi; Zhang, Lei; Liu, Pengchong; Liu, Li; Deng, Hui; Huang, Jinhai

    2015-03-01

    Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have increasingly given rise to human health and food safety. Genetically engineered small molecular antibody is a useful tool in immuno-detection and treatment for clinical illness caused by SEs. In this study, we constructed the V(L)-V(H) tail-parallel genetically engineered antibody against SEs by using the repertoire of rearranged germ-line immunoglobulin variable region genes. Total RNA were extracted from six hybridoma cell lines that stably express anti-SEs antibodies. The variable region genes of light chain (V(L)) and heavy chain (V(H)) were cloned by reverse transcription PCR, and their classical murine antibody structure and functional V(D)J gene rearrangement were analyzed. To construct the eukaryotic V(H)-V(L) tail-parallel co-expression vectors based on the "5'-V(H)-ivs-IRES-V(L)-3'" mode, the ivs-IRES fragment and V(L) genes were spliced by two-step overlap extension PCR, and then, the recombined gene fragment and V(H) genes were inserted into the pcDNA3.1(+) expression vector sequentially. And then the constructed eukaryotic expression clones termed as p2C2HILO and p5C12HILO were transfected into baby hamster kidney 21 cell line, respectively. Two clonal cell lines stably expressing V(L)-V(H) tail-parallel antibodies against SEs were obtained, and the antibodies that expressed intracytoplasma were evaluated by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry. SEs can stimulate the expression of some chemokines and chemokine receptors in porcine IPEC-J2 cells; mRNA transcription level of four chemokines and chemokine receptors can be blocked by the recombinant SE antibody prepared in this study. Our results showed that it is possible to get functional V(L)-V(H) tail-parallel genetically engineered antibodies in same vector using eukaryotic expression system.

  13. 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

    2001-04-01

    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.

  14. 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

    2001-04-01

    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. PMID:11300798

  15. Staphylococcal enterotoxin A-induced fever is associated with increased circulating levels of cytokines in rabbits.

    PubMed Central

    Huang, W T; Lin, M T; Won, S J

    1997-01-01

    Rabbits were injected intravenously with 10 to 100 ng of staphylococcal enterotoxin A (SEA) per kg, and colonic temperatures were monitored. The febrile responses were compared with circulating levels of interferon (IFN), tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-2, and IL-6 just before the injection of SEA. Both colonic temperatures and circulating levels of IFN, TNF, and IL-2 started to rise at 1 to 2 h and reached their peak levels at 3 to 5 h after SEA injection. Both the fever and the increased circulating levels of IFN, TNF, and IL-2 produced by SEA were decreased by pretreatment with indomethacin (a cyclo-oxygenase inhibitor) (15 mg/kg, intraperitoneally), anisomycin (a protein synthesis inhibitor) (15 mg/kg, subcutaneously), or dexamethasone (an effective anti-inflammatory and immunosuppressive agent) (4 mg/kg, intravenously) in rabbits. Rabbits were injected intravenously with 30 ng of SEA per kg on four consecutive days, and colonic temperatures were monitored. Compared to rabbits that received the single injection of SEA, rabbits that received four consecutive injections of SEA showed a lesser increase in circulating levels of IFN, TNF, and IL-2 as well as colonic temperatures in response to an intravenous dose of SEA (30 ng/kg). The data suggest that the prevention of the febrile response elicited by SEA by indomethacin, anisomycin, or dexamethasone is due to prevention by these compounds of the increase in the circulating levels of IFN, TNF, and IL-2. The pyrogenic hyporesponsiveness to repeated injection of SEA is associated with decreased production of these circulating cytokines. PMID:9199433

  16. Evidence that Escherichia coli STb enterotoxin binds to lipidic components extracted from the pig jejunal mucosa.

    PubMed

    Rousset, E; Dubreuil, J D

    1999-11-01

    Escherichia coli strains producing the heat-stable enterotoxin STb cause diarrhoea in pigs, but little is known on the receptor binding step initiating the diarrhoeal process. In the present study, pig jejunal mucosa extracts were tested for the presence of binding component(s) for STb. Jejunal epithelial cells and the mucus layer were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated material was transferred to a polyvinylidene difluoride (PVDF) membrane and overlayed with STb. The results indicated that a band migrating with the tracking dye was bound by STb. This band was not stained by Coomassie blue and was thus regarded as non proteinic but rather as a lipidic component. Thus, total lipid extracts were obtained from the epithelial cells and the mucus layer. Compared to SDS-PAGE on 12% gels, a better separation of the low molecular mass components contained in these extracts was obtained using high-density Phastgel. Most of the components were detected following silver staining but not using Coomassie blue. Interestingly, commercially available pure glycolipids could also be visualized, after separation, only following silver staining. In the total lipid extracts, a band migrating in the 2.5-6.5 kDa range was observed. Using a monoclonal antisulfatide antibody, this band was recognized indicating that sulfatide was, in effect, present in the extract. When pure sulfatide was run on the same gels, it showed the same electrophoretic mobility. In addition, a dose dependent binding of STb to sulfatide could be observed. Taken together, these data suggested that sulfatide present on the jejunal mucosa, could represent a natural target binding molecule for STb. PMID:10482388

  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. 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. PMID:25361922

  19. Staphylococcal enterotoxin B initiates protein kinase C translocation and eicosanoid metabolism while inhibiting thrombin-induced aggregation in human platelets.

    PubMed

    Tran, Uyen; Boyle, Thomas; Shupp, Jeffrey W; Hammamieh, Rasha; Jett, Marti

    2006-08-01

    Staphylococcal enterotoxin (SE) B, a heat-stable toxin secreted by Staphylococcus aureus, has been implicated in the pathogenesis and exacerbation of several critical illnesses. It has been hypothesized that enterotoxins may interact with blood products such as platelets, in addition to T-lymphocytes and renal proximal tubule cells. The aim of this present study was to elucidate whether SEB directly alters human platelet function. Human platelet rich plasma (PRP) was pre-incubated with SEA, SEB, SEC or TSST-1, (at various concentrations and incubation times). After incubation, PRP was exposed to thrombin and aggregation was assessed. Incubation with all toxins tested resulted in decreased aggregation, specifically; exposure to 10mu g/ml of SEB for 30 min caused a 20% decrease and a 49% decrease at 90 min. A similar reduction in aggregation was seen in samples incubated with phorbol myristate acetate, a known stimulator of protein kinase C (PKC). Further, platelets exposed to SEB exhibited an increased plasma membrane PKC activity. Sphingosine, an inhibitor of PKC proved to block the SEB-induced reduction in aggregation. SEB effects on platelet metabolism were investigated using high performance liquid chromatography showing up to a 2-fold increase of active metabolites lipoxin A4 and 12-HETE, as compared to control. These data indicate that SEB is able to induce platelet dysfunction, and these effects may be mediated through activation of PKC.

  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 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.

  2. Large-scale purification of staphylococcal enterotoxins A, B, and C2 by dye ligand affinity chromatography.

    PubMed Central

    Brehm, R D; Tranter, H S; Hambleton, P; Melling, J

    1990-01-01

    A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production. Images PMID:2339869

  3. Comparison of genotypes and enterotoxin genes between Staphylococcus aureus isolates from blood and nasal colonizers in a Korean hospital.

    PubMed

    Peck, Kyong Ran; Baek, Jin Yang; Song, Jae-Hoon; Ko, Kwan Soo

    2009-08-01

    In this study, we investigated the genetic background of 70 Staphylococcus aureus isolates (36 methicillin-resistant S. aureus [MRSA] and 34 methicillin-susceptible S. aureus [MSSA]) obtained from blood at a Korean tertiary-care hospital, using spa typing, multilocus sequence typing, and SCCmec typing. In addition, the prevalence of enterotoxin (sea, seb, sec, sed, see, seg, seh, sei, and sek), tst, and pvl genes among the samples was assessed via polymerase chain reaction, and the results were compared with those of 95 isolates of S. aureus obtained from nasal swabs. All MRSA isolates from blood, except one, belonged to three major clones: sequence type (ST)5-MRSA-II, ST72-MRSA-II (or IVA), and ST239-MRSA-III, among which ST5-MRSA-II was the predominant clone. The prevalence of enterotoxin genes in the S. aureus isolates obtained from blood differed significantly from those from the nasal swabs for the sea, seb, sec, and seh gene. In particular, the seb and sec genes were detected exclusively in the MRSA isolates of ST5 or spa-CC002, thereby suggesting the co-adaptation of virulence genes with the genetic background and their contribution to biological fitness.

  4. Staphylococcal enterotoxin B initiates protein kinase C translocation and eicosanoid metabolism while inhibiting thrombin-induced aggregation in human platelets.

    PubMed

    Tran, Uyen; Boyle, Thomas; Shupp, Jeffrey W; Hammamieh, Rasha; Jett, Marti

    2006-08-01

    Staphylococcal enterotoxin (SE) B, a heat-stable toxin secreted by Staphylococcus aureus, has been implicated in the pathogenesis and exacerbation of several critical illnesses. It has been hypothesized that enterotoxins may interact with blood products such as platelets, in addition to T-lymphocytes and renal proximal tubule cells. The aim of this present study was to elucidate whether SEB directly alters human platelet function. Human platelet rich plasma (PRP) was pre-incubated with SEA, SEB, SEC or TSST-1, (at various concentrations and incubation times). After incubation, PRP was exposed to thrombin and aggregation was assessed. Incubation with all toxins tested resulted in decreased aggregation, specifically; exposure to 10mu g/ml of SEB for 30 min caused a 20% decrease and a 49% decrease at 90 min. A similar reduction in aggregation was seen in samples incubated with phorbol myristate acetate, a known stimulator of protein kinase C (PKC). Further, platelets exposed to SEB exhibited an increased plasma membrane PKC activity. Sphingosine, an inhibitor of PKC proved to block the SEB-induced reduction in aggregation. SEB effects on platelet metabolism were investigated using high performance liquid chromatography showing up to a 2-fold increase of active metabolites lipoxin A4 and 12-HETE, as compared to control. These data indicate that SEB is able to induce platelet dysfunction, and these effects may be mediated through activation of PKC. PMID:16550298

  5. 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. PMID:25311736

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcus aureus is an important bacterial pathogen that produces a range of Staphylococcal Enterotoxins (SEs) which cause gastroenteritis and superantigen activation of T cells, the mechanism of which is not well understood. The ability to rapidly detect and quantify SEs is very important in ...

  7. 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.

  8. 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

  9. Stimulation of intestinal mucosal adenyl cyclase by cholera enterotoxin and prostaglandins

    PubMed Central

    Kimberg, Daniel V.; Field, Michael; Johnson, Judith; Henderson, Antonia; Gershon, Elaine

    1971-01-01

    The effects of several prostaglandins (PG) and a highly purified preparation of cholera enterotoxin (CT) on intestinal mucosal adenyl cyclase activity and the effect of CT on intestinal mucosal cyclic 3′,5′-adenosine monophosphate concentration were determined in guinea pig and rabbit small intestine and were correlated with the effects of the same agents on ion transport. Adenyl cyclase activity, measured in a crude membrane fraction of the mucosa, was found at all levels of the small intestine with the highest activity per milligram protein in the duodenum. The prostaglandins, when added directly to the assay, increased adenyl cyclase activity; the greatest effect (2-fold increase) was obtained with PGE1 (maximal effect at 0.03 mM) and PGE2. The prostaglandins also increased short-circuit current (SCC) in isolated guinea pig ileal mucosa, with PGE1 and PGE2 again giving the greatest effects. The prior addition of theophylline (10 mM) reduced the subsequent SCC response to PGE1 and vice versa. It was concluded, therefore, that the SCC response to PGE1, like the response to theophylline, represented active Cl secretion. CT increased adenyl cyclase activity in guinea pig and rabbit ileal mucosa when preincubated with the mucosa from 1 to 2.5 hr in vitro or for 2.5 hr in vivo but not when added directly to the assay. The increments in activity caused by PGE1 and NaF were the same in CT-treated and control mucosa. Cyclic 3′,5′-AMP concentration in rabbit ileal mucosa was increased 3.5-fold after a 2 hr preincubation with CT in vitro. Phosphodiesterase activity in the crude membrane fraction of the mucosa was unaffected by either CT or PGE1. A variety of other agents including insulin, glucagon, parathormone, thyroid-stimulating hormone, L-thyroxine, thyrocalcitonin, vasopressin, and epinephrine all failed to change adenyl cyclase activity. It is concluded that CT and certain prostaglandins produce small intestinal fluid secretion by increasing mucosal adenyl

  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. Application of MALDI-TOF mass spectrometry for the detection of enterotoxins produced by pathogenic strains of the Bacillus cereus group.

    PubMed

    Tsilia, Varvara; Devreese, Bart; de Baenst, Ilse; Mesuere, Bart; Rajkovic, Andreja; Uyttendaele, Mieke; Van de Wiele, Tom; Heyndrickx, Marc

    2012-10-01

    Enterotoxins produced by different species of the Bacillus cereus group, such as cytotoxin K1 (CytK1) and non-haemolytic enterotoxin (NHE), have been associated with diarrhoeal food poisoning incidents. Detection of CytK1 is not possible with commercial assays while NHE is recognised by an immunological kit (TECRA) that does not specifically target this protein because it is based on polyclonal antibodies. It is evident that the lack of suitable tools for the study of enterotoxins hampers the possibilities for accurate hazard identification and characterisation in microbial food safety risk assessment. We applied matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) for the detection of CytK1 and NHE produced by pathogenic strains of the B. cereus group using protein digests from 1D gel electrophoresis. Secretion of CytK1 and two of the three components of NHE was confirmed in supernatants of different B. cereus cultures. For each protein, we introduce biomarkers that could be used for the screening of food poisoning or food/environmental isolates that can secrete enterotoxins. For example, tryptic peptides of 2,310.2 and 1,192.5 Da (calculated mass) can be indicators for CytK1 and NheA, respectively, although a simultaneous detection of other enterotoxin-specific peptides is recommended to assure the presence of a toxin in an unknown sample. Comparison of MALDI-TOF/MS with the TECRA kit showed that our methodological strategy performed well and it had the competitive advantage of specifically detecting NheA. Therefore, MALDI-TOF/MS can be successfully incorporated into risk assessment procedures in order to determine the involvement of strains of the B. cereus group in foodborne outbreaks, including the recently described cytK1 producing species, Bacillus cytotoxicus.

  12. 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.

  13. DIARRHEA OUTBREAK IN PERNAMBUCO, BRAZIL, ASSOCIATED WITH A HEAT-STABLE CYTOTOXIC ENTEROTOXIN PRODUCED BY Aeromonas caviae

    PubMed Central

    LOPES, Ana Carolina Amaral; MARTINS, Luciano Moura; GATTI, Maria Silvia Viccari; FALAVINA DOS REIS, Cristhiane Moura; HOFER, Ernesto; YANO, Tomomasa

    2015-01-01

    SUMMARY In the present study enterotoxic and cytotoxic activities of twenty Aeromonas caviaestrains were examined. They originated from fecal specimens of patients with acute diarrhea during an outbreak in Brazil in 2004. Culture supernatants of fourteen strains (70%) caused fluid accumulation in rabbit ileal intestinal loops and in suckling mice assays, and also showed a cytotoxic activity in Vero and Caco-2 cells. The enterotoxic and cytotoxic factors were heat-stable after culture supernatants treatment at 100 ºC. The results revealed that A. caviaestrains produce a putative diarrheagenic virulence factor, a heat-stable cytotoxic enterotoxin that could be linked to the diarrhea outbreak that took place in Brazil. PMID:26422161

  14. Correlation of temperature and toxicity in murine studies of staphylococcal enterotoxins and toxic shock syndrome toxin 1.

    PubMed

    Stiles, B G; Campbell, Y G; Castle, R M; Grove, S A

    1999-03-01

    This study describes a quick (<12 h) assay for detecting temperature decreases in BALB/c and C57BL/6 mice injected intraperitoneally (i.p. ) with staphylococcal enterotoxin A (SEA), SEB, or SEC3 or toxic shock syndrome toxin 1 and a potentiating dose of lipopolysaccharide (LPS). Toxin-specific antisera effectively neutralized the temperature fluctuations in this model. Orally administered SEA or SEB (50 microg/animal), with or without LPS, did not have an effect on temperature or lethality. Versus wild-type mice, transgenic knockout mice lacking the p55 receptor for tumor necrosis factor (TNF) or gamma interferon were protected against an i.p. challenge of SEA plus LPS. The p75 receptor for TNF and intercellular adhesion molecule 1 have a negligible role in this toxic shock model.

  15. The use of a sandwich ELISA for the detection of staphylococcal enterotoxin A in foods from outbreaks of food poisoning.

    PubMed Central

    Wieneke, A. A.; Gilbert, R. J.

    1985-01-01

    Foods from outbreaks of food poisoning were examined for the presence of staphylococcal enterotoxin A (SEA) by a sandwich ELISA using microtitre trays as the solid phase and SEA antibodies raised in sheep. The presence of SEA was confirmed by neutralization tests. The toxin was detected in 12 of 15 foods from separate outbreaks of staphylococcal food poisoning; all 15 foods contained a strain of Staphylococcus aureus which produced SEA. For most foods a simple extraction procedure without a concentration step was sufficient to detect the toxin. The method was semi-quantitative and recoveries of SEA added to control foods varied from 30 to 80%. The foods from outbreaks contained between 1 and 10 micrograms of SEA/100 g. SEA was not found in foods from 21 outbreaks in which an SEA-producing strain of Staph. aureus was not isolated. PMID:3926871

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. Detection and Measurement of Staphylococcal Enterotoxin-Like K (SEl-K) Secretion by Staphylococcus aureus Clinical Isolates

    PubMed Central

    Aguilar, Jorge L.; Varshney, Avanish K.; Wang, Xiaobo; Stanford, Lindsay; Scharff, Matthew

    2014-01-01

    Staphylococcal enterotoxin-like K (SEl-K) is a potent mitogen that elicits T-cell proliferation and cytokine production at very low concentrations. However, unlike the classical enterotoxins SEB and toxic shock syndrome toxin 1 (TSST-1), the gene for SEl-K is commonly present in more than half of all Staphylococcus aureus clinical isolates and is present in almost all USA300 community-acquired methicillin-resistant S. aureus (CA-MRSA) isolates. Sequencing of the sel-k gene in over 20 clinical isolates and comparative analysis with all 14 published sel-k sequences indicate that there are at least 6 variants of the sel-k gene, including one that is conserved among all examined USA300 strains. Additionally, we have developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) that specifically detects and measures SEl-K protein in culture supernatants and biological fluids. Quantification of in vitro SEl-K secretion by various S. aureus isolates using this novel capture ELISA revealed detectable amounts of SEl-K secretion by all isolates, with the highest secretion levels being exhibited by MRSA strains that coexpress SEB. In vivo secretion was measured in a murine thigh abscess model, where similar levels of SEl-K accumulation were noted regardless of whether the infecting strain exhibited high or low secretion of SEl-K in vitro. We conclude that SEl-K is commonly expressed in the setting of staphylococcal infection, in significant amounts. SEl-K should be further explored as a target for passive immunotherapy against complicated S. aureus infection. PMID:24808237

  1. 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.

  2. Crystal Structure of Staphylococcal Enterotoxin I (SEI) in Complex with a Human Major Histocompatibility Complex Class II Molecule*

    PubMed Central

    Fernández, Marisa M.; Guan, Rongjin; Swaminathan, Chittoor P.; Malchiodi, Emilio L.; Mariuzza, Roy A.

    2009-01-01

    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 β-chain. However, the basis for promiscuous MHC recognition by zinc-dependent superantigens is not obvious, because the β-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 Å 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 β-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 β-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. PMID:16829512

  3. 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. PMID:27220526

  4. The carboxyl-terminal region of staphylococcal enterotoxin type A is required for a fully active molecule.

    PubMed Central

    Hufnagle, W O; Tremaine, M T; Betley, M J

    1991-01-01

    Staphylococcal enterotoxin type A (SEA) gene (sea+) mutations were constructed by exonuclease III digestion or cassette mutagenesis. Five different sea mutations that had 1, 3, 7, 39, and 65 codons deleted from the 3' end of sea+ were identified and confirmed by restriction enzyme and nucleotide sequence analyses. Each of these sea mutations was constructed in Escherichia coli and transferred to Staphylococcus aureus by using the plasmid vector pC194. Culture supernatants from the parent S. aureus strain that lacked an enterotoxin gene (negative controls) and from derivatives that contained either sea+ (positive control) or a sea mutation were examined for in vitro sensitivity to degradation by monkey stomach lavage fluid, the ability to cause emesis when administered by an intragastric route to rhesus monkeys, and the ability to induce T-cell proliferation and by Western immunoblot analysis and a gel double-diffusion assay with polyclonal antibodies prepared against SEA. Altered SEAs corresponding to the predicted sizes were visualized by Western blot analysis of culture supernatants for each of the staphylococcal derivatives that contained a sea mutation. The altered SEA that lacked the C-terminal amino acid residue behaved like SEA in all of the assays performed. The altered SEA that lacked the three C-terminal residues of SEA caused T-cell proliferation but was not emetic; this altered SEA was degraded in vitro by monkey stomach lavage fluid and did not reach in the gel double diffusion assay. Altered SEAs that lacked 7, 39, or 65 carboxyl-terminal residues were degraded by stomach lavage fluid in vitro, did not produce an emetic response, and did not induce T-cell proliferation or form a visible reaction in the gel double-diffusion assay. Images PMID:1903773

  5. Behavior and enterotoxin production by coagulase negative Staphylococcus in cooked ham, reconstituted skimmed milk, and confectionery cream.

    PubMed

    Oliveira, Ana Maria; Miya, Norma Teruko Nago; Sant'Ana, Anderson S; Pereira, José Luiz

    2010-09-01

    In this study, the behavior and enterotoxin production by 10 different coagulase negative Staphylococcus (CNS) strains inoculated in cooked ham, reconstituted skimmed milk, and confectionery cream in the presence or absence of background microbiota have been investigated. After inoculation (103 CFU/g), foods were incubated at 25, 30, and 37 °C and aerobic mesophilic and CNS counts were carried out at 12, 24, 48, and 72 h. Staphylococcal enterotoxins (SE) detection was performed by SET-RPLA (Oxoid, Basingstoke, U.K.) and mini-Vidas® (bioMérieux, La Balme les Grottes, France). CNS counts increased during incubation and approached 10⁶ to 10⁷ CFU/g after 12 h at 37 °C in the 3 foods studied. At 25 °C, counts reached 10⁶ to 10⁷ CFU/g only after 24 to 48 h. The interference of background microbiota on CNS behavior was only observed when they grew in sliced cooked ham, which presented a high initial total count (10⁵ CFU/g). Significantly higher counts of CNS isolated from raw cow's milk in comparison with food handlers isolates were found in reconstituted milk and confectionery cream. Although CNS strains were able to produce SEA, SEB, and SED in culture media, in foods, in the presence or absence of background microbiota S. chromogenes LE0598 was the only strain able to produce SEs. Despite the scarcity of reports on CNS involvement with foodborne disease outbreaks, the results found here support the CNS growth and SE production in foods even in the presence of background microbiota and may affect food safety. PMID:21535559

  6. 3,39-Diindolylmethane Ameliorates Staphylococcal Enterotoxin B–Induced Acute Lung Injury through Alterations in the Expression of MicroRNA that Target Apoptosis and Cell-Cycle Arrest in Activated T Cells.

    PubMed

    Elliott, David M; Nagarkatti, Mitzi; Nagarkatti, Prakash S

    2016-04-01

    3,39-Diindolylmethane (DIM), a natural indole found in cruciferous vegetables, has significant anti-cancer and anti-inflammatory properties. In this current study, we investigated the effects of DIM on acute lung injury (ALI) induced by exposure to staphylococcal enterotoxin B (SEB). We found that pretreatment of mice with DIM led to attenuation of SEB-induced inflammation in the lungs, vascular leak, and IFN-g secretion. Additionally, DIM could induce cell-cycle arrest and cell death in SEB-activated T cells in a concentration-dependent manner. Interestingly, microRNA (miRNA) microarray analysis uncovered an altered miRNA profile in lung-infiltrating mononuclear cells after DIM treatment of SEB-exposed mice. Moreover, computational analysis of miRNA gene targets and regulation networks indicated that DIM alters miRNA in the cell death and cell-cycle progression pathways. Specifically, DIM treatment significantly downregulated several miRNA and a correlative increase associated gene targets. Furthermore, overexpression and inhibition studies demonstrated that DIM-induced cell death, at least in part, used miR-222. Collectively, these studies demonstrate for the first time that DIM treatment attenuates SEB-induced ALI and may do so through the induction of microRNAs that promote apoptosis and cell-cycle arrest in SEB-activated T cells. PMID:26818958

  7. Evaluation of a commercial enzyme immunoassay kit (RIDASCREEN) for detection of staphylococcal enterotoxins A, B, C, D, and E in foods.

    PubMed Central

    Park, C E; Akhtar, M; Rayman, M K

    1994-01-01

    The RIDASCREEN SET kit (R-Biopharm GmbH, Darmstadt, Germany), a commercial staphylococcal enterotoxin (SE) visual immunoassay kit, was evaluated for its efficacy. The kit utilizes monovalent capture antibodies against SE types A to E (SEA to SEE); therefore, it simultaneously detects and identifies the enterotoxin types. The major advantages of the kit are (i) a high degree of specificity (except for naturally occurring peroxidases, food compositions or ingredients and microbiological products due to growth of nonstaphylococcal microorganisms did not cause false-positive results; additionally, no cross-reactions among reagents of the kits were observed), (ii) excellent sensitivity (minimum detectable limits were 0.20 to 0.30 ng of SEs per ml of extracts of ham, salami, and mushroom and 0.30 to 0.35 ng of SEs per ml of cheese extracts, or 0.50 to 0.75 ng of SEs per g of foods such as noodles, ham, salami, cheese, and turkey), (iii) simplicity (the kit enabled direct assay of SEs in food extracts without the need for lengthy extraction or concentration procedures), (iv) rapidity (it took less than 3 h to complete the analysis of individual enterotoxin types SEA to SEE), and (v) its semiquantitative results (optical density values could be read against a standard curve to estimate the amount of SE in the extract). The RIDASCREEN kit is a convenient, rapid, and reliable tool for the detection and identification of SEs in foods. PMID:8135522

  8. Detection of mecA and enterotoxin genes in Staphylococcus aureus isolates associated with bovine mastitis and characterization of Staphylococcal cassette chromosome mec (SCCmec) in MRSA strains.

    PubMed Central

    Havaei, Seyed Asghar; Assadbeigi, Behnaz; Esfahani, Bahram Nasr; Hoseini, Nafiseh Sadat; Rezaei, Nahid; Havaei, Seyed Rouhollah

    2015-01-01

    Background and Objectives: Staphylococcus aureus is one of the main causatives of bovine mastitis. Resistance of some strains to methicillin, can complicate the treatment of its infections. On the other hand, enterotoxin production is also important. Therefore, the aim of our study was to investigate the methicillin resistance and enterotoxin production in S. aureus isolates caused bovine mastitis. Materials and Methods: Four hundred and fifty milk samples were collected. After isolation of Staphylococcus aureus, MRSA strains were detected by cefoxitin disc diffusion and oxacillin agar screening methods. DNA was extracted by phenol – chloroform method and PCR was applied for mecA, sea and seb genes. SCCmec types of mecA gene were identified using multiplex-PCR. Results: Fifty-four (12%) S. aureus were isolated. Out of these, 10 and 9 MRSA strains identified by cefoxitin disc diffusion and oxacillin agar screening methods, respectively. All 10 MRSA isolates identified by cefoxitin disc diffusion, were positive for mecA gene and all of them belonged to SCCmec type IV. The sea genes were detected in 19 isolates and only two isolates were positive for seb genes. One isolate possessed both sea and seb genes. Conclusion: Findings of this study indicated that results of cefoxitin disc diffusion test is in concordance with the PCR for mecA gene and has a higher sensitivity compared to oxacillin agar screening method. Finally, Our findings suggest that enterotoxin A is the dominant type. PMID:26668704

  9. Prevalence of Enterotoxin Genes and Antibacterial Susceptibility Pattern of Staphylococcus aureus Strains Isolated from Animal Originated Foods in West of Iran

    PubMed Central

    Mashouf, Rasoul Y.; Hosseini, Seyed M.; Mousavi, Seyed M.; Arabestani, Mohammad R.

    2015-01-01

    Objectives The aims of our study were to evaluate the prevalence of Staphylococcus aureus (S. aureus) strains in food samples of animal origin, examine their antibacterial susceptibility pattern, and to detect staphylococcal enterotoxin (SEs) genes and the mecA gene in isolated S. aureus strains using the polymerase chain reaction (PCR).  Methods A total of 1050 food samples including 671 raw milk and dairy products and 379 raw meats were collected between September 2013 and June 2014 in Hamadan, Iran. Food samples were analyzed for S. aureus identification. The antibiotic susceptibility pattern of all isolates was determined using the disk agar diffusion method followed by detecting mecA resistance gene using PCR. In addition, harboring of SE genes were determined using a multiplex PCR assay targeting nine genes.   Results A total of 98 (9.3%) S. aureus strains were isolated from 1050 food samples. Of the 98 isolates examined, the most frequent resistance was observed to erythromycin (30.6%), followed by tetracycline (29.6%), gentamicin (27.6%), clindamycin (26.5%), ciprofloxacin and rifampin (24.5%), trimethoprim-sulfamethoxazole (14.3%), and cefoxitin (5.1%). All cefoxitin resistant isolates were positive for mecA. The prevalence of SEs was 77.6% (n=76). Among the genes that code classic enterotoxins, sea was the most frequent and was carried by 25.5% of isolates, followed by see in 18.4%, sed in 11.2%, sec in 5.1%, and seb in 4.1% of isolates. Among the detected enterotoxins, seg was the predominantly identified enterotoxin gene in isolates with prevalence of 35.7%. The seh gene with prevalence of 1% and sei gene with 3.1% were other detected enterotoxins with low frequencies.   Conclusion The high prevalence of SE genes detected indicates a potential risk for causing animal-originated food poisoning. The increasing prevalence of community-acquired MRSA and its emerging antibiotic resistance in foods is a serious problem for public health. PMID:26366263

  10. Application of a SERS-based lateral flow immunoassay strip for the rapid and sensitive detection of staphylococcal enterotoxin B

    NASA Astrophysics Data System (ADS)

    Hwang, Joonki; Lee, Sangyeop; Choo, Jaebum

    2016-06-01

    A novel surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay (LFA) biosensor was developed to resolve problems associated with conventional LFA strips (e.g., limits in quantitative analysis and low sensitivity). In our SERS-based biosensor, Raman reporter-labeled hollow gold nanospheres (HGNs) were used as SERS detection probes instead of gold nanoparticles. With the proposed SERS-based LFA strip, the presence of a target antigen can be identified through a colour change in the test zone. Furthermore, highly sensitive quantitative evaluation is possible by measuring SERS signals from the test zone. To verify the feasibility of the SERS-based LFA strip platform, an immunoassay of staphylococcal enterotoxin B (SEB) was performed as a model reaction. The limit of detection (LOD) for SEB, as determined with the SERS-based LFA strip, was estimated to be 0.001 ng mL-1. This value is approximately three orders of magnitude more sensitive than that achieved with the corresponding ELISA-based method. The proposed SERS-based LFA strip sensor shows significant potential for the rapid and sensitive detection of target markers in a simplified manner.A novel surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay (LFA) biosensor was developed to resolve problems associated with conventional LFA strips (e.g., limits in quantitative analysis and low sensitivity). In our SERS-based biosensor, Raman reporter-labeled hollow gold nanospheres (HGNs) were used as SERS detection probes instead of gold nanoparticles. With the proposed SERS-based LFA strip, the presence of a target antigen can be identified through a colour change in the test zone. Furthermore, highly sensitive quantitative evaluation is possible by measuring SERS signals from the test zone. To verify the feasibility of the SERS-based LFA strip platform, an immunoassay of staphylococcal enterotoxin B (SEB) was performed as a model reaction. The limit of detection (LOD) for SEB, as

  11. Campomanesia adamantium Peel Extract in Antidiarrheal Activity: The Ability of Inhibition of Heat-Stable Enterotoxin by Polyphenols

    PubMed Central

    Lescano, Caroline Honaiser; de Oliveira, Ivan Pires; Zaminelli, Tiago; Baldivia, Débora da Silva; da Silva, Luan Ramos; Napolitano, Mauro; Silvério, Camila Bitencourt Mendes; Lincopan, Nilton; Sanjinez-Argandoña, Eliana Janet

    2016-01-01

    Campomanesia adamantium (Myrtaceae) is a medicinal plant distributed in Brazilian Cerrado. Different parts of this plant are used in popular medicine for treatment of several diseases like fever, diarrhea, hypercholesterolemia and rheumatism. The aim of this work was to evaluate the inhibition of heat-stable enterotoxin type A (STa) by gallic acid present in the peel of C. adamantium fruit and assays to assess the antidiarrheal activity, anti-inflammatory and cytotoxic properties of peel extract using the T84 cell line model. The possible inhibition exerted by the gallic acid of the peel extract on the STa peptide was inferred by molecular dynamics simulations. The antidiarrheal effects were investigated measuring cGMP accumulation in cells after stimulation by STa toxin and antibacterial activity was assessed. The anti-inflammatory activity was assessed by inhibition of COX-1 and COX-2. MTT and LDH assays were used to evaluate any possible cytotoxic action while the CyQUANT test was used to investigate the effect on cell proliferation. A representation showing how the possible interactions between STa and the gallic acid of the extract might reduce the action of the enterotoxin is presented. C. adamantium peel extract significantly decreased the levels of cGMP in T84 cells. However, no effect on the species of microorganisms was observed. The extract also inhibited COX-1 (IC50 255.70 ± 0.04 ng/mL) and COX-2 (IC50 569.50 ± 0.11 ng/mL) enzymes. Cytotoxicity assay have shown significant changes in cells treated with the extract, which inhibited the cell proliferation until 72 hours of treatment. Direct interactions of phenolic compounds present in the extract with the STa toxin may limit its activity. Curative effect in the diarrhea treatment and its anti-inflammatory action is based on the pharmacological properties, mechanism of action of the C. adamantium peel extract, and no toxic effects of the peel extract presented on this work. PMID:27764241

  12. An alternative explanation for the occurrence of short circuit current increases in the small intestine following challenge by bacterial enterotoxins.

    PubMed

    Lucas, M L

    2013-10-01

    Secretory diarrhoeal disease due to enterotoxins is thought to arise from the enhancement to pathologically high rates of normally occurring chloride ion and therefore fluid secretion from enterocytes. In support of this concept, many enterotoxins increase intestinal short-circuit current, regarded now as faithfully reflecting the increased chloride ion secretion. Contradicting this assumption, STa reduces absorption but does not cause secretion in vivo although short-circuit current is increased in vitro. There is therefore a mismatch between an assumed enterocyte mediated secretory event that should but does not cause net fluid secretion and an undoubtedly increased short-circuit current. It is proposed here that short-circuit current increases are not themselves secretory events but result from interrupted fluid absorption. A noteworthy feature of compounds that inhibit the increase in short-circuit current is that the majority are vasoactive, neuroactive or both. In general, vasodilator substances increase current. An alternative hypothesis for the origin of short-circuit current increases is that these result from reflex induction of electrogenic fluid absorption. This reflex enhances a compensatory response that is also present at a cellular level. An intestinal reflex is therefore proposed by which decreases in interstitial and intravascular volume or pressure within the intestine initiate an electrogenic fluid absorption mechanism that compensates for the loss of electrically neutral fluid absorption. This hypothesis would explain the apparently complex pharmacology of short-circuit current increases since many depressor substances have receptors in common with enterocytes and enteric nerves. The proposed alternative view of the origin of short-circuit current increases assumes that these do not represent chloride secretion from the enterocytes. This view may therefore aid the successful development of anti-diarrhoeal drugs to overcome a major cause of

  13. Studies on the pathogenicity of Yersinia enterocolitica and Y. enterocolitica-like bacteria. 1. Enterotoxin production at 22 degrees C and 37 degrees C by environmental and human isolates from Scandinavia.

    PubMed

    Kapperud, G

    1980-10-01

    Altogether, 412 strains of Yersinia enterocolitica and Y. enterocolitica-like bacteria from environmental and human sources were examined for their ability to produce enterotoxin at 22 degrees C and 37 degrees C using the infant mouse assay. A total of 73 strains were positive at 22 degrees C, only. Another 28 strains were positive both at 22 degrees C and 37 degrees C. Of these 28 strains, 26 were sucrose and aesculin non-fermenting and all were Voges-Proskauer negative. All of these strains were isolated from non-human sources. All of 13 strains belonging to O-serogroup 28 produced enterotoxin at 37 degrees C. The results indicate that enterotoxin production at 22 degrees C is widespread in Y. enterocolitica with the highest prevalence among human clinical isolates. Enterotoxin production at both 22 degrees C and 37 degrees C seems to be common in Y. kristensenii (sucrose non-fermentors). Enterotoxin production is apparently rare in Y. frederiksenii (rhamnose fermentors) and in Y. intermedia (rhamnose and melibiose fermentors).

  14. Localized surface plasmon resonance-based hybrid Au-Ag nanoparticles for detection of Staphylococcus aureus enterotoxin B

    NASA Astrophysics Data System (ADS)

    Zhu, Shaoli; Du, ChunLei; Fu, Yongqi

    2009-09-01

    A triangular hybrid Au-Ag nanoparticles array was proposed for the purpose of biosensing in this paper. Constructing the hybrid nanoparticles, an Au thin film is capped on the Ag nanoparticles which are attached on glass substrate. The hybrid nanoparticles array was designed by means of finite-difference and time-domain (FDTD) algorithm-based computational numerical calculation and optimization. Sensitivity of refractive index of the hybrid nanoparticles array was obtained by the computational calculation and experimental detection. Moreover, the hybrid nanoparticles array can prevent oxidation of the pure Ag nanoparticles from atmosphere environment because the Au protective layer was deposited on top of the Ag nanoparticles so as to isolate the Ag particles from the atmosphere. We presented a novel surface covalent link method between the localized surface plasmon resonance (LSPR) effect-based biosensors with hybrid nanoparticles array and the detected target molecules. The generated surface plasmon wave from the array carries the biological interaction message into the corresponding spectra. Staphylococcus aureus enterotoxin B (SEB), a small protein toxin was directly detected at nanogramme per milliliter level using the triangular hybrid Au-Ag nanoparticles. Hence one more option for the SEB detection is provided by this way.

  15. Protective effect of recombinant staphylococcal enterotoxin A entrapped in polylactic-co-glycolic acid microspheres against Staphylococcus aureus infection.

    PubMed

    Chen, Liben; Li, Shuang; Wang, Zhengfang; Chang, Ruilong; Su, Jingliang; Han, Bo

    2012-03-19

    Staphylococcus aureus is an important cause of nosocomial and community-acquired infections in humans and animals, as well as the cause of mastitis in dairy cattle. Vaccines aimed at preventing S. aureus infection in bovine mastitis have been studied for many years, but have so far been unsuccessful due to the complexity of the bacteria, and the lack of suitable vaccine delivery vehicles. The current study developed an Escherichia coli protein expression system that produced a recombinant staphylococcal enterotoxin A (rSEA) encapsulated into biodegradable microparticles generated by polylactic-co-glycolic acid (PLGA) dissolved in methylene chloride and stabilized with polyvinyl acetate. Antigen loading and surface properties of the microparticles were investigated to optimize particle preparation protocols. The prepared PLGA-rSEA microspheres had a diameter of approximately 5 μm with a smooth and regular surface. The immunogenicity of the PLGA-rSEA vaccine was assessed using mice as an animal model and showed that the vaccine induced a strong humoral immune response and increased the percent survival of challenged mice and bacterial clearance. Histological analysis showed moderate impairment caused by the pathogen upon challenge afforded by immunization with PLGA-rSEA microspheres. Antibody titer in the sera of mice immunized with PLGA-rSEA microparticles was higher than in vaccinated mice with rSEA. In conclusion, the PLGA-rSEA microparticle vaccine developed here could potentially be used as a vaccine against enterotoxigenic S. aureus.

  16. Prophage-Encoded Staphylococcal Enterotoxin A: Regulation of Production in Staphylococcus aureus Strains Representing Different Sea Regions

    PubMed Central

    Zeaki, Nikoleta; Budi Susilo, Yusak; Pregiel, Anna; Rådström, Peter; Schelin, Jenny

    2015-01-01

    The present study investigates the nature of the link between the staphylococcal enterotoxin A (SEA) gene and the lifecycle of Siphoviridae bacteriophages, including the origin of strain variation regarding SEA production after prophage induction. Five strains representing three different genetic lines of the sea region were studied under optimal and prophage-induced growth conditions and the Siphoviridae lifecycle was followed through the phage replicative form copies and transcripts of the lysogenic repressor, cro. The role of SOS response on prophage induction was addressed through recA transcription in a recA-disruption mutant. Prophage induction was found to increase the abundance of the phage replicative form, the sea gene copies and transcripts and enhance SEA production. Sequence analysis of the sea regions revealed that observed strain variances were related to strain capacity for prophage induction, rather than sequence differences in the sea region. The impact of SOS response activation on the phage lifecycle was demonstrated by the absence of phage replicative form copies in the recA-disruption mutant after prophage induction. From this study it emerges that all aspects of SEA-producing strain, the Siphoviridae phage and the food environment must be considered when evaluating SEA-related hazards. PMID:26690218

  17. Crystal Structure of Staphylococcal Enterotoxin G (SEG) in Complex with a Mouse T-cell Receptor Beta Chain

    SciTech Connect

    Fernandez, M.M.; Robinson, H.; Cho, S.; De Marzi, M. C.; Kerzic, M. C.; Mariuzza, R. A.; Malchiodi, E. L.

    2011-01-14

    Superantigens (SAgs) are bacterial or viral toxins that bind MHC class II (MHC-II) molecules and T-cell receptor (TCR) in a nonconventional manner, inducing T-cell activation that leads to inflammatory cytokine production, which may result in acute toxic shock. In addition, the emerging threat of purpura fulminans and community-associated meticillin-resistant Staphylococcus aureus emphasizes the importance of a better characterization of SAg binding to their natural ligands that may allow the development of reagents to neutralize their action. The three-dimensional structure of the complex between a mouse TCR {beta} chain (mV{beta}8.2) and staphylococcal enterotoxin G (SEG) at 2.0 {angstrom} resolution revealed a binding site that does not conserve the 'hot spots' present in mV{beta}8.2-SEC2, mV{beta}8.2-SEC3, mV{beta}8.2-SEB, and mV{beta}8.2-SPEA complexes. Analysis of the mV{beta}8.2-SEG interface allowed us to explain the higher affinity of this complex compared with the others, which may account for the early activation of T-cells bearing mV{beta}8.2 by SEG. This mode of interaction between SEG and mV{beta}8.2 could be an adaptive advantage to bestow on the pathogen a faster rate of colonization of the host.

  18. Cross-linking staphylococcal enterotoxin A bound to major histocompatibility complex class I is required for TNF-alpha secretion

    NASA Technical Reports Server (NTRS)

    Wright, A. D.; Chapes, S. K.

    1999-01-01

    The mechanism of how superantigens function to activate cells has been linked to their ability to bind and cross-link the major histocompatibility complex class II (MHCII) molecule. Cells that lack the MHCII molecule also respond to superantigens, however, with much less efficiency. Therefore, the purpose of this study was to confirm that staphylococcal enterotoxin A (SEA) could bind the MHCI molecule and to test the hypothesis that cross-linking SEA bound to MHCII-deficient macrophages would induce a more robust cytokine response than without cross-linking. We used a capture enzyme-linked immunosorbent assay and an immunprecipitation assay to directly demonstrate that MHCI molecules bind SEA. Directly cross-linking MHCI using monoclonal antibodies or cross-linking bound SEA with an anti-SEA antibody or biotinylated SEA with avidin increased TNF-alpha and IL-6 secretion by MHCII(-/-) macrophages. The induction of a vigorous macrophage cytokine response by SEA/anti-SEA cross-linking of MHCI offers a mechanism to explain how MHCI could play an important role in superantigen-mediated pathogenesis. Copyright 1999 Academic Press.

  19. Prevalence of multi-antimicrobial-agent resistant, shiga toxin and enterotoxin producing Escherichia coli in surface waters of river Ganga.

    PubMed

    Ram, Siya; Vajpayee, Poornima; Shanker, Rishi

    2007-11-01

    The consumption of polluted surface water for domestic and recreational purposes by large populations in developing nations is a major cause of diarrheal disease related mortality. The river Ganga and its tributaries meet 40% of the water requirement for drinking and irrigation in India. In this study, Escherichia coli isolates (n=75) of the river Ganga water were investigated for resistance to antimicrobial agents (n=15) and virulence genes specific to shiga toxin (STEC) and enterotoxin producing E. coli (ETEC). E. coli isolates from the river Ganga water exhibit resistance to multiple antimicrobial agents. The distribution of antimicrobial agent resistance in E. colivaries significantly (chi2: 81.28 at df = 24, p < 0.001) between the sites. Both stx1 and stx2 genes were present in 82.3% of STEC (n=17) while remaining isolates possess either stxl (11.8%) or stx2 (5.9%). The presence of eaeA, hlyA, and chuA genes was observed in 70.6, 88.2, and 58.8% of STEC, respectively. Both LT1 and ST1 genes were positive in 66.7% of ETEC (n=15) while 33.3% of isolates harbor only LT1 gene. The prevalence of multi-antimicrobial-agent resistant E. coli in the river Ganga water poses increased risk of infections in the human population.

  20. Crystal Structure of Staphylococcal Enterotoxin G (SEG) in Complex with a Mouse T-cell Receptor β Chain*

    PubMed Central

    Fernández, Marisa M.; Cho, Sangwoo; De Marzi, Mauricio C.; Kerzic, Melissa C.; Robinson, Howard; Mariuzza, Roy A.; Malchiodi, Emilio L.

    2011-01-01

    Superantigens (SAgs) are bacterial or viral toxins that bind MHC class II (MHC-II) molecules and T-cell receptor (TCR) in a nonconventional manner, inducing T-cell activation that leads to inflammatory cytokine production, which may result in acute toxic shock. In addition, the emerging threat of purpura fulminans and community-associated meticillin-resistant Staphylococcus aureus emphasizes the importance of a better characterization of SAg binding to their natural ligands that may allow the development of reagents to neutralize their action. The three-dimensional structure of the complex between a mouse TCR β chain (mVβ8.2) and staphylococcal enterotoxin G (SEG) at 2.0 Å resolution revealed a binding site that does not conserve the “hot spots” present in mVβ8.2-SEC2, mVβ8.2-SEC3, mVβ8.2-SEB, and mVβ8.2-SPEA complexes. Analysis of the mVβ8.2-SEG interface allowed us to explain the higher affinity of this complex compared with the others, which may account for the early activation of T-cells bearing mVβ8.2 by SEG. This mode of interaction between SEG and mVβ8.2 could be an adaptive advantage to bestow on the pathogen a faster rate of colonization of the host. PMID:21059660

  1. Comparative evaluation of the association among enumeration methods and production of enterotoxins in food-derived Staphylococcus aureus.

    PubMed

    Zhang, Chi; Zhang, Deqin; Yang, Jun; Zhou, Jungui; Hu, Qilong; Ling, Rui; Dong, Mingsheng

    2012-01-01

    Staphylococcal food poisoning is one of the most common foodborne diseases worldwide; it results from the ingestion of staphylococcal enterotoxins (SEs) in food, mainly Staphylococcus aureus. This study investigated the statistical relationships among morphological enumerations of food-derived S. aureus and production of SEs using different methodologies. Food samples naturally contaminated with coagulase-positive S. aureus were submitted for enumeration on Baird-Parker (BP) agar, Rabbit Plasma Fibrinogen agar (RPFA), and Petrifilm Staph Express count system (STX), and the morphologically typical colonies were isolated for VIDAS and real-time (RT) PCR tests. RPFA and STX displayed better performance for the enumeration of SE-positive S. aureus when compared with BP, including higher frequencies of SE-positive isolates and better correlation indices between typical and SE-positive counts. Among all the evaluated culture media, no significant difference (P > 0.05) was shown on the frequencies of typical colonies that carried 11 individual se genes. In addition, results for SE identification between VIDAS and RT-PCR assay were unparalleled. These data will be valuable for the selection of methods for inspection of food-derived S. aureus.

  2. Prevalence of multi-antimicrobial-agent resistant, shiga toxin and enterotoxin producing Escherichia coli in surface waters of river Ganga.

    PubMed

    Ram, Siya; Vajpayee, Poornima; Shanker, Rishi

    2007-11-01

    The consumption of polluted surface water for domestic and recreational purposes by large populations in developing nations is a major cause of diarrheal disease related mortality. The river Ganga and its tributaries meet 40% of the water requirement for drinking and irrigation in India. In this study, Escherichia coli isolates (n=75) of the river Ganga water were investigated for resistance to antimicrobial agents (n=15) and virulence genes specific to shiga toxin (STEC) and enterotoxin producing E. coli (ETEC). E. coli isolates from the river Ganga water exhibit resistance to multiple antimicrobial agents. The distribution of antimicrobial agent resistance in E. colivaries significantly (chi2: 81.28 at df = 24, p < 0.001) between the sites. Both stx1 and stx2 genes were present in 82.3% of STEC (n=17) while remaining isolates possess either stxl (11.8%) or stx2 (5.9%). The presence of eaeA, hlyA, and chuA genes was observed in 70.6, 88.2, and 58.8% of STEC, respectively. Both LT1 and ST1 genes were positive in 66.7% of ETEC (n=15) while 33.3% of isolates harbor only LT1 gene. The prevalence of multi-antimicrobial-agent resistant E. coli in the river Ganga water poses increased risk of infections in the human population. PMID:18044515

  3. Multiple-Strain Approach and Probabilistic Modeling of Consumer Habits in Quantitative Microbial Risk Assessment: A Quantitative Assessment of Exposure to Staphylococcal Enterotoxin A in Raw Milk.

    PubMed

    Crotta, Matteo; Rizzi, Rita; Varisco, Giorgio; Daminelli, Paolo; Cunico, Elena Cosciani; Luini, Mario; Graber, Hans Ulrich; Paterlini, Franco; Guitian, Javier

    2016-03-01

    Quantitative microbial risk assessment (QMRA) models are extensively applied to inform management of a broad range of food safety risks. Inevitably, QMRA modeling involves an element of simplification of the biological process of interest. Two features that are frequently simplified or disregarded are the pathogenicity of multiple strains of a single pathogen and consumer behavior at the household level. In this study, we developed a QMRA model with a multiple-strain approach and a consumer phase module (CPM) based on uncertainty distributions fitted from field data. We modeled exposure to staphylococcal enterotoxin A in raw milk in Lombardy; a specific enterotoxin production module was thus included. The model is adaptable and could be used to assess the risk related to other pathogens in raw milk as well as other staphylococcal enterotoxins. The multiplestrain approach, implemented as a multinomial process, allowed the inclusion of variability and uncertainty with regard to pathogenicity at the bacterial level. Data from 301 questionnaires submitted to raw milk consumers were used to obtain uncertainty distributions for the CPM. The distributions were modeled to be easily updatable with further data or evidence. The sources of uncertainty due to the multiple-strain approach and the CPM were identified, and their impact on the output was assessed by comparing specific scenarios to the baseline. When the distributions reflecting the uncertainty in consumer behavior were fixed to the 95th percentile, the risk of exposure increased up to 160 times. This reflects the importance of taking into consideration the diversity of consumers' habits at the household level and the impact that the lack of knowledge about variables in the CPM can have on the final QMRA estimates. The multiple-strain approach lends itself to use in other food matrices besides raw milk and allows the model to better capture the complexity of the real world and to be capable of geographical

  4. A study on the prevalence of Aeromonas spp. and its enterotoxin genes in samples of well water, tap water, and bottled water

    PubMed Central

    Didugu, Hareesh; Thirtham, Madhavarao; Nelapati, Krishnaiah; Reddy, K Kondal; Kumbhar, Baba Saheb; Poluru, Anusha; Pothanaboyina, Guruvishnu

    2015-01-01

    Aim: The aim of this work was to study the prevalence of Aeromonas spp. and its enterotoxin genes in various water sources. Materials and Methods: 125 samples (50 from well water, 50 from tap water, and 25 from bottled water) were collected from various sources in and around Greater Hyderabad Municipal Corporation and examined for the presence of aeromonads by both cultural and polymerase chain reaction (PCR) assay. Alkaline peptone water with ampicillin was used as enrichment. Aeromonas isolation medium and ampicillin dextrin agar were used as selective media. The boiling and snap chilling method was used for DNA extraction. Primers targeted against 16S rRNA, aer, and ast were used to identify aeromonads and its enterotoxins. Results: 48%, 18%, and 12% of well water, tap water, and bottled water samples were found positive by cultural assay with an overall prevalence of 28.8%. Aeromonads were detected in 32 % (52% in well water, 20% in tap water, and 16% in bottled water) of samples by PCR assay. Aerolysin (aer) gene was noticed in 34.6%, 20%, and 0% of well water, tap water, and bottled water samples, respectively, with an overall prevalence of 27.5%. Thermostable cytotonic enterotoxin (ast) was observed in 37.5% (42.3% in well water, 30% in tap water, and 25% in bottled mineral water) of samples. Conclusions: Presence of aeromonads and its toxin genes in various sources of water is of public health concern and emphasizes the need for necessary preventive measures to tackle the problem. PMID:27047024

  5. Detection of biofilm related genes, classical enterotoxin genes and agr typing among Staphylococcus aureus isolated from bovine with subclinical mastitis in southwest of Iran.

    PubMed

    Khoramrooz, Seyed Sajjad; Mansouri, Fariba; Marashifard, Masoud; Malek Hosseini, Seyed Ali Asghar; Akbarian Chenarestane-Olia, Fereshteh; Ganavehei, Banafsheh; Gharibpour, Farzaneh; Shahbazi, Ardavan; Mirzaii, Mehdi; Darban-Sarokhalil, Davood

    2016-08-01

    Staphylococcus aureus by producing biofilm and facilitating chronic infection is a common cause of mastitis in cows and thereby can cause food poisoning by production of enterotoxins in milk. The agr typing method is an important tool for epidemiological investigation about S. aureus. The aims of the present study were to detect biofilm related genes, 5 classical enterotoxin genes and the agr types among S. aureus isolates. The ability of S. aureus isolates to produce biofilm was evaluated by modified CRA plate. Six biofilm related adhesion genes (icaD, icaA, fnbA, bap, clfA and cna), five classical enterotoxin genes (sea, seb, sec, sed and see) and tst-1 gene were detected by PCR methods. Multiplex-PCR was used to determination of the agr groups. 55 out of 80(68.8%) S. aureus isolates were biofilm producer. The icaD gene was detected in 70 (87.5%) of isolates. The prevalence rates of fnbA, icaA, clfA, cna and bap were 72.5, 56.25, 50, 22.5, and 5% respectively. The agr group I and III were detected in 57.5% 25% of studied isolates. The sea, sed and tst-1 genes were found in 10%, 7.5% and 1.25% of isolates respectively. The majority of S. aureus were able to produce biofilm. Significant associations were observed between presence of the icaD, icaA, fnbA, clfA and the cna genes as well as biofilm formation. The present study revealed that isolates with the agr type III are more potent for biofilm production. Our data supported a possible link between the agr types and certain SE genes. PMID:27251096

  6. Interlaboratory Profiency Testing trial on the Detection of Staphylococcal Enterotoxins types SEA to SEE in food in Germany 2013.

    PubMed

    Fetsch, Alexandra; Steege, Katja; Leeser, Daniel; Krause, Gladys

    2016-01-01

    In the autumn 2013, the National Reference Laboratory for coagulase positive staphylococci (CPS) including Staphylococcus (S.) aureus (NRL-Staph) at the Federal Institute for Risk Assessment has organized its first interlaboratory profiency testing (ILPT) trial for the detection of staphylococcal enterotoxins (SE) types SEA to SEE in food. The purpose of the ILPT was to assess the analytical competence of the official laboratories of the Federal German "Länder"authorities. Moreover, it was the intention to gain an overview of the standard methods implemented in the participating laboratories for the purpose of SE detection in food. Five cream cheese samples at three different contamination levels (blank, low, and high) were sent to each participant. In total, 15 laboratories participated to the ILPT: 14 laboratories from 11 Federal German "Länder", and the European Reference Laboratory for CPS including S. aureus (EU-RL for CPS). Data sets from 14 participating laboratories were included in the analysis. Overall, a specificity of 100% (14/14 true negative results), a sensitivity of 55% (31/56 true positive results), and an accuracy of 64% (45/60 true results) was achieved. The majority of participants (9/15) used other analytical methods for the detection of SE in food than the suggested European Screening Method (ESM) v5. To conclude on the ILPT in general it is to state that the majority of participating laboratories failed to correctly identify SE-low-contaminated samples. Further efforts are necessary to improve the analytical capacity and sensitivity as regards the detection of SE in food in Germany. PMID:27529990

  7. Antibody Binding Studies Reveal Conformational Flexibility of the Bacillus cereus Non-Hemolytic Enterotoxin (Nhe) A-Component

    PubMed Central

    Märtlbauer, E.

    2016-01-01

    The non-hemolytic enterotoxin complex (Nhe) is supposed to be the main virulence factor of B. cereus causing a diarrheal outcome of food poisoning. This tripartite toxin consists of the single components NheA, -B and -C all of them being necessary for maximum toxicity. In the past, research activities aiming to elucidate the mode-of-action of Nhe were mostly focused on the B- and C-component. In this study the generation of novel monoclonal antibodies (mAb) and their thorough characterization enabled the determination of key features for NheA. By the means of immunoaffinity chromatography it could be shown that NheA does not interact with -B and -C in solution. Additionally, the establishment of a highly sensitive sandwich-EIA now enables the detection of NheA in B. cereus supernatants down to 20 pg ml-1.Peptide-based epitope mapping in combination with partially deleted recombinant NheA fragments allowed the allocation of the binding regions for the three mAbs under study. Furthermore, by different EIA set-ups the conformational flexibility of NheA could be shown. For two of the antibodies under study different mechanisms of NheA neutralization were proven. Due to prevention of complete pore formation by one of the antibodies, NheA could be detected in an intermediate stage of the tripartite complex on the cell surface. Taken together, the results obtained in the present study allow a refinement of the mode-of-action for the Nhe toxin-complex. PMID:27768742

  8. Screening of tea extract and theaflavins for inhibitory effects on the biological activity and production of staphylococcal enterotoxin A.

    PubMed

    Shimamura, Yuko; Aoki, Natsumi; Sugiyama, Yuka; Nakayama, Tsutomu; Masuda, Shuichi

    2014-11-01

    This study aimed to develop a novel method with tea extracts and its components, to reduce the risk of foodborne illnesses caused by the bacterial toxin staphylococcal enterotoxin A (SEA). The potential effect of tea extracts, theaflavins, and epitheaflagallin on staphylococcal growth was studied. A broth microdilution method was used to determine the minimum inhibitory concentration of these samples against an SEA-producing strain, Staphylococcus aureus C-29. The following assays were performed to evaluate various effects on concentrations of no effect on staphylococcal growth. The interactions of theaflavin-rich green tea extracts (TGE), theaflavins, and epitheaflagallin to cultured S. aureus C-29 were determined using Western blot analysis. As a result, all samples suppressed the binding affinity of the anti-SEA antibody to SEA. Since these samples could react directly with SEA, we examined whether they could bind to SEA. Our results demonstrated that binding of the anti-SEA antibody to 4 theaflavins-treated SEA was inhibited in a dose-dependent manner. On the other hand, the production of SEA was significantly decreased by treatment with TGE and epitheaflagallin. Based on the finding that TGE and epitheaflagallin inhibit the production of SEA, we further examined the relative expression levels of sea toxin-encoding genes after treatment with TGE and epitheaflagallin with real-time RT-PCR. TGE and epitheaflagallin significantly supressed the gene transcription of SEA in S. aureus C-29. We then tested whether the samples block the biological activity of SEA in murine spleen cells. TGE, theaflavins, and epitheaflagallin became inactivated the biological activity of SEA. These results suggest that edible and safe compounds in tea can be used to inactivate both pathogens and toxins.

  9. Shigella enterotoxin-2 is a type III effector that participates in Shigella-induced interleukin 8 secretion by epithelial cells.

    PubMed

    Farfán, Mauricio J; Toro, Cecilia S; Barry, Eileen M; Nataro, James P

    2011-04-01

    We have previously described a protein termed Shigella enterotoxin 2 (ShET-2), which induces rises in short-circuit current in rabbit ileum mounted in the Ussing chamber. Published reports have postulated that ShET-2 may be secreted by the Shigella type III secretion system (T3SS). In this study, we show that ShET-2 secretion into the extracellular space requires the T3SS in Shigella flexneri 2a strain 2457T and a ShET-2-TEM fusion was translocated into epithelial cells in a T3SS-dependent manner. The ShET-2 gene, sen, is encoded downstream of the ospC1 gene of S. flexneri, and we show that sen is cotranscribed with this T3SS-secreted product. Considering that T3SS effectors have diverse roles in Shigella infection and that vaccine constructs lacking ShET-2 are attenuated in volunteers, we asked whether ShET-2 has a function other than its enterotoxic activity. We constructed a ShET-2 mutant in 2457T and tested its effect on epithelial cell invasion, plaque formation, guinea pig keratoconjunctivitis and interleukin 8 (IL-8) secretion from infected monolayers. Although other phenotypes were not different compared with the wild-type parent, we found that HEp-2 and T84 cells infected with the ShET-2 mutant exhibited significantly reduced IL-8 secretion into the basolateral compartment, suggesting that ShET-2 might participate in the Shigella-induced inflammation of epithelial cells.

  10. Spray-dried animal plasma prevents the effects of Staphylococcus aureus enterotoxin B on intestinal barrier function in weaned rats.

    PubMed

    Pérez-Bosque, Anna; Amat, Concepció; Polo, Javier; Campbell, Joy M; Crenshaw, Joe; Russell, Louis; Moretó, Miquel

    2006-11-01

    In this study, we investigated intestinal barrier function during inflammation as well as the effects of dietary supplementation with porcine spray-dried animal plasma (SDAP) proteins and porcine immunoglobulin concentrate (IC). Wistar Lewis rats were fed from d 21 (weaning) until d 34 or 35 either a control diet or a diet containing SDAP or IC. On d 30 and d 33, rats received an intraperitoneal dose of Staphylococcus aureus enterotoxin B (SEB; 0.5 mg/kg body wt; groups SEB, SEB-SDAP, and SEB-IC). SEB reduced the potential difference across the jejunum by 60%, the short-circuit current by 70%, and Na-K-ATPase activity in intestinal mucosa (all P < 0.05). The fluxes of dextran flux (4 kDa) and horseradish peroxidase (HRP, 40 kDa) across the intestinal wall also increased in SEB-treated rats (P < 0.01, P = 0.068, respectively). SEB also increased HRP flux across the paracellular space (P < 0.05). Moreover, SEB-treated rats had a reduced expression of tight junction proteins, such as ZO-1 (10% reduction; P < 0.05) and beta-catenin (20% reduction; P < 0.05). Dietary supplementation with SDAP or IC prevented dextran (P < 0.05) and HRP (P < 0.05) paracellular flux across the intestinal epithelium. SDAP supplementation also prevented SEB effects on Na-K-ATPase activity (P < 0.05). In our model of SEB-induced intestinal inflammation, the increased permeability across the intestinal mucosa was due to the lower expression of tight junction proteins, an effect that can be prevented by both SDAP and IC supplementation.

  11. Review Over a 3-Year Period of European Union Proficiency Tests for Detection of Staphylococcal Enterotoxins in Food Matrices.

    PubMed

    Nia, Yacine; Mutel, Isabelle; Assere, Adrien; Lombard, Bertrand; Auvray, Frederic; Hennekinne, Jacques-Antoine

    2016-04-01

    Staphylococcal food poisoning outbreaks are a major cause of foodborne illnesses in Europe and their notifications have been mandatory since 2005. Even though the European regulation on microbiological criteria for food defines a criterion on staphylococcal enterotoxin (SE) only in cheese and dairy products, European Food Safety Authority (EFSA) data reported that various types of food matrices are involved in staphylococcal food poisoning outbreaks. The European Screening Method (ESM) of European Union Reference Laboratory for Coagulase Positive Staphylococci (EURL CPS) was validated in 2011 for SE detection in food matrices and is currently the official method used for screening purposes in Europe. In this context, EURLCPS is annually organizing Inter-Laboratory Proficiency Testing Trials (ILPT) to evaluate the competency of the European countries' National Reference Laboratories (NRLs) to analyse SE content in food matrices. A total of 31 NRLs representing 93% of European countries participated in these ILPTs. Eight food matrices were used for ILPT over the period 2013-2015, including cheese, freeze-dried cheese, tuna, mackerel, roasted chicken, ready-to-eat food, milk, and pastry. Food samples were spiked with four SE types (i.e., SEA, SEC, SED, and SEE) at various concentrations. Homogeneity and stability studies showed that ILPT samples were both homogeneous and stable. The analysis of results obtained by participants for a total of 155 blank and 620 contaminated samples allowed for evaluation of trueness (>98%) and specificity (100%) of ESM. Further to the validation study of ESM carried out in 2011, these three ILPTs allowed for the assessment of the proficiency of the NRL network and the performance of ESM on a large variety of food matrices and samples. The ILPT design presented here will be helpful for the organization of ILPT on SE detection by NRLs or other expert laboratories. PMID:27089364

  12. Identification of HLA-DR1 beta chain residues critical for binding staphylococcal enterotoxins A and E

    PubMed Central

    1992-01-01

    Superantigens are thought to make external contacts with major histocompatibility complex (MHC) class II molecules and with the V beta portion of a T cell antigen receptor (TCR), thereby stimulating entire families of T cells. The precise mapping of superantigen binding sites on class II molecules may provide valuable information on how TCR and MHC molecules interact. Two bacterial superantigens, staphylococcal enterotoxins A and E (SEA/SEE) bind well to most HLA-DR alleles, but poorly to HLA-DRw53. The sequences responsible for this binding were localized to the putative alpha helix of the DR beta chain by measuring toxin binding to a panel of chimeric class II molecules expressed on transfected cells. Binding of SEA/SEE to the DRw14 (Dw9) molecule suggested that the conserved histidine 81 in the beta chain of most DR molecules was important, whereas the tyrosine 81 in the DRw53 beta chain was detrimental for high-affinity binding. To prove this, reciprocal point mutations were introduced in the DR1 and DRw53 beta chains. Mutation of histidine 81 in the DR1 beta chain to tyrosine reduced SEA/SEE binding, but did not prevent recognition of two DR1- restricted peptides by six of eight antigen-specific T cell lines. Conversely, introduction to histidine at position 81 in the DRw53 beta chain restored normal levels of SEA/SEE binding. These data suggest that a binding site of SEA and SEE lies on the outer face of the beta chain alpha helix, pointing away from the antigen-binding groove. PMID:1370684

  13. Interlaboratory Profiency Testing trial on the Detection of Staphylococcal Enterotoxins types SEA to SEE in food in Germany 2013.

    PubMed

    Fetsch, Alexandra; Steege, Katja; Leeser, Daniel; Krause, Gladys

    2016-01-01

    In the autumn 2013, the National Reference Laboratory for coagulase positive staphylococci (CPS) including Staphylococcus (S.) aureus (NRL-Staph) at the Federal Institute for Risk Assessment has organized its first interlaboratory profiency testing (ILPT) trial for the detection of staphylococcal enterotoxins (SE) types SEA to SEE in food. The purpose of the ILPT was to assess the analytical competence of the official laboratories of the Federal German "Länder"authorities. Moreover, it was the intention to gain an overview of the standard methods implemented in the participating laboratories for the purpose of SE detection in food. Five cream cheese samples at three different contamination levels (blank, low, and high) were sent to each participant. In total, 15 laboratories participated to the ILPT: 14 laboratories from 11 Federal German "Länder", and the European Reference Laboratory for CPS including S. aureus (EU-RL for CPS). Data sets from 14 participating laboratories were included in the analysis. Overall, a specificity of 100% (14/14 true negative results), a sensitivity of 55% (31/56 true positive results), and an accuracy of 64% (45/60 true results) was achieved. The majority of participants (9/15) used other analytical methods for the detection of SE in food than the suggested European Screening Method (ESM) v5. To conclude on the ILPT in general it is to state that the majority of participating laboratories failed to correctly identify SE-low-contaminated samples. Further efforts are necessary to improve the analytical capacity and sensitivity as regards the detection of SE in food in Germany.

  14. Review Over a 3-Year Period of European Union Proficiency Tests for Detection of Staphylococcal Enterotoxins in Food Matrices

    PubMed Central

    Nia, Yacine; Mutel, Isabelle; Assere, Adrien; Lombard, Bertrand; Auvray, Frederic; Hennekinne, Jacques-Antoine

    2016-01-01

    Staphylococcal food poisoning outbreaks are a major cause of foodborne illnesses in Europe and their notifications have been mandatory since 2005. Even though the European regulation on microbiological criteria for food defines a criterion on staphylococcal enterotoxin (SE) only in cheese and dairy products, European Food Safety Authority (EFSA) data reported that various types of food matrices are involved in staphylococcal food poisoning outbreaks. The European Screening Method (ESM) of European Union Reference Laboratory for Coagulase Positive Staphylococci (EURL CPS) was validated in 2011 for SE detection in food matrices and is currently the official method used for screening purposes in Europe. In this context, EURLCPS is annually organizing Inter-Laboratory Proficiency Testing Trials (ILPT) to evaluate the competency of the European countries’ National Reference Laboratories (NRLs) to analyse SE content in food matrices. A total of 31 NRLs representing 93% of European countries participated in these ILPTs. Eight food matrices were used for ILPT over the period 2013–2015, including cheese, freeze-dried cheese, tuna, mackerel, roasted chicken, ready-to-eat food, milk, and pastry. Food samples were spiked with four SE types (i.e., SEA, SEC, SED, and SEE) at various concentrations. Homogeneity and stability studies showed that ILPT samples were both homogeneous and stable. The analysis of results obtained by participants for a total of 155 blank and 620 contaminated samples allowed for evaluation of trueness (>98%) and specificity (100%) of ESM. Further to the validation study of ESM carried out in 2011, these three ILPTs allowed for the assessment of the proficiency of the NRL network and the performance of ESM on a large variety of food matrices and samples. The ILPT design presented here will be helpful for the organization of ILPT on SE detection by NRLs or other expert laboratories. PMID:27089364

  15. The structure of Escherichia coli heat-stable enterotoxin b by nuclear magnetic resonance and circular dichroism.

    PubMed Central

    Sukumar, M.; Rizo, J.; Wall, M.; Dreyfus, L. A.; Kupersztoch, Y. M.; Gierasch, L. M.

    1995-01-01

    The heat-stable enterotoxin b (STb) is secreted by enterotoxigenic Escherichia coli that cause secretory diarrhea in animals and humans. It is a 48-amino acid peptide containing two disulfide bridges, between residues 10 and 48 and 21 and 36, which are crucial for its biological activity. Here, we report the solution structure of STb determined by two- and three-dimensional NMR methods. Approximate interproton distances derived from NOE data were used to construct structures of STb using distance-geometry and simulated annealing procedures. The NMR-derived structure shows that STb is helical between residues 10 and 22 and residues 38 and 44. The helical structure in the region 10-22 is amphipathic and exposes several polar residues to the solvent, some of which have been shown to be important in determining the toxicity of STb. The hydrophobic residues on the opposite face of this helix make contacts with the hydrophobic residues of the C-terminal helix. The loop region between residues 21 and 36 has another cluster of hydrophobic residues and exposes Arg 29 and Asp 30, which have been shown to be important for intestinal secretory activity. CD studies show that reduction of disulfide bridges results in a dramatic loss of structure, which correlates with loss of function. Reduced STb adopts a predominantly random-coil conformation. Chromatographic measurements of concentrations of native, fully reduced, and single-disulfide species in equilibrium mixtures of STb in redox buffers indicate that the formation of the two disulfide bonds in STb is only moderately cooperative. Similar measurements in the presence of 8 M urea suggest that the native secondary structure significantly stabilizes the disulfide bonds. PMID:8528070

  16. Influence of carvacrol and thymol on the physiological attributes, enterotoxin production and surface characteristics of Staphylococcus aureus strains isolated from foods

    PubMed Central

    Souza, E.L.; Oliveira, C.E.V.; Stamford, T.L.M.; Conceição, M.L.; Neto, N.J. Gomes

    2013-01-01

    This study evaluated the influence of the phenolic compounds carvacrol (CAR) and thymol (THY) on some physiological characteristics and on the modulation of the secretion of some staphylococcal virulence factors, that is, coagulase and enterotoxin. This study also investigated possible mechanisms for the establishment of the anti-staphylococcal activity of these compounds. Sublethal concentrations (0.3 and 0.15 μL/mL) of CAR and THY inhibited the activity of the enzymes coagulase and lipase and led to a decrease in salt tolerance. At the tested sublethal concentrations, both CAR and THY led to a total suppression of enterotoxin production. The loss of a 260-nm-absorbing material and an efflux of potassium ions occurred immediately after the addition of CAR and THY at 0.6 and 1.2 μL/mL and increased up to 120 min of exposure. Electron microscopy of cells exposed to CAR and THY (0.6 μL/mL) revealed that individual cells appeared to be deformed, with projections of cellular material. The observations of leakage of cellular material and an altered cell surface suggest that gross damage to a cell’s cytoplasmic membrane, which results in a disruption in protein secretion, could be responsible for the anti-staphylococcal properties of CAR and THY. PMID:24159280

  17. Prevalence of genes for enterotoxins, toxic shock syndrome toxin 1 and exfoliative toxin among clinical isolates of Staphylococcus pseudintermedius from canine origin.

    PubMed

    Yoon, Jang W; Lee, Gi-Jong; Lee, So-Young; Park, Chul; Yoo, Jong-Hyun; Park, Hee-Myung

    2010-10-01

    A total of 74 Staphylococcus pseudintermedius strains were isolated from the 99 clinical cases of canine pyoderma or chronic otitis in our veterinary teaching hospital during May 2006-February 2008. In this study, we examined the genetic distribution of staphylococcal pyogenic toxins such as staphylococcal enterotoxins A (sea), B (seb), C (sec), D (sed), E (see), and toxic shock syndrome toxin 1 (tst) as well as the previously characterized S. intermedius exfoliative toxin (siet) among those isolates. The polymerase chain reaction analyses with the toxin gene-specific primers revealed that 18 (24.3%) of 74 S. pseudintermedius isolates carried the sec genes, but none of the sea, seb, sed, see and tst genes. Further DNA sequencing analysis of the amplified sec genes revealed that they all belonged to the canine type C staphylococcal enterotoxin (SEC(canine) ) whose superantigenic activity has been demonstrated. In addition to the sec(canine) genes, our polymerase chain reaction results showed that all the 74 isolates carried the siet gene. Since both SEC(canine) and SIET toxins are known to be biologically active, it would be interesting to investigate how those toxins are involved in the pathogenesis of the canine diseases by S. pseudintermedius such as pyoderma or chronic otitis.

  18. Modulation of Endotoxin- and Enterotoxin-Induced Cytokine Release by In Vivo Treatment with β-(1,6)-Branched β-(1,3)-Glucan

    PubMed Central

    Soltys, Jindrich; Quinn, Mark T.

    1999-01-01

    Leukocytes activated by endotoxin or enterotoxins release proinflammatory cytokines, thereby contributing to the cascade of events leading to septic shock. In the present studies, we analyzed the effects of in vivo administration of a soluble immunomodulator, β-(1,6)-branched β-(1,3)-glucan (soluble β-glucan), on toxin-stimulated cytokine production in monocytes and lymphocytes isolated from treated mice. In vitro stimulation of lymphocytes isolated from soluble β-glucan-treated mice with lipopolysaccharide (LPS) resulted in enhanced production of interleukin-6 (IL-6) and suppressed production of tumor necrosis factor alpha (TNF-α), while stimulation of these cells with staphylococcal enterotoxin B (SEB) or toxic shock syndrome toxin 1 (TSST-1) resulted in enhanced production of gamma interferon (IFN-γ) and suppressed production of IL-2 and TNF-α compared to that in cells isolated from untreated mice. In vitro stimulation of monocytes isolated from soluble β-glucan-treated mice with LPS also resulted in suppressed TNF-α production, while stimulation of these cells with SEB or TSST-1 resulted in suppressed IL-6 and TNF-α production compared to that in cells isolated from untreated mice. Thus, the overall cytokine pattern of leukocytes from soluble β-glucan-treated mice reflects suppressed production of proinflammatory cytokines, especially TNF-α. Taken together, our results suggest that treatment with soluble β-glucan can modulate the induction cytokines during sepsis, resulting in an overall decrease in host mortality. PMID:9864222

  19. Prevalence and diversity of enterotoxin genes with genetic background of Staphylococcus aureus isolates from different origins in China.

    PubMed

    Chao, Guoxiang; Bao, Guangyu; Cao, Yongzhong; Yan, Wenguang; Wang, Yan; Zhang, Xiaorong; Zhou, Liping; Wu, Yantao

    2015-10-15

    Staphylococcal enterotoxins (SE) induce toxin-mediated diseases, such as food poisoning. In the present study, 568 isolates from different sources were tested for the prevalence of 18 SE genes and performed spa typing. In addition, we characterized the relationships between the distribution of SE genes and molecular clones based on multilocus sequence typing (MLST), spa and staphylococcal cassette chromosome mec (SCCmec) typing in selected 250 isolates. Approximately 54.40% of the isolates from different sources harbored one or more SE genes forming 120 distinct gene profiles. Seven genes, sea, seb, seg, seo, sem, seq, and sel were more frequently detected. The distributions of the SE genes among the isolates from human, animals, and foodborne origins were highly different with isolates from environments (P<0.01). The classic SE genes in both foodborne and human origin isolates were significantly higher than that in animal origin isolates (P<0.01), whereas the prevalence of genes of egc cluster and the other genes was similar in human, animal, and foodborne origin isolates (P>0.05). We identified two important gene clusters, sea-sek-seq, which is closely related to hospital-acquired (HA) methicillin-resistant Staphylococcus aureus (MRSA)-III, and the egc cluster, which accounts for nearly half of all genes. Approximately 71% isolates could be typed by spa, yielding 103 spa types, of which 18 spa types were primary types. In clonal complex (CC) 239, an important Asian HA-MRSA-III clone from humans, nearly all isolates harbored complete or partial sea-sek-seq cluster; the main spa types were t030 and t037. In CC630, an important new community-associated (CA) MRSA-V CC in China, only sporadic SE genes, three main spa types, t4549, t2196, and t377 were observed. The egc cluster coexisting with other genes was present in isolates of CC5, CC9, CC1281, CC1301, CC30 and sequence type (ST) 25, but completely absent in isolates of CC239, CC59, CC7, and CC88. The results

  20. Comparative evaluation of three 64Cu-labeled E. coli heat-stable enterotoxin analogues for PET imaging of colorectal cancer.

    PubMed

    Liu, Dijie; Overbey, Douglas; Watkinson, Lisa D; Smith, Charles J; Daibes-Figueroa, Said; Hoffman, Timothy J; Forte, Leonard R; Volkert, Wynn A; Giblin, Michael F

    2010-07-21

    Analogues of the E. coli heat-stable enterotoxin (STh) are currently under study as both imaging and therapeutic agents for colorectal cancer. Studies have shown that the guanylate cyclase C (GC-C) receptor is commonly expressed in colorectal cancers. It has also been shown that STh peptides inhibit the growth of tumor cells expressing GC-C. The ability to determine GC-C status of tumor tissue using in vivo molecular imaging techniques would provide a useful tool for the optimization of GC-C-targeted therapeutics. In this work, we have compared receptor binding affinities, internalization/efflux rates, and in vivo biodistribution patterns of an STh analogue linked to N-terminal DOTA, TETA, and NOTA chelating moieties and radiolabeled with Cu-64. The peptide F(19)-STh(2-19) was N-terminally labeled with three different chelating groups via NHS ester activation and characterized by RP-HPLC, ESI-MS, and GC-C receptor binding assays. The purified conjugates were radiolabeled with Cu-64 and used for in vitro internalization/efflux, in vivo biodistribution, and in vivo PET imaging studies. In vivo experiments were carried out using SCID mice bearing T84 human colorectal cancer tumor xenografts. Incorporation of DOTA-, TETA-, and NOTA-chelators at the N-terminus of the peptide F(19)-STh(2-19) resulted in IC(50)s between 1.2 and 3.2 nM. In vivo, tumor localization was similar for all three compounds, with 1.2-1.3%ID/g at 1 h pi and 0.58-0.83%ID/g at 4 h pi. The principal difference between the three compounds related to uptake in nontarget tissues, principally kidney and liver. At 1 h pi, (64)Cu-NOTA-F(19)-STh(2-19) demonstrated significantly (p < 0.05) lower uptake in liver than (64)Cu-DOTA-F(19)-STh(2-19) (0.36 +/- 0.13 vs 1.21 +/- 0.65%ID/g) and significantly (p < 0.05) lower uptake in kidney than (64)Cu-TETA-F(19)-STh(2-19) (3.67 +/- 1.60 vs 11.36 +/- 2.85%ID/g). Use of the NOTA chelator for coordination of Cu-64 in the context of E. coli heat-stable enterotoxin

  1. The J beta segment of the T cell receptor contributes to the V beta-specific T cell expansion caused by staphylococcal enterotoxin B and Urtica dioica superantigens.

    PubMed

    Musette, P; Galelli, A; Truffa-Bachi, P; Peumans, W; Kourilsky, P; Gachelin, G

    1996-03-01

    We have used a new polymerase chain reaction-based technique to analyze at the clonal level the CDR3 diversity and the J beta usage associated with the V beta-dependent T cell receptor (TCR) recognition of two superantigens: the staphylococcal enterotoxin B and the Urtica dioica agglutinin. Our results show that subset of J beta elements is preferentially expanded in a given V beta family, independently of the nature of the superantigen. By contrast, the CDR3 loop does not contribute significantly to the T cell expansion induced by the superantigens. We conclude that the J beta segment of the TCR beta chain, but not the CDR3 region, participates in superantigen binding, presumably by influencing the quaternary structure of the TCR beta chain. PMID:8605929

  2. Quantitative Analysis of Staphylococcal Enterotoxins A and B in Food Matrices Using Ultra High-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS)

    PubMed Central

    Zuberovic Muratovic, Aida; Hagström, Thomas; Rosén, Johan; Granelli, Kristina; Hellenäs, Karl-Erik

    2015-01-01

    A method that uses mass spectrometry (MS) for identification and quantification of protein toxins, staphylococcal enterotoxins A and B (SEA and SEB), in milk and shrimp is described. The analysis was performed using a tryptic peptide, from each of the toxins, as the target analyte together with the corresponding 13C-labeled synthetic internal standard peptide. The performance of the method was evaluated by analyzing spiked samples in the quantification range 2.5–30 ng/g (R2 = 0.92–0.99). The limit of quantification (LOQ) in milk and the limit of detection (LOD) in shrimp was 2.5 ng/g, for both SEA and SEB toxins. The in-house reproducibility (RSD) was 8%–30% and 5%–41% at different concentrations for milk and shrimp, respectively. The method was compared to the ELISA method, used at the EU-RL (France), for milk samples spiked with SEA at low levels, in the quantification range of 2.5 to 5 ng/g. The comparison showed good coherence for the two methods: 2.9 (MS)/1.8 (ELISA) and 3.6 (MS)/3.8 (ELISA) ng/g. The major advantage of the developed method is that it allows direct confirmation of the molecular identity and quantitative analysis of SEA and SEB at low nanogram levels using a label and antibody free approach. Therefore, this method is an important step in the development of alternatives to the immune-assay tests currently used for staphylococcal enterotoxin analysis. PMID:26378579

  3. Protective effects of recombinant staphylococcal enterotoxin type C mutant vaccine against experimental bovine infection by a strain of Staphylococcus aureus isolated from subclinical mastitis in dairy cattle.

    PubMed

    Chang, Byoung Sun; Moon, Jin San; Kang, Hyun-Mi; Kim, Young-In; Lee, Hong-Kyun; Kim, Jong-Duk; Lee, Byung-Saeng; Koo, Hye Cheong; Park, Yong Ho

    2008-04-16

    Staphylococcus aureus is one of the main etiological agents of bovine mastitis; however, antibiotics that are effective against bovine strains of S. aureus are not currently available. Staphylococcal enterotoxin type C (SEC), a superantigen, is the enterotoxin most frequently expressed by bovine strains of S. aureus and one of immunogenic determinants. The purpose of this study was to evaluate the protective effectiveness of recombinant SEC mutant vaccine (MastaVactrade mark) against experimentally induced bovine infection. Three representative SEC secreting strains were selected from 9 candidate isolates that showed various intensities of pathogenicity on mice and inoculated into 5 lactating dairy cattle at a concentration of 50-5.0x10(8) CFU per quarter. The optimal experimental bovine subclinical mastitis model was produced by inoculation with 50 CFU of S. aureus 409 per quarter, a level which was not lethal to mice. After the experimental model was determined, other 3 cattle were intramuscularly administered three doses of vaccine at day 0, at 2 wks and at 6 wks. Nine quarters of 3 vaccinated cattle and 8 quarters of 3 control cattle were then challenged with S. aureus 409. An SEC-specific ELISA test conducted at 4 wks post-immunization confirmed the presence of a high antibody titer against SEC in all vaccinated cattle. The somatic cell counts from the vaccinated group remained relatively low, whereas those of control group increased significantly after challenge with S. aureus. After challenge, S. aureus was not isolated from any cattle in the vaccinated group, whereas it was isolated from 75% of the cattle in the control group. These results indicate that recombinant SEC mutant vaccine had a protective effect against S. aureus intramammary infection in lactating cattle.

  4. Nucleotide sequence of yst, the Yersinia enterocolitica gene encoding the heat-stable enterotoxin, and prevalence of the gene among pathogenic and nonpathogenic yersiniae.

    PubMed

    Delor, I; Kaeckenbeeck, A; Wauters, G; Cornelis, G R

    1990-09-01

    The gene encoding the heat-stable enterotoxin (yst) was cloned from the chromosome of Yersinia enterocolitica W1024 (serotype O:9), and the nucleotide sequence was determined. The yst gene encodes a 71-amino-acid polypeptide. The C-terminal 30 amino acids of the predicted protein exactly correspond to the amino acid sequence of the toxin extracted from culture supernatants (T. Takao, N. Tominaga, and Y. Shimonishi, Biochem. Biophys. Res. Commun. 125:845-851, 1984). The N-terminal 18 amino acids have the properties of a signal sequence. The central 22 residues are removed during or after the secretion process. This organization in three domains (Pre, Pro, and mature Yst) resembles that of the enterotoxin STa of Escherichia coli. The degree of conservation between the E. coli and Y. enterocolitica toxins is much lower in the Pre and the Pro domains than in the mature proteins. The mature toxin of Y. enterocolitica is much larger than that of E. coli, but the active domain appears to be highly conserved. The yst gene of Y. enterocolitica introduced in E. coli K-12 directed the secretion of an active toxin. The cloned yst gene was used as an epidemiological probe among a collection of 174 strains representative of all Yersinia species except Yersinia pestis and numerous Y. enterocolitica subgroups. In Y. enterocolitica, there was a clear-cut difference between pathogenic and nonpathogenic strains: 89 of 89 pathogenic and none of 51 nonpathogenic strains contained yst-homologous DNA, suggesting that Yst is involved in pathogenesis. Among the other Yersinia species, only four strains of Yersinia kristensenii had DNA homologous to yst.

  5. Nucleotide sequence of yst, the Yersinia enterocolitica gene encoding the heat-stable enterotoxin, and prevalence of the gene among pathogenic and nonpathogenic yersiniae.

    PubMed Central

    Delor, I; Kaeckenbeeck, A; Wauters, G; Cornelis, G R

    1990-01-01

    The gene encoding the heat-stable enterotoxin (yst) was cloned from the chromosome of Yersinia enterocolitica W1024 (serotype O:9), and the nucleotide sequence was determined. The yst gene encodes a 71-amino-acid polypeptide. The C-terminal 30 amino acids of the predicted protein exactly correspond to the amino acid sequence of the toxin extracted from culture supernatants (T. Takao, N. Tominaga, and Y. Shimonishi, Biochem. Biophys. Res. Commun. 125:845-851, 1984). The N-terminal 18 amino acids have the properties of a signal sequence. The central 22 residues are removed during or after the secretion process. This organization in three domains (Pre, Pro, and mature Yst) resembles that of the enterotoxin STa of Escherichia coli. The degree of conservation between the E. coli and Y. enterocolitica toxins is much lower in the Pre and the Pro domains than in the mature proteins. The mature toxin of Y. enterocolitica is much larger than that of E. coli, but the active domain appears to be highly conserved. The yst gene of Y. enterocolitica introduced in E. coli K-12 directed the secretion of an active toxin. The cloned yst gene was used as an epidemiological probe among a collection of 174 strains representative of all Yersinia species except Yersinia pestis and numerous Y. enterocolitica subgroups. In Y. enterocolitica, there was a clear-cut difference between pathogenic and nonpathogenic strains: 89 of 89 pathogenic and none of 51 nonpathogenic strains contained yst-homologous DNA, suggesting that Yst is involved in pathogenesis. Among the other Yersinia species, only four strains of Yersinia kristensenii had DNA homologous to yst. Images PMID:2201642

  6. Prevalence and Characteristics of Enterotoxin B-Producing Staphylococcus aureus Isolated from Food Sources: A Particular Cluster of ST188 Strains was Identified.

    PubMed

    Song, Qifa; Zhu, Zhenhua; Chang, Yanzi; Shen, Xuanyi; Gao, Hong; Yang, Yuanbin

    2016-03-01

    The aim of this study was to investigate the prevalence and characteristics of staphylococcal enterotoxin B (SEB) producing Staphylococcus aureus (S. aureus) isolated from food sources. A total of 412 S. aureus isolates were recovered from 1970 milk and dairy samples (n = 236) and 2450 meat samples (n = 176) in China from 2009 to 2014. Of the 412 isolates, 124 isolates were tested positive for 1 or more classical staphylococcal enterotoxin (SE) genes using PCR, and 31 isolates were positive for seb gene and further proved to be SEB-producing. Four SE profiles were observed among 31 SEB-producing isolates when investigated using ELISA kit, that is, SEB (16 isolates), SEA+SEB (6 isolates), SEB+SEC (6 isolates), and SEB+SED (3 isolates). Thirteen sequence types (STs) were identified in the 31 SEB-producing S. aureus isolates using multilocus sequence typing (MLST). The 3 most detected STs were ST1 (7 isolates), ST188 (6 isolates), ST59 (3 isolates). Two distinct clusters were identified by pulsed-field gel electrophoresis (PFGE), each of which showed excellent consistency with ST188 and ST1 achieved by MLST, respectively. In summary, this study reveals that various SE profiles are observed in SEB-producing S. aureus isolates and the great part of SEB-producing S. aureus isolates are showed as clusters. Especially, a particular cluster of ST188 strains was observed in SEB-producing S. aureus isolates which was associated with outbreaks of SFP and needs further attention.

  7. Epitope mapping and characterization of antigenic determinants of heat-stable enterotoxin (STh) of enterotoxigenic Escherichia coli by using monoclonal antibodies.

    PubMed Central

    Takeda, T; Nair, G B; Suzuki, K; Zhe, H X; Yokoo, Y; De Mol, P; Hemelhof, W; Butzler, J P; Takeda, Y; Shimonishi, Y

    1993-01-01

    A panel of monoclonal antibodies (MAbs) specific for the heat-stable enterotoxin (STh) of enterotoxigenic Escherichia coli was produced. All four MAbs (8G7, 53-4, 11C, and SH1) bound to native STh in an enzyme-linked immunosorbent assay to various degrees, with clone SH1 showing the best affinity. The MAbs were screened for neutralizing and guanylate cyclase-inhibiting activities by the suckling mouse assay and the cyclic GMP assay using T84 cells, respectively. The contact amino acid residues governing the reactivity of the four MAbs were precisely determined by using several chemically synthesized analogs of the various heat-stable enterotoxins (STa's). Three distinct antigenic sites of STh sufficiently removed from each other, one near the N terminus, another in the core functional region of the toxin, and the third in the C-terminal region, were recognized by the different MAbs. MAb SH1, which recognized Asn at position 4 and Tyr at position 5 from the N terminus was 100 times more potent in neutralizing the bioactivity of STh in the suckling mouse assay than was MAb 11C, which recognized Thr at position 16 and Tyr at position 19 from the N terminus of the STh molecule. The MAbs which recognized Leu at position 9 from the N terminus (MAb 53-4) and Tyr at position 19 from the N terminus (MAb 8G7) showed intermediate activities in the neutralization assay. The guanylate cyclase-inhibiting activities of SH1 and 11C essentially paralleled the results for the neutralization of bioactivity, while MAbs 53-4 and 8G7 exhibited reverse activity. These results indicate that MAbs that recognize the N-terminal residues which have been shown not to be essential for toxic activity have a potent protective capacity. None of the MAbs reacted with reduced and carboxy-methylated native STh. This suggests that all of the MAbs mediate their effect by reacting with conformation-dependent antigenic determinants. PMID:7678100

  8. Characteristics of enterotoxin distribution, hemolysis, lecithinase, and starch hydrolysis of Bacillus cereus isolated from infant formulas and ready-to-eat foods.

    PubMed

    Hwang, Ji-Yeon; Park, Jong-Hyun

    2015-03-01

    Bacillus cereus is a ubiquitous environmental microbe implicated as a main cause of food poisoning with various symptoms, depending on the strain type and the isolation source. In this study, the potential virulence factors and biochemical properties of B. cereus isolated from infant formulas and ready-to-eat (RTE) foods were analyzed and compared. A total of 347 B. cereus strains were isolated and identified from 687 infant food formulas and RTE food samples. All the isolates had one or more enterotoxin genes, and one-half of the strains had all 3 enterotoxin genes (hbl, nhe, and cytK) that are involved in food poisoning in humans. Here, all the 3 genes were detected in 50% of the B. cereus isolates from RTE foods and only 14% of the isolates were identified from infant formulas. The latter harbored low cytK and bceT, and very low hbl genes. Most B. cereus isolates possessed the hemolysis gene, but not the ces gene. The infant formula isolates showed stronger hemolysis activity than the other isolates. In addition, 26% of the total isolates showed low lecithinase activities and 10% showed high lecithinase activities. A greater number of isolates from the infant formula showed high lecithinase activity than those from the RTE foods. Approximately 83% of the isolates were positive and 17% were negative for starch hydrolysis. Over 90% of the RTE food isolates and only 35% of the infant formula isolates were positive for starch hydrolysis. However, all the strains possessed nhe, but their harboring patterns of hbl and cytK were significantly different. Most starch-hydrolyzing strains possessed hbl, but only 23% nonstarch-hydrolyzing isolates possessed this gene. Moreover, very low nonstarch hydrolyzing strains harbored cytK. Most nonstarch-hydrolyzing isolates showed high lecithinase and strong hemolysis activities, and very low hbl and cytK harboring. In summary, most infant formula isolates showed stronger hemolysis and higher lecithinase activities with lower

  9. Assessment of Enterotoxin Production and Cross-Contamination of Staphylococcus aureus between Food Processing Materials and Ready-To-Eat Cooked Fish Paste.

    PubMed

    Tango, Charles Nkufi; Hong, Sung-Sam; Wang, Jun; Oh, Deog-Hwan

    2015-12-01

    This study evaluated Staphylococcus aureus growth and subsequent staphylococcal enterotoxin A production in tryptone soy broth and on ready-to-eat cooked fish paste at 12 to 37 °C, as well as cross-contamination between stainless steel, polyethylene, and latex glove at room temperature. A model was developed using Barany and Roberts's growth model, which satisfactorily described the suitable growth of S. aureus with R(2)-adj from 0.94 to 0.99. Except at 12 °C, S. aureus cells in TSB presented a lag time lower (14.64 to 1.65 h), grew faster (0.08 to 0.31 log CFU/h) and produced SEA at lower cell density levels (5.65 to 6.44 log CFU/mL) compare to those inoculated on cooked fish paste with data of 16.920 to 1.985 h, 0.02 to 0.23 log CFU/h, and 6.19 to 7.11 log CFU/g, respectively. Staphylococcal enterotoxin type A (SEA) visual immunoassay test showed that primary SEA detection varied considerably among different storage temperature degrees and media. For example, it occurred only during exponential phase at 30 and 37 °C in TSB, but in cooked fish paste it took place at late exponential phase of S. aureus growth at 20 and 25 °C. The SEA detection test was negative on presence of S. aureus on cooked fish paste stored at 12 and 15 °C, although cell density reached level of 6.12 log CFU/g at 15 °C. Cross-contamination expressed as transfer rate of S. aureus from polyethylene surface to cooked fish paste surface was slower than that observed with steel surface to cooked fish paste under same conditions. These results provide helpful information for controlling S. aureus growth, SEA production and cross-contamination during processing of cooked fish paste.

  10. Assessment of Enterotoxin Production and Cross-Contamination of Staphylococcus aureus between Food Processing Materials and Ready-To-Eat Cooked Fish Paste.

    PubMed

    Tango, Charles Nkufi; Hong, Sung-Sam; Wang, Jun; Oh, Deog-Hwan

    2015-12-01

    This study evaluated Staphylococcus aureus growth and subsequent staphylococcal enterotoxin A production in tryptone soy broth and on ready-to-eat cooked fish paste at 12 to 37 °C, as well as cross-contamination between stainless steel, polyethylene, and latex glove at room temperature. A model was developed using Barany and Roberts's growth model, which satisfactorily described the suitable growth of S. aureus with R(2)-adj from 0.94 to 0.99. Except at 12 °C, S. aureus cells in TSB presented a lag time lower (14.64 to 1.65 h), grew faster (0.08 to 0.31 log CFU/h) and produced SEA at lower cell density levels (5.65 to 6.44 log CFU/mL) compare to those inoculated on cooked fish paste with data of 16.920 to 1.985 h, 0.02 to 0.23 log CFU/h, and 6.19 to 7.11 log CFU/g, respectively. Staphylococcal enterotoxin type A (SEA) visual immunoassay test showed that primary SEA detection varied considerably among different storage temperature degrees and media. For example, it occurred only during exponential phase at 30 and 37 °C in TSB, but in cooked fish paste it took place at late exponential phase of S. aureus growth at 20 and 25 °C. The SEA detection test was negative on presence of S. aureus on cooked fish paste stored at 12 and 15 °C, although cell density reached level of 6.12 log CFU/g at 15 °C. Cross-contamination expressed as transfer rate of S. aureus from polyethylene surface to cooked fish paste surface was slower than that observed with steel surface to cooked fish paste under same conditions. These results provide helpful information for controlling S. aureus growth, SEA production and cross-contamination during processing of cooked fish paste. PMID:26556562

  11. The three-dimensional crystal structure of cholera toxin

    SciTech Connect

    Zhang, Rong-Guang; Westbrook, M.L.; Nance, S.; Spangler, B.D.; Scott, D.L.; Westbrook, E.M.

    1996-02-01

    The clinical manifestations of cholera are largely attributable to the actions of a secreted hexameric AB{sub 5} enterotoxin (choleragen). We have solved the three-dimensional structure of choleragen at 2.5 {Angstrom} resolution and compared the refined coordinates with those of choleragenoid (isolated B pentamer) and the heat-labile enterotoxin from Escherichia coli (LT). The crystalline coordinates provide a detailed view of the stereochemistry implicated in binding to GM1 gangliosides and in carrying out ADP-ribosylation. The A2 chain of choleragen, in contrast to that of LT, is a nearly continuous {alpha}-helix with an interpretable carboxyl tail.

  12. Chimeric anti-staphylococcal enterotoxin B antibodies and lovastatin act synergistically to provide in vivo protection against lethal doses of SEB.

    PubMed

    Tilahun, Mulualem E; Kwan, Alan; Natarajan, Kannan; Quinn, Megan; Tilahun, Ashenafi Y; Xie, Chen; Margulies, David H; Osborne, Barbara A; Goldsby, Richard A; Rajagopalan, Govindarajan

    2011-01-01

    Staphylococcal enterotoxin B (SEB) is one of a family of toxins secreted by Staphylococcus aureus that act as superantigens, activating a large fraction of the T-cell population and inducing production of high levels of inflammatory cytokines that can cause toxic shock syndrome (TSS) and death. Extracellular engagement of the TCR of T-cells and class II MHC of antigen presenting cells by SEB triggers the activation of many intracellular signaling processes. We engineered chimeric antibodies to block the extracellular engagement of cellular receptors by SEB and used a statin to inhibit intracellular signaling. Chimeric human-mouse antibodies directed against different neutralizing epitopes of SEB synergistically inhibited its activation of human T-cells in vitro. In the in vivo model of lethal toxic shock syndrome (TSS) in HLA-DR3 transgenic mice, two of these antibodies conferred significant partial protection when administered individually, but offered complete protection in a synergistic manner when given together. Similarly, in vivo, lovastatin alone conferred only partial protection from TSS similar to single anti-SEB antibodies. However, used in combination with one chimeric neutralizing anti-SEB antibody, lovastatin provided complete protection against lethal TSS in HLA-DR3 transgenic mice. These experiments demonstrate that in vivo protection against lethal doses of SEB can be achieved by a statin of proven clinical safety and chimeric human-mouse antibodies, agents now widely used and known to be of low immunogenicity in human hosts.

  13. Overexpression of Metastatic Related MicroRNAs, Mir-335 and Mir-10b, by Staphylococcal Enterotoxin B in the Metastatic Breast Cancer Cell Line

    PubMed Central

    Heidary, Mohammad Foad; Mahmoodzadeh Hosseini, Hamideh; Mehdizadeh Aghdam, Elnaz; Nourani, Mohammad Reza; Ranjbar, Reza; Mirnejad, Reza; Imani Fooladi, Abbas Ali

    2015-01-01

    Purpose: One of the advanced cancer therapy strategies is immune-stimulating compound based immunotherapy Staphylococcal enterotoxin B (SEB) is one of the potent superantigens, which can efficiently activate antitumor immune response to eradicate tumor growth and inhibit metastasis. Herein, we evaluated the effect of SEB on the expression of two master microRNAs, mir-335 and mir-10b, involved in metastasis. Methods: A metastatic breast cancer cell line MDA-MB231was treated with four different concentrations of SEB, including 10, 102, 103 and 104 ng/ml, for 24 and 48 hours. To identify the cytotoxic effect of SEB, treated cells were examined by MTT assay. The stem loop RT-PCR (TaqMan) was used to analyze the mir-335 and mir-10b expression. Results: Results showed that SEB significantly increased the expression of mir-335 both after 24 and 48 hours (pv < 0.001 and pv < 0.05, respectively). No significant differences were found in the mir-10b expression. Conclusion: Moreover, our findings demonstrated no cytotoxic effect of SEB on the treated cells. Our results suggest that SEB probably induces its anti-metastatic effect via the expression regulation of the main genes which contributes to metastasis. PMID:26236665

  14. Involvement of protein kinase C in the mechanism of action of Escherichia coli heat-stable enterotoxin (STa) in a human colonic carcinoma cell line, COLO-205

    SciTech Connect

    Gupta, Dyuti Datta; Saha, Subhrajit; Chakrabarti, Manoj K. . E-mail: mkc_niced@yahoo.co.in

    2005-08-01

    The present study was undertaken to determine the involvement of calcium-protein kinase C pathway in the mechanism of action of Escherichia coli heat stable enterotoxin (STa) apart from STa-induced activation of guanylate cyclase in human colonic carcinoma cell line COLO-205, which was used as a model cultured cell line to study the mechanism of action of E. coli STa. In response to E. coli STa, protein kinase C (PKC) activity was increased in a time-dependent manner with its physical translocation from cytosol to membrane. Inhibition of the PKC activity in membrane fraction and inhibition of its physical translocation in response to IP{sub 3}-mediated calcium release inhibitor dantrolene suggested the involvement of intracellular store depletion in the regulation of PKC activity. Among different PKC isoforms, predominant involvement of calcium-dependent protein kinase C (PKC{alpha}) was specified using isotype-specific pseudosubstrate, which showed pronounce enzyme activity. Inhibition of enzyme activity by PKC{alpha}-specific inhibitor Goe6976 and immunoblott study employing isotype-specific antibody further demonstrated the involvement of calcium-dependent isoform of PKC in the mechanism of action of E. coli STa. Moreover, inhibition of guanylate cyclase activity by PKC{alpha}-specific inhibitor Goe6976 suggested the involvement of PKC{alpha} in the regulation of guanylate cyclase activity.

  15. Modeling the Effect of Water Activity, pH, and Temperature on the Probability of Enterotoxin A Production by Staphylococcus aureus.

    PubMed

    Ding, Tian; Yu, Yan-Yan; Hwang, Cheng-An; Dong, Qing-Li; Chen, Shi-Guo; Ye, Xing-Qian; Liu, Dong-Hong

    2016-01-01

    The objectives of this study were to develop a probability model of Staphylococcus aureus enterotoxin A (SEA) production as affected by water activity (a(w)), pH, and temperature in broth and assess its applicability for milk. The probability of SEA production was assessed in tryptic soy broth using 24 combinations of a(w) (0.86 to 0.99), pH (5.0 to 7.0), and storage temperature (10 to 30°C). The observed probabilities were fitted with a logistic regression to develop a probability model. The model had a concordant value of 97.5% and concordant index of 0.98, indicating that the model satisfactorily describes the probability of SEA production. The model showed that a(w), pH, and temperature were significant factors affecting the probability of toxin production. The model predictions were in good agreement with the observed values obtained from milk. The model may help manufacturers in selecting product pH and a(w) and storage temperatures to prevent SEA production. PMID:26735042

  16. Multiple B-cell epitope vaccine induces a Staphylococcus enterotoxin B-specific IgG1 protective response against MRSA infection.

    PubMed

    Zhao, Zhuo; Sun, He-Qiang; Wei, Shan-Shan; Li, Bin; Feng, Qiang; Zhu, Jiang; Zeng, Hao; Zou, Quan-Ming; Wu, Chao

    2015-01-01

    No vaccine against methicillin-resistant Staphylococcus aureus (MRSA) has been currently approved for use in humans. Staphylococcus enterotoxin B (SEB) is one of the most potent MRSA exotoxins. In the present study, we evaluated the efficacy and immunologic mechanisms of an SEB multiple B-cell epitope vaccine against MRSA infection. Synthetic overlapping peptide ELISA identified three novel B-cell immunodominant SEB epitopes (in addition to those previously known): SEB31-48, SEB133-150, and SEB193-210. Six B-cell immunodominant epitopes (amino acid residues 31-48, 97-114, 133-150, 193-210, 205-222, and 247-261) were sufficient to induce robust IgG1/IgG2b-specific protective responses against MRSA infection. Therefore, we constructed a recombinant MRSA SEB-specific multiple B-cell epitope vaccine Polypeptides by combining the six SEB immunodominant epitopes and demonstrated its ability to induce a robust SEB-specific IgG1 response to MRSA, as well as a Th2-directing isotype response. Moreover, Polypeptides-induced antisera stimulated synergetic opsonophagocytosis killing of MRSA. Most importantly, Polypeptides was more effective at clearing the bacteria in MRSA-infected mice than the whole SEB antigen, and was able to successfully protect mice from infection by various clinical MRSA isolates. Altogether, these results support further evaluation of the SEB multiple B-cell epitope-vaccine to address MRSA infection in humans.

  17. Multiple B-cell epitope vaccine induces a Staphylococcus enterotoxin B-specific IgG1 protective response against MRSA infection

    PubMed Central

    Zhao, Zhuo; Sun, He-Qiang; Wei, Shan-Shan; Li, Bin; Feng, Qiang; Zhu, Jiang; Zeng, Hao; Zou, Quan-Ming; Wu, Chao

    2015-01-01

    No vaccine against methicillin-resistant Staphylococcus aureus (MRSA) has been currently approved for use in humans. Staphylococcus enterotoxin B (SEB) is one of the most potent MRSA exotoxins. In the present study, we evaluated the efficacy and immunologic mechanisms of an SEB multiple B-cell epitope vaccine against MRSA infection. Synthetic overlapping peptide ELISA identified three novel B-cell immunodominant SEB epitopes (in addition to those previously known): SEB31–48, SEB133–150, and SEB193–210. Six B-cell immunodominant epitopes (amino acid residues 31–48, 97–114, 133–150, 193–210, 205–222, and 247–261) were sufficient to induce robust IgG1/IgG2b-specific protective responses against MRSA infection. Therefore, we constructed a recombinant MRSA SEB-specific multiple B-cell epitope vaccine Polypeptides by combining the six SEB immunodominant epitopes and demonstrated its ability to induce a robust SEB-specific IgG1 response to MRSA, as well as a Th2-directing isotype response. Moreover, Polypeptides-induced antisera stimulated synergetic opsonophagocytosis killing of MRSA. Most importantly, Polypeptides was more effective at clearing the bacteria in MRSA-infected mice than the whole SEB antigen, and was able to successfully protect mice from infection by various clinical MRSA isolates. Altogether, these results support further evaluation of the SEB multiple B-cell epitope-vaccine to address MRSA infection in humans. PMID:26201558

  18. Molecular typing of toxic shock syndrome toxin-1- and Enterotoxin A-producing methicillin-sensitive Staphylococcus aureus isolates from an outbreak in a neonatal intensive care unit.

    PubMed

    Layer, Franziska; Sanchini, Andrea; Strommenger, Birgit; Cuny, Christiane; Breier, Ann-Christin; Proquitté, Hans; Bührer, Christoph; Schenkel, Karl; Bätzing-Feigenbaum, Jörg; Greutelaers, Benedikt; Nübel, Ulrich; Gastmeier, Petra; Eckmanns, Tim; Werner, Guido

    2015-10-01

    Outbreaks of Staphylococcus aureus are common in neonatal intensive care units (NICUs). Usually they are documented for methicillin-resistant strains, while reports involving methicillin-susceptible S. aureus (MSSA) strains are rare. In this study we report the epidemiological and molecular investigation of an MSSA outbreak in a NICU among preterm neonates. Infection control measures and interventions were commissioned by the Local Public Health Authority and supported by the Robert Koch Institute. To support epidemiological investigations molecular typing was done by spa-typing and Multilocus sequence typing; the relatedness of collected isolates was further elucidated by DNA SmaI-macrorestriction, microarray analysis and bacterial whole genome sequencing. A total of 213 neonates, 123 healthcare workers and 205 neonate parents were analyzed in the period November 2011 to November 2012. The outbreak strain was characterized as a MSSA spa-type t021, able to produce toxic shock syndrome toxin-1 and Enterotoxin A. We identified seventeen neonates (of which two died from toxic shock syndrome), four healthcare workers and three parents putatively involved in the outbreak. Whole-genome sequencing permitted to exclude unrelated cases from the outbreak and to discuss the role of healthcare workers as a reservoir of S. aureus on the NICU. Genome comparisons also indicated the presence of the respective clone on the ward months before the first colonized/infected neonates were detected.

  19. Development of a highly specific assay for rapid identification of pathogenic strains of Yersinia enterocolitica based on PCR amplification of the Yersinia heat-stable enterotoxin gene (yst).

    PubMed

    Ibrahim, A; Liesack, W; Griffiths, M W; Robins-Browne, R M

    1997-06-01

    The chromosomal gene yst, which encodes a heat-stable enterotoxin of Yersinia enterocolitica, is a useful diagnostic marker because it occurs only in invasive strains of this species. A homologous gene also occurs in some strains of Yersinia kristensenii. Sequence analysis of the yst genes from two different strains of Y. enterocolitica and from Y. kristensenii revealed a substantial number of mismatches at the 3' ends of the yst genes of the so-called American and European biotypes of Y. enterocolitica. Moreover, several mismatches and a deletion of 5 codons were found in the yst of Y. kristensenii. These findings were used to develop a PCR-based assay for yst of Y. enterocolitica which yielded a detectable product in as little as 50 min. The assay was 100% specific in terms of its ability to identify potentially pathogenic strains of Y. enterocolitica regardless of biotype or serotype. The PCR yielded an amplicon that was visible on agarose gel electrophoresis from as few as 100 CFU, or 10 CFU when the PCR was combined with dot blot hybridization with a digoxigenin-labeled oligonucleotide probe that corresponded to an internal sequence of yst. These results establish the value of the yst gene as a target for the identification of pathogenic bioserotypes of Y. enterocolitica and the usefulness of PCR for this purpose.

  20. Development of a highly specific assay for rapid identification of pathogenic strains of Yersinia enterocolitica based on PCR amplification of the Yersinia heat-stable enterotoxin gene (yst).

    PubMed Central

    Ibrahim, A; Liesack, W; Griffiths, M W; Robins-Browne, R M

    1997-01-01

    The chromosomal gene yst, which encodes a heat-stable enterotoxin of Yersinia enterocolitica, is a useful diagnostic marker because it occurs only in invasive strains of this species. A homologous gene also occurs in some strains of Yersinia kristensenii. Sequence analysis of the yst genes from two different strains of Y. enterocolitica and from Y. kristensenii revealed a substantial number of mismatches at the 3' ends of the yst genes of the so-called American and European biotypes of Y. enterocolitica. Moreover, several mismatches and a deletion of 5 codons were found in the yst of Y. kristensenii. These findings were used to develop a PCR-based assay for yst of Y. enterocolitica which yielded a detectable product in as little as 50 min. The assay was 100% specific in terms of its ability to identify potentially pathogenic strains of Y. enterocolitica regardless of biotype or serotype. The PCR yielded an amplicon that was visible on agarose gel electrophoresis from as few as 100 CFU, or 10 CFU when the PCR was combined with dot blot hybridization with a digoxigenin-labeled oligonucleotide probe that corresponded to an internal sequence of yst. These results establish the value of the yst gene as a target for the identification of pathogenic bioserotypes of Y. enterocolitica and the usefulness of PCR for this purpose. PMID:9163505

  1. Methicillin-Susceptible Staphylococcus aureus Endocarditis Isolates Are Associated With Clonal Complex 30 Genotype and a Distinct Repertoire of Enterotoxins and Adhesins

    PubMed Central

    Nienaber, Juhsien J.C.; Sharma Kuinkel, Batu K.; Clarke-Pearson, Michael; Lamlertthon, Supaporn; Park, Lawrence; Rude, Thomas H.; Barriere, Steve; Woods, Christopher W.; Chu, Vivian H.; Marín, Mercedes; Bukovski, Suzana; Garcia, Patricia; Corey, G.Ralph; Korman, Tony; Doco-Lecompte, Thanh; Murdoch, David R.; Reller, L. Barth

    2011-01-01

    Background. Using multinational collections of methicillin-susceptible Staphylococcus aureus (MSSA) isolates from infective endocarditis (IE) and soft tissue infections (STIs), we sought to (1) validate the finding that S. aureus in clonal complex (CC) 30 is associated with hematogenous complications and (2) test the hypothesis that specific genetic characteristics in S. aureus are associated with infection severity. Methods. IE and STI isolates from 2 cohorts were frequency matched by geographic origin. Isolates underwent spa typing to infer CC and multiplex polymerase chain reaction for presence of virulence genes. Results. 114 isolate pairs were genotyped. IE isolates were more likely to be CC30 (19.5% vs 6.2%; P = .005) and to contain 3 adhesins (clfB, cna, map/eap; P < .0001 for all) and 5 enterotoxins (tst, sea, sed, see, and sei; P ≤ .005 for all). CC30 isolates were more likely to contain cna, tst, sea, see, seg, and chp (P < .05 for all). Conclusions. MSSA IE isolates were significantly more likely to be CC30 and to possess a distinct repertoire of virulence genes than MSSA STI isolates from the same region. The genetic basis of this association requires further study. PMID:21844296

  2. Molecular typing of toxic shock syndrome toxin-1- and Enterotoxin A-producing methicillin-sensitive Staphylococcus aureus isolates from an outbreak in a neonatal intensive care unit.

    PubMed

    Layer, Franziska; Sanchini, Andrea; Strommenger, Birgit; Cuny, Christiane; Breier, Ann-Christin; Proquitté, Hans; Bührer, Christoph; Schenkel, Karl; Bätzing-Feigenbaum, Jörg; Greutelaers, Benedikt; Nübel, Ulrich; Gastmeier, Petra; Eckmanns, Tim; Werner, Guido

    2015-10-01

    Outbreaks of Staphylococcus aureus are common in neonatal intensive care units (NICUs). Usually they are documented for methicillin-resistant strains, while reports involving methicillin-susceptible S. aureus (MSSA) strains are rare. In this study we report the epidemiological and molecular investigation of an MSSA outbreak in a NICU among preterm neonates. Infection control measures and interventions were commissioned by the Local Public Health Authority and supported by the Robert Koch Institute. To support epidemiological investigations molecular typing was done by spa-typing and Multilocus sequence typing; the relatedness of collected isolates was further elucidated by DNA SmaI-macrorestriction, microarray analysis and bacterial whole genome sequencing. A total of 213 neonates, 123 healthcare workers and 205 neonate parents were analyzed in the period November 2011 to November 2012. The outbreak strain was characterized as a MSSA spa-type t021, able to produce toxic shock syndrome toxin-1 and Enterotoxin A. We identified seventeen neonates (of which two died from toxic shock syndrome), four healthcare workers and three parents putatively involved in the outbreak. Whole-genome sequencing permitted to exclude unrelated cases from the outbreak and to discuss the role of healthcare workers as a reservoir of S. aureus on the NICU. Genome comparisons also indicated the presence of the respective clone on the ward months before the first colonized/infected neonates were detected. PMID:26321006

  3. PROMOTING RESILIENCE.

    PubMed

    Desjardins, Eric; Barker, Gillian; Lindo, Zoë; Dieleman, Catherine; Dussault, Antoine C

    2015-06-01

    Broadening contingents of ecologists and environmental scientists have recently begun to promote ecological resilience both as a conceptual framework and as a practical goal. As some critics have noted, this growing interest has brought with it a multiplication of notions of ecological resilience. This paper reviews how and why the notion of ecological resilience has been adopted, used, and defended in ecology since its introduction by C. S. Holling in 1973. We highlight the many faces of ecological resilience, but unlike other reviewers who see these as disunified and confused, we interpret ecological resilience as an evolving, multidimensional, theoretical concept unified by its role in guiding practical response to ecological and environmental challenges. This perspective informs a review of some of the factors often recognized as favoring resilience (structural and response diversity, functional redundancy, modularity, and spatial heterogeneity); we show how the roles and relationships of these factors can be clarified by considering them in the theoretical framework of Complex Adaptive Systems (CASs).

  4. The Mutation Glu151Asp in the B-Component of the Bacillus cereus Non-Hemolytic Enterotoxin (Nhe) Leads to a Diverging Reactivity in Antibody-Based Detection Systems

    PubMed Central

    Didier, Andrea; Jeßberger, Nadja; Krey, Victoria; Dietrich, Richard; Scherer, Siegfried; Märtlbauer, Erwin

    2015-01-01

    The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection. For the diarrhea-associated enterotoxins, the assessment of the non-hemolytic enterotoxin B (NheB) titer determined by a sandwich enzyme immunoassay (EIA) correlates best with in vitro cytotoxicity. In general, the regulation of enterotoxin expression of B. cereus is a coordinately-regulated process influenced by environmental, and probably also by host factors. As long as these factors are not completely understood, the currently-applied diagnostic procedures are based on indirect approaches to assess the potential virulence of an isolate. To date, sandwich EIA results serve as a surrogate marker to categorize isolates as either potentially low or highly toxic. Here, we report on a single amino acid exchange in the NheB sequence leading to an underestimation of the cytotoxic potential in a limited number of strains. During the screening of a large panel of B. cereus isolates, six showed uncommon features with low sandwich EIA titers despite high cytotoxicity. Sequence analysis revealed the point-mutation Glu151Asp in the potential binding region of the capture antibody. Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots. A commercially-available assay based on a lateral flow device detects all strains correctly as NheB producers in a qualitative manner. In conclusion, isolates showing low NheB titers should additionally be assayed in an indirect EIA or for their in vitro cytotoxicity to ensure a correct classification as either low or highly toxic. PMID:26569304

  5. The mutation Glu151Asp in the B-component of the Bacillus cereus non-hemolytic enterotoxin (Nhe) leads to a diverging reactivity in antibody-based detection systems.

    PubMed

    Didier, Andrea; Jeßberger, Nadja; Krey, Victoria; Dietrich, Richard; Scherer, Siegfried; Märtlbauer, Erwin

    2015-11-09

    The ability of Bacillus cereus to cause foodborne toxicoinfections leads to increasing concerns regarding consumer protection. For the diarrhea-associated enterotoxins, the assessment of the non-hemolytic enterotoxin B (NheB) titer determined by a sandwich enzyme immunoassay (EIA) correlates best with in vitro cytotoxicity. In general, the regulation of enterotoxin expression of B. cereus is a coordinately-regulated process influenced by environmental, and probably also by host factors. As long as these factors are not completely understood, the currently-applied diagnostic procedures are based on indirect approaches to assess the potential virulence of an isolate. To date, sandwich EIA results serve as a surrogate marker to categorize isolates as either potentially low or highly toxic. Here, we report on a single amino acid exchange in the NheB sequence leading to an underestimation of the cytotoxic potential in a limited number of strains. During the screening of a large panel of B. cereus isolates, six showed uncommon features with low sandwich EIA titers despite high cytotoxicity. Sequence analysis revealed the point-mutation (Glu)151(Asp) in the potential binding region of the capture antibody. Application of this antibody also results in low titers in an indirect EIA format and shows variable detection intensities in Western-immunoblots. A commercially-available assay based on a lateral flow device detects all strains correctly as NheB producers in a qualitative manner. In conclusion, isolates showing low NheB titers should additionally be assayed in an indirect EIA or for their in vitro cytotoxicity to ensure a correct classification as either low or highly toxic.

  6. Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr-activated sepharose-4B affinity chromatography.

    PubMed

    Hedayati Ch, Mojtaba; Amani, Jafar; Sedighian, Hamid; Amin, Mohsen; Salimian, Jafar; Halabian, Raheleh; Imani Fooladi, Abbas Ali

    2016-09-01

    Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA-aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (kd  = 2.3 × 10(-11) ). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27091327

  7. Antitumor x anti-CD3 bifunctional antibodies redirect T-cells activated in vivo with staphylococcal enterotoxin B to neutralize pulmonary metastases.

    PubMed

    Penna, C; Dean, P A; Nelson, H

    1994-05-15

    T-cell antitumor activities are limited by the requirement of two specific major histocompatibility complex restricted steps, T-helper cell activation and cytotoxic T-lymphocyte targeting. The aim of this study was to investigate whether bypassing these major histocompatibility complex restricted steps using nonspecific in vivo activation of T-cells with staphylococcal enterotoxin-B (SE-B) and retargeting with antitumor x anti-CD3 bifunctional antibody (BFA) could provide an effective antitumor response. C3H/HeN mice were injected i.v. with CL-62 melanoma cells, which express the human melanoma antigen p97, and were treated 10 min later with SE-B and/or anti-CD3 (500A2) x anti-p97 (96.5) BFA. Pulmonary metastases were counted 14 days following injections. SE-B alone induced a dose-dependent activation of T-cells as measured by increased interleukin-2 receptor expression and enhanced proliferative responses. SE-B doses greater than 10 micrograms significantly reduced the number of pulmonary metastases versus control (P < 0.01). Combined treatment with SE-B (50 micrograms) and BFA (5 to 50 micrograms) significantly decreased pulmonary metastases compared to treatment with SE-B alone (P < 0.05). Similar reductions in metastases were observed with the F(ab')2 BFA but not with the unconjugated antitumor component of the BFA. Combined treatments with SE-B plus BFA accomplished better tumor neutralization than adoptively transferred in vitro activated splenocytes (4 x 10(7)) retargeted with BFA (5-100 micrograms; P < 0.05). These studies demonstrate that T-cells can be activated in vivo by SE-B and retargeted with small doses of BFA. In this immunocompetent syngeneic pulmonary metastasis model, SE-B plus BFA provided a dramatic antitumor response. PMID:8168104

  8. T cell-mediated lethal shock triggered in mice by the superantigen staphylococcal enterotoxin B: critical role of tumor necrosis factor

    PubMed Central

    1992-01-01

    Because mice are more resistant than humans to the pathogenic effects of bacterial toxins, we used D-Galactosamine- (D-Gal) sensitized mice as a model system to evaluate potential toxic shock symptoms triggered by the superantigen staphylococcal enterotoxin B (SEB). We show that similar to endotoxin (lipopolysaccharide) [LPS], the exotoxin SEB causes lethal shock within 8 h in D-Gal-sensitized mice, inducing 100% and about 50% lethality with 20 and 2 micrograms SEB, respectively. The lethal shock triggered by the superantigen SEB is mediated by T cells, a conclusion based on the observation that T cell repopulation of SCID mice conferred sensitivity to SEB. Since CSA also conferred protection, the role of T cell-derived lymphokines in mediating lethal shock was evaluated. Within 30-60 min after SEB injection, serum tumor necrosis factor (TNF) levels peaked, followed immediately by interleukin-2 (IL- 2). Serum-borne lymphokines were detected well in advance of signs of T cell activation, as assessed by IL-2 receptor expression of SEB- reactive V beta 8+ T cells. Passive immunization with anti-TNF- alpha/beta-neutralizing monoclonal antibody also conferred protection, indicating that it is TNF which is critical for initiating toxic shock symptoms. Taken together, this study defines basic differences between endotoxin (LPS)- and exotoxin (SEB)-mediated lethal shock, in that the former is mediated by macrophages and the latter by T cells. Yet the pathogenesis distal to the lymphokine/cytokine-producing cells appears surprisingly similar in that TNF represents a key mediator in inducing shock. PMID:1730929

  9. The novel heat-stable enterotoxin subtype gene (ystB) of Yersinia enterocolitica: nucleotide sequence and distribution of the yst genes.

    PubMed

    Ramamurthy, T; Yoshino, K i; Huang, X; Balakrish Nair, G; Carniel, E; Maruyama, T; Fukushima, H; Takeda, T

    1997-10-01

    The gene (ystB) encoding the novel subtype of the heat-stable enterotoxin (Y-STb) was cloned from the chromosome of a clinical isolate of Yersinia enterocolitica 84-50 (serotype O:5, biotype 1A) and the nucleotide sequence was determined. The ystB contained 216 base pairs that encoded a protein of 71 amino acid residues. The C-terminal 30 residues of the precursor protein exactly corresponded to the amino acid sequence of the Y-STb toxin, purified from the culture supernatant of the wild strain. Homology search revealed that there are 76.9% nucleotide sequence similarity between ystB and the Yersinia kristensenii ST gene, and 73.5% with the Y. enterocolitica prototype sequence of yst (ystA). When tested with the PCR generated ystB specific probe, 36 of 304 Y. enterocolitica strains from 18 countries hybridized with the probe. All the ystB probe positive strains belonged to biotype 1A and mostly to the so-called non-pathogenic serotype O:5, O:6, O:7,8 O:7,13 and O:10, while ystA was predominantly found among the pathogenic serotypes (78.5%). Out of 36 ystB gene positive strains, 18 were clinical origin from six countries, which were also positive in the suckling mice assay suggesting that ystB may play an important role in the pathogenesis, and the so-called non-pathogenic serotypes could be virulent for human.

  10. Detection of the staphylococcal enterotoxin D-like gene from staphylococcal food poisoning isolates over the last two decades in Tokyo.

    PubMed

    Suzuki, Yasunori; Kobayashi, Makiko; Matsushita, Shigeru; Uehara, Satomi; Kato, Rei; Sato'o, Yusuke; Ono, Hisaya K; Sadamasu, Kenji; Kai, Akemi; Kamata, Yoichi

    2015-08-01

    The plasmid is a very well-known mobile genetic element that participates in the acquisition of virulence genes, such as staphylococcal enterotoxins (SEs), via horizontal transfer. SEs are emetic toxins and causative agents in staphylococcal food poisoning (SFP). We herein identified the types of plasmids harbored by seven SFP isolates and examined their production of plasmid-related SE/SEl to determine whether the new types of plasmid-related SE or SE-like (SEl) toxins (i.e. SElJ and SER) were involved in SFP. These isolates harbored pIB485-like plasmids, and all, except for one isolate, produced SElJ and SER. The amount of SER produced by each isolate accounted for the highest or second highest percentage of the total amount of SE/SEl produced. These new types of plasmid-related SE/SEls as well as classical SE may play a role in SFP. The seven isolates were classified into two SED-production types; a high SED-production type (>500 ng/ml) and no SED-production type. A nucleotide sequencing analysis revealed that three plasmids harbored by the SED-non-producing isolates had a single-base deletion in the sed gene with a resulting stop codon (from 233 amino acids of the intact SED to 154 amino acids of the mutant SED (mSED)). A real-time reverse transcription-PCR analysis showed that the mRNA of the msed gene was transcribed in the isolates. If the msed gene was translated as a protein, mSED may act as an emetic toxin instead of intact SED.

  11. Conjugates of group A and W135 capsular polysaccharides of neisseria meningitidis bound to recombinant Staphylococcus aureus enterotoxin C1: preparation, physicochemical characterization, and immunological properties in mice.

    PubMed

    Jin, Zhigang; Bohach, Gregory A; Shiloach, Joseph; Norris, Scott E; Freedberg, Darón I; Deobald, Claudia; Coxon, Bruce; Robbins, John B; Schneerson, Rachel

    2005-12-01

    Neisseria meningitidis groups A (GAM) and W135 capsular polysaccharides (CPs) were bound to recombinant Staphylococcus aureus enterotoxin C1 (rSEC). The CPs were activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate and then bound to adipic acid dihydrazide derivatives of rSEC. Syntheses were conducted with native GAM CP (GAMP), W135 CP (W135P), and ultrasonicated or hydrazine-treated W135P at various concentrations of reactants, pHs, and ionic strengths. The conjugates were characterized by compositional and serologic analyses, high-performance size-exclusion chromatography with multi-angle laser light scattering detection, and immunogenicity in 5- to 6-week-old mice. Conjugates injected subcutaneously in phosphate-buffered saline elicited immunoglobulin G (IgG) responses against their respective CPs and rSEC, whereas GAMP and W135P alone did not induce detectable CP antibodies. The O-acetyl content of W135P was low, and its removal had no adverse effect upon the conjugate's immunogenicity. Reduction of the molecular size of W135P by treatment with hydrazine improved the immunogenicity of W135P-rSEC. IgG anti-CP elicited by the conjugates showed complement-dependent bactericidal activity against their respective organisms, and IgG anti-rSEC neutralized the T-cell proliferative activity of native SEC. A bivalent formulation of GAMP-rSEC and W135P-rSEC elicited IgG anti-CP at comparable levels to those induced by the conjugates administered separately. PMID:16299279

  12. Clostridium perfringens enterotoxin C-terminal domain labeled to fluorescent dyes for in vivo visualization of micrometastatic chemotherapy-resistant ovarian cancer.

    PubMed

    Cocco, Emiliano; Shapiro, Erik M; Gasparrini, Sara; Lopez, Salvatore; Schwab, Carlton L; Bellone, Stefania; Bortolomai, Ileana; Sumi, Natalia J; Bonazzoli, Elena; Nicoletti, Roberta; Deng, Yang; Saltzman, W Mark; Zeiss, Caroline J; Centritto, Floriana; Black, Jonathan D; Silasi, Dan-Arin; Ratner, Elena; Azodi, Masoud; Rutherford, Thomas J; Schwartz, Peter E; Pecorelli, Sergio; Santin, Alessandro D

    2015-12-01

    Identification of micrometastatic disease at the time of surgery remains extremely challenging in ovarian cancer patients. We used fluorescence microscopy, an in vivo imaging system and a fluorescence stereo microscope to evaluate fluorescence distribution in Claudin-3- and -4-overexpressing ovarian tumors, floating tumor clumps isolated from ascites and healthy organs. To do so, mice harboring chemotherapy-naïve and chemotherapy-resistant human ovarian cancer xenografts or patient-derived xenografts (PDXs) were treated with the carboxyl-terminal binding domain of the Clostridium perfringens enterotoxin (c-CPE) conjugated to FITC (FITC-c-CPE) or the near-infrared (NIR) fluorescent tag IRDye CW800 (CW800-c-CPE) either intraperitoneally (IP) or intravenously (IV). We found tumor fluorescence to plateau at 30 min after IP injection of both the FITC-c-CPE and the CW800-c-CPE peptides and to be significantly higher than in healthy organs (p < 0.01). After IV injection of CW800-c-CPE, tumor fluorescence plateaued at 6 hr while the most favorable tumor-to-background fluorescence ratio (TBR) was found at 48 hr in both mouse models. Importantly, fluorescent c-CPE was highly sensitive for the in vivo visualization of peritoneal micrometastatic tumor implants and the identification of ovarian tumor spheroids floating in malignant ascites that were otherwise not detectable by conventional visual observation. The use of the fluorescent c-CPE peptide may represent a novel and effective optical approach at the time of primary debulking surgery for the real-time detection of micrometastatic ovarian disease overexpressing the Claudin-3 and -4 receptors or the identification of residual disease at the time of interval debulking surgery after neoadjuvant chemotherapy treatment.

  13. T cell stimulation by staphylococcal enterotoxins. Clonally variable response and requirement for major histocompatibility complex class II molecules on accessory or target cells

    PubMed Central

    1988-01-01

    Staphylococcal enterotoxins (SE) are the most potent mitogens for T lymphocytes known; concentrations of less than 10(-9) M are sufficient for T cell activation. The mechanism of T cell activation by SE is unknown. We have used cloned human cytotoxic and proliferative T lymphocytes to dissect the molecular mechanism of T cell activation by SE. With rare exceptions, all TCR alpha/beta chain-expressing T cell clones of CD4+ or CD8+ phenotype, as well as CD4-8- TCR alpha/beta chain negative chain-expressing T lymphocyte clones, respond with proliferation and/or cytotoxicity to SE. For triggering of all these clones, the presence of autologous or allogeneic MHC class II molecules on accessory or target cells is necessary. This requirement for class II antigens is not due to an immunological recognition of processed SE, since inhibition of antigen processing has no influence on the T cell response to SE. SE acts on the T cells directly since (a) they stimulate a rise in intracellular calcium concentration in T cell lines or purified T cells, and (b) accessory cells can be replaced by phorbolesters in the proliferative activation of resting T cells by SE. Furthermore, the T cell response to SE shows extensive clonal heterogeneity. These results suggest that SE are functionally bivalent mitogens binding highly selectively to HLA class II molecules and the TCR. Thus, compared with other polyclonal T cell activating agents, activation with SE most closely mimicks the physiological way of MHC- restricted antigen recognition by T lymphocytes. PMID:3259256

  14. Therapeutic Inhibition of Pro-Inflammatory Signaling and Toxicity to Staphylococcal Enterotoxin B by a Synthetic Dimeric BB-Loop Mimetic of MyD88

    PubMed Central

    Kissner, Teri L.; Ruthel, Gordon; Alam, Shahabuddin; Mann, Enrique; Ajami, Dariush; Rebek, Mitra; Larkin, Eileen; Fernandez, Stefan; Ulrich, Robert G.; Ping, Sun; Waugh, David S.; Rebek, Julius; Saikh, Kamal U.

    2012-01-01

    Staphylococcal enterotoxin B (SEB) exposure triggers an exaggerated pro-inflammatory cytokine response that often leads to toxic shock syndrome (TSS) associated with organ failure and death. MyD88 mediates pro-inflammatory cytokine signaling induced by SEB exposure and MyD88−/− mice are resistant to SEB intoxication, suggesting that MyD88 may be a potential target for therapeutic intervention. We targeted the BB loop region of the Toll/IL-1 receptor (TIR) domain of MyD88 to develop small-molecule therapeutics. Here, we report that a synthetic compound (EM-163), mimic to dimeric form of BB-loop of MyD88 attenuated tumor necrosis factor (TNF)- α, interferon (IFN)-γ, interleukin (IL)-1β, IL-2 and IL-6 production in human primary cells, whether administered pre- or post-SEB exposure. Results from a direct binding assay, and from MyD88 co-transfection/co-immunoprecipitation experiments, suggest that EM-163 inhibits TIR-TIR domain interaction. Additional results indicate that EM-163 prevents MyD88 from mediating downstream signaling. In an NF-kB-driven reporter assay of lipopolysaccharide-stimulated MyD88 signaling, EM-163 demonstrated a dose-dependent inhibition of reporter activity as well as TNF-α and IL-1β production. Importantly, administration of EM-163 pre- or post exposure to a lethal dose of SEB abrogated pro-inflammatory cytokine responses and protected mice from toxic shock-induced death. Taken together, our results suggest that EM-163 exhibits a potential for therapeutic use against SEB intoxication. PMID:22848400

  15. Detection and genetic analysis of the enteroaggregative Escherichia coli heat-stable enterotoxin (EAST1) gene in clinical isolates of enteropathogenic Escherichia coli (EPEC) strains

    PubMed Central

    2014-01-01

    Background The enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) encoded by astA gene has been found in enteropathogenic E. coli (EPEC) strains. However, it is not sufficient to simply probe strains with an astA gene probe due to the existence of astA mutants (type 1 and type 2 SHEAST) and EAST1 variants (EAST1 v1-4). In this study, 222 EPEC (70 typical and 152 atypical) isolates were tested for the presence of the astA gene sequence by PCR and sequencing. Results The astA gene was amplified from 54 strains, 11 typical and 43 atypical. Sequence analysis of the PCR products showed that 25 strains, 7 typical and 18 atypical, had an intact astA gene. A subgroup of 7 atypical strains had a variant type of the astA gene sequence, with four non-synonymous nucleotide substitutions. The remaining 22 strains had mutated astA gene with nucleotide deletions or substitutions in the first 8 codons. The RT-PCR results showed that the astA gene was transcribed only by the strains carrying either the intact or the variant type of the astA gene sequence. Southern blot analysis indicated that astA is located in EAF plasmid in typical strains, and in plasmids of similar size in atypical strains. Strains carrying intact astA genes were more frequently found in diarrheic children than in non-diarrheic children (p < 0.05). Conclusion In conclusion, our data suggest that the presence of an intact astA gene may represent an additional virulence determinant in both EPEC groups. PMID:24884767

  16. Disruption of the guanylyl cyclase-C gene leads to a paradoxical phenotype of viable but heat-stable enterotoxin-resistant mice.

    PubMed Central

    Schulz, S; Lopez, M J; Kuhn, M; Garbers, D L

    1997-01-01

    Heat-stable enterotoxins (STa), which cause an acute secretory diarrhea, have been suggested to mediate their actions through the guanylyl cyclase-C (GC-C) receptor. The GC-C gene was disrupted by insertion of neo into exon 1 and subsequent homologous recombination. GC-C null mice contained no detectable GC-C protein. Intestine mucosal guanylyl cyclase activity was approximately 16-fold higher in wild-type mice than in the GC-C null mice, and STa-stimulable guanylyl cyclase activity was absent in the null animals. Thus, GC-C is the major cyclase activity present in the intestine, and also completely accounts for the STa-induced elevations of cGMP. Gavage with STa resulted in marked fluid accumulation within the intestine of wild-type and heterozygous suckling mice, but GC-C null animals were resistant. In addition, infection with enterotoxigenic bacteria that produce STa led to diarrhea and death in wild-type and heterozygous mice, while the null mice were protected. Cholera toxin, in contrast, continued to cause diarrhea in GC-C null mice, demonstrating that the cAMP signaling pathway remained intact. Markedly different diets (high carbohydrate, fat, or protein) or the inclusion of high salt (K+, Na+) in the drinking water or diet also did not severely affect the null animals. Given that GC-C is a major intestinal receptor in all mammals, the pressure to retain a functional GC-C in the face of diarrhea-inflicted mortality remains unexplained. Therefore, GC-C likely provides a protective effect against stressors not yet tested, possibly pathogens other than noninvasive enterotoxigenic bacteria. PMID:9294128

  17. Formation of small transmembrane pores: An intermediate stage on the way to Bacillus cereus non-hemolytic enterotoxin (Nhe) full pores in the absence of NheA.

    PubMed

    Zhu, Kui; Didier, Andrea; Dietrich, Richard; Heilkenbrinker, Uta; Waltenberger, Eva; Jessberger, Nadja; Märtlbauer, Erwin; Benz, Roland

    2016-01-15

    The non-hemolytic enterotoxin (Nhe) of Bacillus cereus is a three-partite toxin formed of the components NheA, -B and -C. Pore formation and subsequent lysis of target cells caused by Nhe is an orchestrated process comprising three steps: (i) formation of NheB/C oligomers in solution, (ii) attachment of the oligomers to the cell membrane, (iii) binding of NheA to the oligomers. The present study aimed to characterize the properties of the NheB/C complex and the fate of the target cell upon binding. An enzyme immunoassay allowing kinetic measurements and surface plasmon resonance revealed the fast and high affinity formation of the NheB/C oligomers. The benefit of these complexes is a more stable cell binding as well as stronger and earlier cytotoxic effect. High molecular mass hetero-oligomers (620 kDa) probably consisting of one NheC and up to 15 NheB were detected by size-exclusion chromatography and on native PAGE immunoblots. Due to the NheBC application the morphology and membrane permeability of Vero cells is partly disturbed. Formation of stable transmembrane channels with a conductance of about 870 pS and a diameter of about 2 nm due to the application of NheBC could be demonstrated in lipid bilayer experiments. Thus, the NheBC complex itself has a tendency to increase the membrane permeability prior to the emergence of full pores containing also NheA.

  18. Dietary n-3 PUFAs augment caspase 8 activation in Staphylococcal aureus enterotoxin B stimulated T-cells.

    PubMed

    Gill, R; Jen, K L; McCabe, M J J; Rosenspire, A

    2016-10-15

    Epidemiological studies have linked consumption of n-3 PUFAs with a variety of beneficial health benefits, particularly with respect to putative anti-inflammatory effects. Unfortunately, many of these results remain somewhat controversial because in most instances there has not been a linkage to specific molecular mechanisms. For instance, dietary exposure to low levels of mercury has been shown to be damaging to neural development, but concomitant ingestion of n-3 PUFAs as occurs during consumption of fish, has been shown to counteract the detrimental effects. As the mechanisms mediating the neurotoxicity of environmental mercury are not fully delineated, it is difficult to conceptualize a testable molecular mechanism explaining how n-3 PUFAs negate its neurotoxic effects. However, environmental exposure to mercury also has been linked to increased autoimmunity. By way of a molecular understanding of this immuno-toxic association, disruption of CD95 signaling is well established as a triggering factor for autoimmunity, and we have previously shown that environmentally relevant in vitro and dietary exposures to mercury interfere with CD95 signaling. In particular we have shown that activation of caspase 8, as well as downstream activation of caspase 3, in response to CD95 agonist stimulation is depressed by mercury. More recently we have shown in vitro that the n-3 PUFA docosahexaenoic acid counteracts the negative effect of mercury on CD95 signaling by restoring caspase activity. We hypothesized that concomitant ingestion of n-3 PUFAs with mercury might be protective from the immuno-toxic effects of mercury, as it is with mercury's neuro-toxic effects, and in the case of immuno-toxicity this would be related to restoration of CD95 signal strength. We now show that dietary ingestion of n-3 PUFAs generally promotes CD95 signaling by upregulating caspase 8 activation. Apart from accounting for the ability of n-3 PUFAs to specifically counteract autoimmune sequelae of

  19. Dietary n-3 PUFAs augment caspase 8 activation in Staphylococcal aureus enterotoxin B stimulated T-cells.

    PubMed

    Gill, R; Jen, K L; McCabe, M J J; Rosenspire, A

    2016-10-15

    Epidemiological studies have linked consumption of n-3 PUFAs with a variety of beneficial health benefits, particularly with respect to putative anti-inflammatory effects. Unfortunately, many of these results remain somewhat controversial because in most instances there has not been a linkage to specific molecular mechanisms. For instance, dietary exposure to low levels of mercury has been shown to be damaging to neural development, but concomitant ingestion of n-3 PUFAs as occurs during consumption of fish, has been shown to counteract the detrimental effects. As the mechanisms mediating the neurotoxicity of environmental mercury are not fully delineated, it is difficult to conceptualize a testable molecular mechanism explaining how n-3 PUFAs negate its neurotoxic effects. However, environmental exposure to mercury also has been linked to increased autoimmunity. By way of a molecular understanding of this immuno-toxic association, disruption of CD95 signaling is well established as a triggering factor for autoimmunity, and we have previously shown that environmentally relevant in vitro and dietary exposures to mercury interfere with CD95 signaling. In particular we have shown that activation of caspase 8, as well as downstream activation of caspase 3, in response to CD95 agonist stimulation is depressed by mercury. More recently we have shown in vitro that the n-3 PUFA docosahexaenoic acid counteracts the negative effect of mercury on CD95 signaling by restoring caspase activity. We hypothesized that concomitant ingestion of n-3 PUFAs with mercury might be protective from the immuno-toxic effects of mercury, as it is with mercury's neuro-toxic effects, and in the case of immuno-toxicity this would be related to restoration of CD95 signal strength. We now show that dietary ingestion of n-3 PUFAs generally promotes CD95 signaling by upregulating caspase 8 activation. Apart from accounting for the ability of n-3 PUFAs to specifically counteract autoimmune sequelae of

  20. Staphylococcal Enterotoxin B (SEB) Induces Memory CD4 T Cell Anergy in vivo and Impairs Recall Immunity to Unrelated Antigens

    PubMed Central

    Janik, David K; Lee, William T

    2015-01-01

    Introduction Naïve and memory T cells can utilize unique regulatory pathways to promote protection but prevent self-reactivity. A bacterial superantigen SEB exploits unique TCR proximal signaling processes in memory CD4 T cells to induce clonal anergy. The aim of this study was to determine if SEB could antagonize memory CD4 T cells in vivo and whether there would be consequences on recall immune responses. We evaluated Ab responses to a T-dependent antigen as a measurement of memory T cell helper function. Method BALB/c mice were primed with TNP-RGG to elicit memory B cells and also immunized with an ovalbumin peptide to elicit memory helper T cells. Another group of TNP-RGG immunized mice were used as adoptive transfer recipients of exogenous DO11.10 memory T cells. Mice were challenged with TNP-OVA with or without prior administration of SEB. B cells secreting IgM or IgG TNP-specific Ab were enumerated by ELISPOT as indicators of primary versus secondary humoral immunity. Results Comparing the SEB and non-SEB-treated groups, the SEB-treated group failed to produce TNP-specific IgG in response to challenge with TNP-OVA, even if they were previously immunized with OVA. All groups produced IgM, indicating that the primary Ab responses and naïve helper T cells were not impacted by SEB. SEB had no negative impact when DO11.10 × Fyn−/− memory T cells were used as donor cells. Conclusion The present study indicated that SEB selectively targeted memory CD4 T cells in vivo and prevented helper function. Consequently, recall humoral immunity was lost. The data are most consistent with in vivo T cell anergy as opposed to indirect suppression as elimination of Fyn kinase restored helper function. These data suggest that bacterial superantigens can impair post-vaccination memory cell responses to unrelated antigens via their ability to target Vb families and antagonize memory cell activation. PMID:26807307

  1. Enterotoxin Gene Profile and Molecular Characterization of Staphylococcus aureus Isolates from Bovine Bulk Milk and Milk Products of Tigray Region, Northern Ethiopia.

    PubMed

    Tarekgne, Enquebaher K; Skjerdal, Taran; Skeie, Siv; Rudi, Knut; Porcellato, Davide; Félix, Benjamin; Narvhus, Judith A

    2016-08-01

    Staphylococcal food poisoning (SFP) is an important foodborne disease worldwide, and milk and milk products are commonly associated with SFP outbreaks. The objectives of this study were to investigate the distribution of staphylococcal enterotoxin (se) genes in Staphylococcus aureus from raw cow's milk and milk products and to assess their genetic background with the spa typing method. Of the 549 samples (297 bulk milk and 162 milk product samples) collected from Tigray region, Northern Ethiopia, 160 (29.1%) were positive for S. aureus, of which 82 (51%) were found to harbor se genes by a modified multiplex PCR. Nine se genes were identified: sea (n = 12), seb (n = 3), sec (n = 3), sed(n = 4), seg (n = 49), seh (n = 2), sei (n = 40), sej (n = 1), and tsst-1 (n = 24). The classical type of genes accounted for 27%. Of the 82 enterotoxigenic isolates, 41.5 and 12.4% harbored two or more se genes, respectively. The highest gene association was observed between sei and seg, whereas sea and seb were always found together with the new types of se genes. Altogether, 18 genotypes of toxin genes were identified, and 33% of the samples contained > 5 log CFU ml(-1) S. aureus. spa typing identified 22 spa types and three novel spa sequences, which showed the high genetic diversity of the isolates. No apparent relationship was observed between spa type and se genes. Of the 25 spa types, 13 (52%) were from raw milk, 3 (12%) from milk products, and 9 (36%) from both types of sample. Types t314 (20.7%,n = 17), t458 (18.3%, n = 15), and t6218 (9.8%, n= 8) were the most common spa types identified and were widely distributed in three of the eight studied localities. This is the first study from the Tigray region to report the high distribution of enterotoxigenic S. aureus with a diversified genetic background from dairy food. The study may provide valuable data for microbial food safety risk assessment, molecular epidemiology, and phylogenetic studies of S. aureus in Ethiopia.

  2. Natural indoles, indole-3-carbinol and 3,3′-diindolymethane, inhibit T cell activation by staphylococcal enterotoxin B through epigenetic regulation involving HDAC expression

    SciTech Connect

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

    2014-01-01

    Staphylococcal enterotoxin B (SEB) is a potent exotoxin produced by the Staphylococcus aureus. This toxin is classified as a superantigen because of its ability to directly bind with MHC-II class molecules followed by activation of a large proportion of T cells bearing specific Vβ-T cell receptors. Commonly associated with classic food poisoning, SEB has also been shown to induce toxic shock syndrome, and is also considered to be a potential biological warfare agent because it is easily aerosolized. In the present study, we assessed the ability of indole-3-carbinol (I3C) and one of its byproducts, 3,3′-diindolylmethane (DIM), found in cruciferous vegetables, to counteract the effects of SEB-induced activation of T cells in mice. Both I3C and DIM were found to decrease the activation, proliferation, and cytokine production by SEB-activated Vβ8{sup +} T cells in vitro and in vivo. Interestingly, inhibitors of histone deacetylase class I (HDAC-I), but not class II (HDAC-II), showed significant decrease in SEB-induced T cell activation and cytokine production, thereby suggesting that epigenetic modulation plays a critical role in the regulation of SEB-induced inflammation. In addition, I3C and DIM caused a decrease in HDAC-I but not HDAC-II in SEB-activated T cells, thereby suggesting that I3C and DIM may inhibit SEB-mediated T cell activation by acting as HDAC-I inhibitors. These studies not only suggest for the first time that plant-derived indoles are potent suppressors of SEB-induced T cell activation and cytokine storm but also that they may mediate these effects by acting as HDAC inhibitors. - Highlights: • I3C and DIM reduce SEB-induced T cell activation and inflammatory cytokines. • Inhibiting class I HDACs reduces T cell activation and inflammatory cytokines. • Inhibiting class II HDACs increases T cell activation and inflammatory cytokines. • I3C and DIM selectively reduce mRNA expression of class I HDACs. • Novel use and mechanism to counteract

  3. Enterotoxin Gene Profile and Molecular Characterization of Staphylococcus aureus Isolates from Bovine Bulk Milk and Milk Products of Tigray Region, Northern Ethiopia.

    PubMed

    Tarekgne, Enquebaher K; Skjerdal, Taran; Skeie, Siv; Rudi, Knut; Porcellato, Davide; Félix, Benjamin; Narvhus, Judith A

    2016-08-01

    Staphylococcal food poisoning (SFP) is an important foodborne disease worldwide, and milk and milk products are commonly associated with SFP outbreaks. The objectives of this study were to investigate the distribution of staphylococcal enterotoxin (se) genes in Staphylococcus aureus from raw cow's milk and milk products and to assess their genetic background with the spa typing method. Of the 549 samples (297 bulk milk and 162 milk product samples) collected from Tigray region, Northern Ethiopia, 160 (29.1%) were positive for S. aureus, of which 82 (51%) were found to harbor se genes by a modified multiplex PCR. Nine se genes were identified: sea (n = 12), seb (n = 3), sec (n = 3), sed(n = 4), seg (n = 49), seh (n = 2), sei (n = 40), sej (n = 1), and tsst-1 (n = 24). The classical type of genes accounted for 27%. Of the 82 enterotoxigenic isolates, 41.5 and 12.4% harbored two or more se genes, respectively. The highest gene association was observed between sei and seg, whereas sea and seb were always found together with the new types of se genes. Altogether, 18 genotypes of toxin genes were identified, and 33% of the samples contained > 5 log CFU ml(-1) S. aureus. spa typing identified 22 spa types and three novel spa sequences, which showed the high genetic diversity of the isolates. No apparent relationship was observed between spa type and se genes. Of the 25 spa types, 13 (52%) were from raw milk, 3 (12%) from milk products, and 9 (36%) from both types of sample. Types t314 (20.7%,n = 17), t458 (18.3%, n = 15), and t6218 (9.8%, n= 8) were the most common spa types identified and were widely distributed in three of the eight studied localities. This is the first study from the Tigray region to report the high distribution of enterotoxigenic S. aureus with a diversified genetic background from dairy food. The study may provide valuable data for microbial food safety risk assessment, molecular epidemiology, and phylogenetic studies of S. aureus in Ethiopia. PMID

  4. Heat-labile IgG2a antibodies affect cure of Trypanosoma musculi infection in C57BL/6 mice.

    PubMed

    Wechsler, D S; Kongshavn, P A

    1986-11-01

    Immune plasma (IP) obtained from mice cured of Trypanosoma musculi infection is able to mediate trypanosome clearance both in vivo and in vitro. A protein A-derived immunoglobulin fraction of IP containing primarily IgG2a and IgG3 shares this curative activity. Additional purification of IP with the use of anti-IgG2a and anti-IgG3 coupled to Sepharose beads demonstrates that the curative activity of IP resides solely in the IgG2a fraction; IP depleted of IgG2a is no longer able to effect T. musculi removal. Furthermore, this curative IgG2a is labile to heat treatment for 30 min at 56 degrees C. Enzyme-linked immunosorbent assays show that trypanosome-specific IgG2a builds up gradually over the course of infection, and temporarily drops slightly at the time of parasite clearance.

  5. Identification of enterotoxigenic Escherichia coli (ETEC) clades with long-term global distribution.

    PubMed

    von Mentzer, Astrid; Connor, Thomas R; Wieler, Lothar H; Semmler, Torsten; Iguchi, Atsushi; Thomson, Nicholas R; Rasko, David A; Joffre, Enrique; Corander, Jukka; Pickard, Derek; Wiklund, Gudrun; Svennerholm, Ann-Mari; Sjöling, Åsa; Dougan, Gordon

    2014-12-01

    Enterotoxigenic Escherichia coli (ETEC), a major cause of infectious diarrhea, produce heat-stable and/or heat-labile enterotoxins and at least 25 different colonization factors that target the intestinal mucosa. The genes encoding the enterotoxins and most of the colonization factors are located on plasmids found across diverse E. coli serogroups. Whole-genome sequencing of a representative collection of ETEC isolated between 1980 and 2011 identified globally distributed lineages characterized by distinct colonization factor and enterotoxin profiles. Contrary to current notions, these relatively recently emerged lineages might harbor chromosome and plasmid combinations that optimize fitness and transmissibility. These data have implications for understanding, tracking and possibly preventing ETEC disease. PMID:25383970

  6. The influence of headspace and dissolved oxygen level on growth and haemolytic BL enterotoxin production of a psychrotolerant Bacillus weihenstephanensis isolate on potato based ready-to-eat food products.

    PubMed

    Samapundo, S; Everaert, H; Wandutu, J N; Rajkovic, A; Uyttendaele, M; Devlieghere, F

    2011-04-01

    The major objective of this study was to determine the influence of the initial headspace and dissolved O(2) level and vacuum packaging on growth and diarrhoeal enterotoxin production by Bacillus weihenstephanensis on potato based ready-to-eat food products. In general, the lower the initial headspace or dissolved O(2) level the slower the maximum growth rate (μ(max), log(10) CFU g(-1) d(-1)), the longer the lag phase duration (λ, d) and the smaller the maximum population density (N(max), log(10) CFU g(-1)) became. The slowest μ(max), the longest λ and the smallest N(max) were generally found for growth under vacuum packaging. This implies shorter shelf-lives will occur at higher initial headspace or dissolved O(2) levels as the growth of B. weihenstephanensis to the infective dose of 10(5) CFU g(-1) in such atmospheres takes a shorter time. Significant consumption of dissolved O(2) only occurred when growth shifted from the lag to the exponential phase and growth generally transitioned from the exponential to the stationary phase when the dissolved O(2) levels fell below ca. 75 ppb. Diarrhoeal enterotoxin production (determined via detection of the L2 component of haemolytic BL) was similar for growth under initial headspace O(2) levels of 1-20.9%, and was only reduced when growth took place under vacuum packaging. The reduction in L2 production when growth took place under vacuum was most probably related to the low final cell densities observed under this condition. Both growth and L2 production were inhibited over a 32-day incubation period at 7 °C by 40% CO(2) irrespective of the headspace or dissolved O(2) levels. The results illustrate the importance of residual O(2) and CO(2) on the shelf-stability and safety of modified atmosphere packaged potato based ready-to-eat food products with regards to B. weihenstephanensis.

  7. Developing a Promotional Video

    ERIC Educational Resources Information Center

    Epley, Hannah K.

    2014-01-01

    There is a need for Extension professionals to show clientele the benefits of their program. This article shares how promotional videos are one way of reaching audiences online. An example is given on how a promotional video has been used and developed using iMovie software. Tips are offered for how professionals can create a promotional video and…

  8. Skin exposure promotes a Th2-dependent sensitization to peanut allergens.

    PubMed

    Tordesillas, Leticia; Goswami, Ritobrata; Benedé, Sara; Grishina, Galina; Dunkin, David; Järvinen, Kirsi M; Maleki, Soheila J; Sampson, Hugh A; Berin, M Cecilia

    2014-11-01

    Sensitization to foods often occurs in infancy, without a known prior oral exposure, suggesting that alternative exposure routes contribute to food allergy. Here, we tested the hypothesis that peanut proteins activate innate immune pathways in the skin that promote sensitization. We exposed mice to peanut protein extract on undamaged areas of skin and observed that repeated topical exposure to peanut allergens led to sensitization and anaphylaxis upon rechallenge. In mice, this epicutaneous peanut exposure induced sensitization to the peanut components Ara h 1 and Ara h 2, which is also observed in human peanut allergy. Both crude peanut extract and Ara h 2 alone served as adjuvants, as both induced a bystander sensitization that was similar to that induced by the atopic dermatitis-associated staphylococcal enterotoxin B. In cultured human keratinocytes and in murine skin, peanut extract directly induced cytokine expression. Moreover, topical peanut extract application induced an alteration dependent on the IL-33 receptor ST2 in skin-draining DCs, resulting in Th2 cytokine production from T cells. Together, our data support the hypothesis that peanuts are allergenic due to inherent adjuvant activity and suggest that skin exposure to food allergens contributes to sensitization to foods in early life. PMID:25295541

  9. Skin exposure promotes a Th2-dependent sensitization to peanut allergens

    PubMed Central

    Tordesillas, Leticia; Goswami, Ritobrata; Benedé, Sara; Grishina, Galina; Dunkin, David; Järvinen, Kirsi M.; Maleki, Soheila J.; Sampson, Hugh A.; Berin, M. Cecilia

    2014-01-01

    Sensitization to foods often occurs in infancy, without a known prior oral exposure, suggesting that alternative exposure routes contribute to food allergy. Here, we tested the hypothesis that peanut proteins activate innate immune pathways in the skin that promote sensitization. We exposed mice to peanut protein extract on undamaged areas of skin and observed that repeated topical exposure to peanut allergens led to sensitization and anaphylaxis upon rechallenge. In mice, this epicutaneous peanut exposure induced sensitization to the peanut components Ara h 1 and Ara h 2, which is also observed in human peanut allergy. Both crude peanut extract and Ara h 2 alone served as adjuvants, as both induced a bystander sensitization that was similar to that induced by the atopic dermatitis-associated staphylococcal enterotoxin B. In cultured human keratinocytes and in murine skin, peanut extract directly induced cytokine expression. Moreover, topical peanut extract application induced an alteration dependent on the IL-33 receptor ST2 in skin-draining DCs, resulting in Th2 cytokine production from T cells. Together, our data support the hypothesis that peanuts are allergenic due to inherent adjuvant activity and suggest that skin exposure to food allergens contributes to sensitization to foods in early life. PMID:25295541

  10. Health promotion in Brazil.

    PubMed

    Buss, Paulo Marchiori; de Carvalho, Antonio Ivo

    2007-01-01

    The evolution of health promotion within the Brazilian health system is examined, including an assessment of the intersectoral and development policies that have influenced the process. Particular attention is paid to the legal characteristics of the Unified Health System. Human resources formation and research initiatives in health promotion are outlined, with a summary of the obstacles that need to be overcome in order to ensure the effective implementation of health promotion in the future. Up to the end of the 20th Century health promotion was not used as a term in the Brazilian public heath context. Health promoting activities were concentrated in the area of health education, although targeting the social determinants of health and the principle of intersectoral action were part of the rhetoric. The situation has changed during the last decade, with the publication of a national policy of health promotion, issued by the Ministry of Health and jointly implemented with the States and Municipals Health Secretaries. More recently there has been a re-emergence of the discourse on the social determinants of health and the formation of intersectoral public policies as the basis of a comprehensive health promotion. Health promotion infrastructure, particularly around human resources and financing, requires strengthening in order to ensure capacity and sustainability in health promotion practice. PMID:18372870

  11. Health promotion in Brazil.

    PubMed

    Buss, Paulo Marchiori; de Carvalho, Antonio Ivo

    2007-01-01

    The evolution of health promotion within the Brazilian health system is examined, including an assessment of the intersectoral and development policies that have influenced the process. Particular attention is paid to the legal characteristics of the Unified Health System. Human resources formation and research initiatives in health promotion are outlined, with a summary of the obstacles that need to be overcome in order to ensure the effective implementation of health promotion in the future. Up to the end of the 20th Century health promotion was not used as a term in the Brazilian public heath context. Health promoting activities were concentrated in the area of health education, although targeting the social determinants of health and the principle of intersectoral action were part of the rhetoric. The situation has changed during the last decade, with the publication of a national policy of health promotion, issued by the Ministry of Health and jointly implemented with the States and Municipals Health Secretaries. More recently there has been a re-emergence of the discourse on the social determinants of health and the formation of intersectoral public policies as the basis of a comprehensive health promotion. Health promotion infrastructure, particularly around human resources and financing, requires strengthening in order to ensure capacity and sustainability in health promotion practice.

  12. Investigating potential exogenous tumor initiating and promoting factors for Cutaneous T-Cell Lymphomas (CTCL), a rare skin malignancy.

    PubMed

    Litvinov, Ivan V; Shtreis, Anna; Kobayashi, Kenneth; Glassman, Steven; Tsang, Matthew; Woetmann, Anders; Sasseville, Denis; Ødum, Niels; Duvic, Madeleine

    2016-07-01

    Most skin malignancies are caused by external and often preventable environmental agents. Multiple reports demonstrated that cutaneous T-cell lymphomas (CTCL) can occur in married couples and cluster in families. Furthermore, recent studies document geographic clustering of this malignancy in Texas as well as in other areas of the United States. Multiple infectious, occupational, and medication causes have been proposed as triggers or promoters of this malignancy including hydrochlorothiazide diuretics, Staphylococcus aureus, dermatophytes, Mycobacterium leprae, Chlamydia pneumoniae, human T-Cell lymphotropic virus type 1 (HTLV1), Epstein-Barr virus (EBV), and herpes simplex virus (HSV). In this report, we review recent evidence evaluating the involvement of these agents in cancer initiation/progression. Most importantly, recent molecular experimental evidence documented for the first time that S. aureus can activate oncogenic STAT3 signaling in malignant T cells. Specifically, S. aureus Enterotoxin type A (SEA) was recently shown to trigger non-malignant infiltrating T cells to release IL-2 and other cytokines. These signals upon binging to their cognate receptors on malignant T cells are then able to activate STAT3 and STAT5 oncogenic signaling and promote cancer progression and IL-17 secretion. In light of these findings, it might be important for patients with exacerbation of their CTCL symptoms to maintain high index of suspicion and treat these individuals for S. aureus colonization and/or sepsis with topical and systemic antibiotics. PMID:27622024

  13. High expression Zymomonas promoters

    DOEpatents

    Viitanen, Paul V.; Tao, Luan; Zhang, Yuying; Caimi, Perry G.; McCole, Laura : Zhang, Min; Chou, Yat-Chen; McCutchen, Carol M.; Franden, Mary Ann

    2011-08-02

    Identified are mutants of the promoter of the Z. mobilis glyceraldehyde-3-phosphate dehydrogenase gene, which direct improved expression levels of operably linked heterologous nucleic acids. These are high expression promoters useful for expression of chimeric genes in Zymomonas, Zymobacter, and other related bacteria.

  14. Health Promotion Program.

    ERIC Educational Resources Information Center

    McClary, Cheryl

    The Health Promotion Program began with establishment of a one-credit course in health promotion and wellness and the training of family practice residents at the Mountain Area Health Education Center to serve as lab leaders in the course. The course later became part of the university's general education requirements. In addition, a health…

  15. Minimizing Promotion Trauma.

    ERIC Educational Resources Information Center

    Darling, LuAnn W.; McGrath, Loraine

    1983-01-01

    Nursing administrators can minimize promotion trauma and its unnecessary cost by building awareness of the transition process, clarifying roles and expectations, and attending to the promoted employee's needs. This article will help nursing administrators develop a concept of manager care combined with programs for orientation of new managers,…

  16. 77 FR 47820 - Invention Promoters/Promotion Firms Complaints

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-10

    ... United States Patent and Trademark Office Invention Promoters/Promotion Firms Complaints ACTION: Proposed... concerning invention promoters and responses from the invention promoters to these complaints. An individual may submit a complaint concerning an invention promoter to the USPTO, which will forward the...

  17. IscR Regulates Synthesis of Colonization Factor Antigen I Fimbriae in Response to Iron Starvation in Enterotoxigenic Escherichia coli

    PubMed Central

    Haines, Sara; Arnaud-Barbe, Nadège; Poncet, David; Reverchon, Sylvie; Wawrzyniak, Julien; Nasser, William

    2015-01-01

    ABSTRACT Iron availability functions as an environmental cue for enteropathogenic bacteria, signaling arrival within the human host. As enterotoxigenic Escherichia coli (ETEC) is a major cause of human diarrhea, the effect of iron on ETEC virulence factors was evaluated here. ETEC pathogenicity is directly linked to production of fimbrial colonization factors and secretion of heat-labile enterotoxin (LT) and/or heat-stable enterotoxin (ST). Efficient colonization of the small intestine further requires at least the flagellin binding adhesin EtpA. Under iron starvation, production of the CFA/I fimbriae was increased in the ETEC H10407 prototype strain. In contrast, LT secretion was inhibited. Furthermore, under iron starvation, gene expression of the cfa (CFA/I) and etp (EtpBAC) operons was induced, whereas transcription of toxin genes was either unchanged or repressed. Transcriptional reporter fusion experiments focusing on the cfa operon further showed that iron starvation stimulated cfaA promoter activity in ETEC, indicating that the impact of iron on CFA/I production was mediated by transcriptional regulation. Evaluation of cfaA promoter activity in heterologous E. coli single mutant knockout strains identified IscR as the regulator responsible for inducing cfa fimbrial gene expression in response to iron starvation, and this was confirmed in an ETEC ΔiscR strain. The global iron response regulator, Fur, was not implicated. IscR binding sites were identified in silico within the cfaA promoter and fixation confirmed by DNase I footprinting, indicating that IscR directly binds the promoter region to induce CFA/I. IMPORTANCE Pathogenic enterobacteria modulate expression of virulence genes in response to iron availability. Although the Fur transcription factor represents the global regulator of iron homeostasis in Escherichia coli, we show that several ETEC virulence factors are modulated by iron, with expression of the major fimbriae under the control of the iron

  18. Organization and ELISA-Based Results of the First Proficiency Testing to Evaluate the Ability of European Union Laboratories to Detect Staphylococcal Enterotoxin Type B (SEB) in Buffer and Milk

    PubMed Central

    Nia, Yacine; Rodriguez, Mélanie; Zeleny, Reinhard; Herbin, Sabine; Auvray, Frédéric; Fiebig, Uwe; Avondet, Marc-André; Munoz, Amalia; Hennekinne, Jacques-Antoine

    2016-01-01

    The aim of this work was to organize the first proficiency test (PT) dedicated to staphylococcal enterotoxin B (SEB) detection in milk and buffer solutions. This paper describes the organization of the PT trial according to EN ISO 17043 requirements. Characterization of the SEB stock solution was performed using SDS-PAGE and SE-specific ELISA, and amino acid analysis was used to assign its protein concentration. The solution was then used to prepare six PT materials (four milk and two buffer batches) at a ng/g toxin level, which included one blank and one SEA-containing milk as specificity control. Suitable material homogeneity and stability were assessed using screening and quantitative ELISAs. Among the methods used by the participants, ELISA-based methods demonstrated their efficiency for the detection of SEB in both simple and complex matrices. The results serve as a basis for further improving the detection capabilities in expert laboratories and can therefore be considered as a contribution to biopreparedness. PMID:27649244

  19. Comparison of Dissociation-Enhanced Lanthanide Fluorescent Immunoassays to Enzyme-Linked Immunosorbent Assays for Detection of Staphylococcal Enterotoxin B, Yersinia pestis-Specific F1 Antigen, and Venezuelan Equine Encephalitis Virus

    PubMed Central

    Smith, Darci R.; Rossi, Cynthia A.; Kijek, Todd M.; Henchal, Erik A.; Ludwig, George V.

    2001-01-01

    The dissociation-enhanced lanthanide fluorescent immunoassays (DELFIA) were developed for the detection of staphylococcal enterotoxin B, Yersinia pestis-specific F1 antigen, and Venezuelan equine encephalitis virus. These assays were compared to previously developed enzyme-linked immunosorbent assays (ELISAs) by determining the sensitivity or limit of detection (LOD), the dynamic range, and the reproducibility of each assay in a number of different sample matrices. The sensitivity and specificity of each assay were then determined by using a small panel of blinded spiked and nonspiked samples. All three DELFIAs demonstrated at least 1 log greater sensitivity than corresponding ELISAs utilizing the same reagents and showed an increase in dynamic range of at least 2 log10 concentrations. This increased LOD resulted in higher sensitivity rates for the DELFIA. The specificity of all of the assays evaluated was 100%, and no sample matrix effects were observed in either format. However, the reproducibility of the DELFIA was poor due to randomly distributed wells exhibiting excessive background signal (hot wells), which occurred throughout the evaluation. As this technology matures, the reproducibility of these assays should improve, as will the ability to identify hot wells. Despite its sensitivity, the logistical burden associated with the DELFIA and the technical expertise required to complete assays and interpret the data limit the application of this technology to reference or large clinical laboratories. PMID:11687442

  20. Organization and ELISA-Based Results of the First Proficiency Testing to Evaluate the Ability of European Union Laboratories to Detect Staphylococcal Enterotoxin Type B (SEB) in Buffer and Milk.

    PubMed

    Nia, Yacine; Rodriguez, Mélanie; Zeleny, Reinhard; Herbin, Sabine; Auvray, Frédéric; Fiebig, Uwe; Avondet, Marc-André; Munoz, Amalia; Hennekinne, Jacques-Antoine

    2016-01-01

    The aim of this work was to organize the first proficiency test (PT) dedicated to staphylococcal enterotoxin B (SEB) detection in milk and buffer solutions. This paper describes the organization of the PT trial according to EN ISO 17043 requirements. Characterization of the SEB stock solution was performed using SDS-PAGE and SE-specific ELISA, and amino acid analysis was used to assign its protein concentration. The solution was then used to prepare six PT materials (four milk and two buffer batches) at a ng/g toxin level, which included one blank and one SEA-containing milk as specificity control. Suitable material homogeneity and stability were assessed using screening and quantitative ELISAs. Among the methods used by the participants, ELISA-based methods demonstrated their efficiency for the detection of SEB in both simple and complex matrices. The results serve as a basis for further improving the detection capabilities in expert laboratories and can therefore be considered as a contribution to biopreparedness. PMID:27649244

  1. Claudin-4 Overexpression in Epithelial Ovarian Cancer Is Associated with Hypomethylation and Is a Potential Target for Modulation of Tight Junction Barrier Function Using a C-Terminal Fragment of Clostridium perfringens Enterotoxin1

    PubMed Central

    Litkouhi, Babak; Kwong, Joseph; Lo, Chun-Min; Smedley, James G; McClane, Bruce A; Aponte, Margarita; Gao, Zhijian; Sarno, Jennifer L; Hinners, Jennifer; Welch, William R; Berkowitz, Ross S; Mok, Samuel C; Garner, Elizabeth I O

    2007-01-01

    Background Claudin-4, a tight junction (TJ) protein and receptor for the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), is overexpressed in epithelial ovarian cancer (EOC). Previous research suggests DNA methylation is a mechanism for claudin-4 overexpression in cancer and that C-CPE acts as an absorption-enhancing agent in claudin-4-expressing cells. We sought to correlate claudin-4 overexpression in EOC with clinical outcomes and TJ barrier function, investigate DNA methylation as a mechanism for overexpression, and evaluate the effect of C-CPE on the TJ. Methods Claudin-4 expression in EOC was quantified and correlated with clinical outcomes. Claudin-4 methylation status was determined, and claudin-4-negative cell lines were treated with a demethylating agent. Electric cell-substrate impedance sensing was used to calculate junctional (paracellular) resistance (Rb) in EOC cells after claudin-4 silencing and after C-CPE treatment. Results Claudin-4 overexpression in EOC does not correlate with survival or other clinical endpoints and is associated with hypomethylation. Claudin-4 overexpression correlates with Rb and C-CPE treatment of EOC cells significantly decreased Rb in a dose- and claudin-4-dependent noncytotoxic manner. Conclusions C-CPE treatment of EOC cells leads to altered TJ function. Further research is needed to determine the potential clinical applications of C-CPE in EOC drug delivery strategies. PMID:17460774

  2. Promoting People's Participation.

    ERIC Educational Resources Information Center

    Fraser, Colin

    1981-01-01

    Discusses problems associated with communication in rural areas to promote participation in development programs. Suggests that success of such programs depends on continued government policy in favor of citizen participation in agricultural and rural development. (SK)

  3. [Health promotion. Concept development].

    PubMed

    Sito, A; Berkowska, M

    2000-01-01

    The development of health promotion in theory and practice is presented-from the Ottawa Charter in 1986, to the community based health promotion programmes, as the vision for the 21 Century. The historical mile stones in the process of change and the conceptualisation of health promotion are discussed with reference to the World WHO Conferences and documents from these conferences. These events and documents have been vital as guidelines for member countries, both for implementation of community based programmes, as well as for healthy public policy and for training, especially concerning evaluation. The paper also discusses the main trends in research; definitions of principal concepts are highlighted, concerning planning, implementation and evaluation of health promotion programmes.

  4. Promoting Your Web Site.

    ERIC Educational Resources Information Center

    Raeder, Aggi

    1997-01-01

    Discussion of ways to promote sites on the World Wide Web focuses on how search engines work and how they retrieve and identify sites. Appropriate Web links for submitting new sites and for Internet marketing are included. (LRW)

  5. Promoting Global Health

    PubMed Central

    Winker, Margaret A.; Ferris, Lorraine E.

    2015-01-01

    The Editor-in-Chief of the International Journal of MCH and AIDS (IJMA) is a member of the World Association of Medical Editors (WAME). The Editorial Board of IJMA believes it is important that the statement on promoting global health and this accompanying editorial is brought to the attention of our readers. Medical journal editors have a social responsibility to promote global health by publishing, whenever possible, research that furthers health worldwide.

  6. Health promotion in Brazil.

    PubMed

    Ivo de Carvalho, Antonio; Westphal, Marcia Faria; Pereira Lima, Vera Lucia Góes

    2007-01-01

    Brazil, a Latin American country of continental proportions and contrasts, demographic inequalities, and social inequities, concomitantly faces the challenge of preventing and controlling infectious diseases, injuries, and non-communicable diseases. The loss of strength of the biomedical paradigm, the change in epidemiological profile, and the sociopolitical and cultural challenges of recent decades have fostered the emergence of new formulations about public health thinking and practice. Among them, are the paradigms of Brazilian Collective Health and Health Promotion. The former provides philosophical support for Brazil's Unified Health System (SUS). The aim of this article is to discuss the development of public health within the country's history, and to analyze and compare the theoretical assumptions of Health Promotion and Collective Health. We conclude that health promotion, based on the principles and values disseminated by the international Charters and concerned with social actors and social determinants of the health-disease process, has significant potential to promote the improvement of living and health conditions of the population. This frame of reference guided the formulation of the National Policy of Health Promotion within the Unified Health System, which was institutionalized by a ministerial decree. The importance and application of evaluating the effectiveness of health promotion processes and methodologies in Brazil have been guided by various frames of reference, which we clarify in this article through describing historical processes.

  7. Health promotion in Brazil.

    PubMed

    Ivo de Carvalho, Antonio; Westphal, Marcia Faria; Pereira Lima, Vera Lucia Góes

    2007-01-01

    Brazil, a Latin American country of continental proportions and contrasts, demographic inequalities, and social inequities, concomitantly faces the challenge of preventing and controlling infectious diseases, injuries, and non-communicable diseases. The loss of strength of the biomedical paradigm, the change in epidemiological profile, and the sociopolitical and cultural challenges of recent decades have fostered the emergence of new formulations about public health thinking and practice. Among them, are the paradigms of Brazilian Collective Health and Health Promotion. The former provides philosophical support for Brazil's Unified Health System (SUS). The aim of this article is to discuss the development of public health within the country's history, and to analyze and compare the theoretical assumptions of Health Promotion and Collective Health. We conclude that health promotion, based on the principles and values disseminated by the international Charters and concerned with social actors and social determinants of the health-disease process, has significant potential to promote the improvement of living and health conditions of the population. This frame of reference guided the formulation of the National Policy of Health Promotion within the Unified Health System, which was institutionalized by a ministerial decree. The importance and application of evaluating the effectiveness of health promotion processes and methodologies in Brazil have been guided by various frames of reference, which we clarify in this article through describing historical processes. PMID:17596091

  8. Purification and characterisation of a fimbrial haemagglutinin from Bordetella pertussis for use in an enzyme-linked immunosorbent assay.

    PubMed

    Askelöf, P; Granström, M; Gillenius, P; Lindberg, A A

    1982-02-01

    The fimbrial haemagglutinin (F-HA) of Bordetella pertussis grown on solid medium was extracted with 1M sodium acetate for 72 h at 20 degree C, and partially purified by Sephacryl S-300 gel chromatography. A pooled fraction with fimbrial haemmagglutinating activity was shown to contain fimbriae haemagglutinating activity was shown to contain fimbriae of the expected morphology by electron microscopy. Chemical and biological assays showed that the F-HA fraction contained some heat-labile agglutinogen and lipopolysaccharide but no measureable lymphocytosis-promoting factor or heat-labile toxin. The F-HA fraction used as antigen in an enzyme-linked immunosorbent assay (ELISA) permitted the detection of antibodies in convalescent serum from a patient with whooping cough. The impurities, heat-labile agglutinogens and lipopolysaccharide, did not contribute to the ELISA activity. The method for preparation of the F-HA antigen is simple, reproducible and gives a high yield. PMID:6292428

  9. [Growth promoting antimicrobials].

    PubMed

    Hoogkamp-Korstanje, J A

    1999-06-19

    The committee 'Growth promoting antimicrobials' of the Health Council of the Netherlands in 1998 advised immediate prohibition of all growth promoting antimicrobials related to human drugs and decrease of use of other growth promoting antimicrobials during the next three years in Europe. It is clear that frequent use of antibiotics is associated with development of resistance by selection in animals (and man), but it is not proven that this is an explanation of resistance in the human community. We know only little about the mechanisms and conditions of transfer of bacteria to man. Other questions raised are: what about the resulting possible increase of therapeutic application of antibiotics in animals, how to handle the increase of dung, nitrogen and phosphate in the environment and how farmers can survive with a decrease in income, sometimes by as much as 50%? Although many will feel sympathy for the report and its recommendations, implementing them will be difficult and possibly premature. PMID:10416481

  10. [Advances in new vaccines against human enterotoxigenic Escherichia coli--A review].

    PubMed

    Xia, Pengpeng; Meng, Xianchen; Zhu, Guoqiang

    2016-02-01

    Enterotoxigenic Escherichia coli (ETEC) is the most common cause of diarrhea, which is a second leading cause of death for the children under five years old from all over the world. The key factors of ETEC contain both colonization factors (CFs) and enterotoxins including heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST). CFs mediated the binding of bacteria to the host intestinal epithelial cells, whereas LT and ST stimulated the over-secretion of body fluids and electrolytes, resulting in the destruction of the host fluid balance and leading diarrhea. The vaccine against CFs and enterotoxins could stimulate the host immune response, blocking ETEC adhesion and neutralizing enterotoxins, which is effective in the prevention of ETEC diarrhea. For the moment, depending on the stimulated immune response against LT, a cholera vaccine called Dukoral has been approved for use in some countries for the short-term protection and prevention of travelers' diarrhea. ETEC candidate vaccines are still in progress, which is designed to provide a long and wide-spectrum protection for ETEC infections. This paper briefly summarizes the advanced findings and key problems of vaccine development, and discusses prospects for future research. PMID:27373068

  11. [Advances in new vaccines against human enterotoxigenic Escherichia coli--A review].

    PubMed

    Xia, Pengpeng; Meng, Xianchen; Zhu, Guoqiang

    2016-02-01

    Enterotoxigenic Escherichia coli (ETEC) is the most common cause of diarrhea, which is a second leading cause of death for the children under five years old from all over the world. The key factors of ETEC contain both colonization factors (CFs) and enterotoxins including heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST). CFs mediated the binding of bacteria to the host intestinal epithelial cells, whereas LT and ST stimulated the over-secretion of body fluids and electrolytes, resulting in the destruction of the host fluid balance and leading diarrhea. The vaccine against CFs and enterotoxins could stimulate the host immune response, blocking ETEC adhesion and neutralizing enterotoxins, which is effective in the prevention of ETEC diarrhea. For the moment, depending on the stimulated immune response against LT, a cholera vaccine called Dukoral has been approved for use in some countries for the short-term protection and prevention of travelers' diarrhea. ETEC candidate vaccines are still in progress, which is designed to provide a long and wide-spectrum protection for ETEC infections. This paper briefly summarizes the advanced findings and key problems of vaccine development, and discusses prospects for future research.

  12. Preventing Injuries, Promoting Creativity

    ERIC Educational Resources Information Center

    Maloney, Betsy

    2004-01-01

    Simultaneously promoting a safe learning environment that encourages students to express their creative voices while exploring the rudiments of dance technique can be difficult. This article discusses how the author has learned to do it effectively by employing a few simple classroom strategies. To prevent injuries during class, the author offers…

  13. Partners: Promoting Accessible Recreation.

    ERIC Educational Resources Information Center

    Sable, Janet; Gravink, Jill

    1995-01-01

    The Promoting Accessible Recreation through Networking, Education, Resources and Services (PARTNERS) Project, a partnership between Northeast Passage, the University of New Hampshire, and Granite State Independent Living Foundation, helps create barrier-free recreation for individuals with physical disabilities. The paper describes PARTNERS and…

  14. Promoting Continuing Education Programs.

    ERIC Educational Resources Information Center

    Hendrickson, Gayle A.

    This handbook is intended for use by institutions in marketing their continuing education programs. A section on "Devising Your Strategy" looks at identifying a target audience, determining the marketing approach, and developing a marketing plan and promotional techniques. A discussion of media options looks at the advantages and disadvantages of…

  15. Dynamics of Promotion Decisions.

    ERIC Educational Resources Information Center

    Rix, Elizabeth Ann

    This study attempted to identify the criteria educational administrators use when selecting individuals for promotion. Researchers compared "operative criteria" (those actually used) with "posted criteria" (those espoused). Interviews were conducted with 92 school administrators. Brown's Form PROM, derived from Kelly's role construct repertory…

  16. Promoting La Cultura Hispana

    ERIC Educational Resources Information Center

    Pluviose, David

    2007-01-01

    Launched in 1985 at Arizona State University, the Hispanic Research Center's (HRC) efforts to promote Latino and Chicano art and issues have flourished in recent years. In 2004, the HRC hosted the Arizona International Latina/o Arts Festival in collaboration with the Mesa Southwest Museum. The HRC has also founded a mentoring institute for…

  17. Health Promotion Interventions.

    ERIC Educational Resources Information Center

    Jason, Leonard A.; Curie, Carrie J.; Townsend, Stephanie M.; Pokorny, Steven B.; Katz, Richard B.; Sherk, Joseph L.

    2002-01-01

    Reviews four areas from the prevention science field, including: promoting healthy behavior; preventing substance abuse; preventing high-risk sexual behaviors; and preventing child abuse and sexual abuse. Recommendations are made regarding strategies for implementing empirically validated programs, supplementing school programs with ecological…

  18. Etiology of summer diarrhea among the Navajo.

    PubMed

    Hughes, J M; Rouse, J D; Barada, F A; Guerrant, R L

    1980-07-01

    The etiology of diarrhea in children and adults on the Navajo Indian Reservation was investigated in August 1975. Fifty-six ill individuals and 37 controls were included in the study. Shigella was most commonly associated with diarrhea, and was isolated from 32% of ill children and adults. Fifty percent of Shigella isolates tested were resistant to ampicillin. Heat-stable enterotoxin-(ST)-producing organisms were associated with noninflammatory diarrhea in adults (27% of these cases had ST-producing strains) but not in children. Heat-labile enterotoxin-producing organisms were found among controls as well as individuals with diarrhea. No children had evidence of rotavirus infection. These findings suggest that ST-producing organisms are important causes of sporadic cases of noninflammatory summer diarrhea among Navajo adults and confirm the importance of Shigella in inflammatory diarrhea among adults and children in this setting. PMID:7406112

  19. Diagnosis and Treatment of Acute or Persistent Diarrhea

    PubMed Central

    Pawlowski, Sean W; Warren, Cirle Alcantara; Guerrant, Richard

    2009-01-01

    Studies of microbial pathogens and the toxins they produce are important for determining the mechanisms by which they cause disease and spread throughout a population. Some bacteria produce secretory enterotoxins (such as choleratoxin or the heat-labile or stable enterotoxins produced by E. coli) that invade cells directly. Others produce cytotoxins (such as those produced by Shigella, enteroinvasive E. coli, or C. difficile) that damage cells or trigger host responses that cause small or large bowel diseases (such as enteroaggregative or enteropathogenic E. coli or Salmonella). Viruses (such as noroviruses and rotaviruses) and protozoa (such as Cryptosporidium, Giardia or Entameba histolytica) disrupt cell functions and cause short- or long-term disease. Much epidemiological data about these pathogens have been collected from community- and hospital-acquired settings, as well from patients with traveler’s or persistent diarrhea. These studies have led to practical approaches for prevention, diagnosis and treatment. PMID:19457416

  20. The promotion of breastfeeding.

    PubMed

    Tuluhungwa, R R; Yung, W

    1979-01-01

    To reverse the current trend of a significant decline worldwide in breast feeding means reeducation of medical and health personnel as well as the general public. Programs to promote breast feeding require the commitment of governments, with support from various ministries including health, education, labor, community development and judiciary. Examples of what 3 developing countries--Jamaica, Colombia and Thailand--are doing to promote breast feeding are reported. A large scale breast feeding campaign was launched in Jamaica in October 1977. The 3 phases of the campaign were: 1) preliminary surveys and research and motivation of professional, voluntary and extension groups through training seminars, panel discussions, and meetings; 2) promotion of breast feeding via mass media and motivation of target groups by trained personnel; and 3) evaluation of the campaign. A survey undertaken in 1978 showed that the breast feeding messages had achieved the desired effect--more mothers practiced breast feeding. In Colombia the breast feeding campaign emphasized non-formal education through the use of games and pictures. A game is used which is usually initiated by a health worker in the waiting room of a health center and involves the mothers, the general public, and sometimes the professional personnel. Through reading and interpreting rhymed breast feeding messages, the participants exchange opinions and experiences. Before starting a campaign to encourage low-income urban and semi-urban mothers to breast feed, the National Food and Nutrition Committee of Thailand pretested slogans and posters designed for the promotion of breast feeding. Posters develpoed in accordance with the suggestions made by the women were tested among 126 pregnant and lactating women. The Committee decided which picture to print for low-income and rural audiences and which to print for middle-class audiences. PMID:12336781

  1. Bicycle Promotion Plan

    SciTech Connect

    Simone, G. A.

    1981-03-09

    The objective of this Bicycle Promotion Plan is to outline a set of recommendations and supporting strategies for implementation by the US DOE toward increased use of the bicycle for energy conservation. The recommendations are designed in such a way as to function in concert with: (1) bicycle programs administered by other Federal government agencies; and (2) related programs and activities already sponsored by DOE. The approach to preparation of the Plan involved a review of all current and planned bicycle promotion programs at the Federal level as well as a review of the array of lierature on the subject. The UniWorld project staff also interacted with several DOE program offices, in order to determine the extent to which they might appropriately contribute to the implementation of bicycle promotional efforts. A synthesis of all the information gathered was published in January of 1981 as a part of the project (The Bicycle Program Review). Based upon this information and an examination of the barriers to bicycle use identified by bicycle transportation specialists in the field, UniWorld developed a series of the most potentially effective recommendations and program strategies for implementation by DOE. The recommendations address activities that could be undertaken in conjunction with existing DOE programs, new developments that might be considered to fulfill critical needs in the field, and interagency efforts that DOE could play a role in.

  2. Promoter propagation in prokaryotes

    PubMed Central

    Matus-Garcia, Mariana; Nijveen, Harm; van Passel, Mark W. J.

    2012-01-01

    Transcriptional activation or ‘rewiring’ of silent genes is an important, yet poorly understood, phenomenon in prokaryotic genomes. Anecdotal evidence coming from experimental evolution studies in bacterial systems has shown the promptness of adaptation upon appropriate selective pressure. In many cases, a partial or complete promoter is mobilized to silent genes from elsewhere in the genome. We term hereafter such recruited regulatory sequences as Putative Mobile Promoters (PMPs) and we hypothesize they have a large impact on rapid adaptation of novel or cryptic functions. Querying all publicly available prokaryotic genomes (1362) uncovered >4000 families of highly conserved PMPs (50 to 100 long with ≥80% nt identity) in 1043 genomes from 424 different genera. The genomes with the largest number of PMP families are Anabaena variabilis (28 families), Geobacter uraniireducens (27 families) and Cyanothece PCC7424 (25 families). Family size varied from 2 to 93 homologous promoters (in Desulfurivibrio alkaliphilus). Some PMPs are present in particular species, but some are conserved across distant genera. The identified PMPs represent a conservative dataset of very recent or conserved events of mobilization of non-coding DNA and thus they constitute evidence of an extensive reservoir of recyclable regulatory sequences for rapid transcriptional rewiring. PMID:22933716

  3. The dynamics of mobile promoters: Enhanced stability in promoter regions.

    PubMed

    Rabbani, Mahnaz; Wahl, Lindi M

    2016-10-21

    Mobile promoters are emerging as a new class of mobile genetic elements, first identified by examining prokaryote genome sequences, and more recently confirmed by experimental observations in bacteria. Recent datasets have identified over 40,000 putative mobile promoters in sequenced prokaryote genomes, however only one-third of these are in regions of the genome directly upstream from coding sequences, that is, in promoter regions. The presence of many promoter sequences in non-promoter regions is unexplained. Here we develop a general mathematical model for the dynamics of mobile promoters, extending previous work to capture the dynamics both within and outside promoter regions. From this general model, we apply rigorous model selection techniques to identify which parameters are statistically justified in describing the available mobile promoter data, and find best-fit values of these parameters. Our results suggest that high rates of horizontal gene transfer maintain the population of mobile promoters in promoter regions, and that once established at these sites, mobile promoters are rarely lost, but are commonly copied to other genomic regions. In contrast, mobile promoter copies in non-promoter regions are more numerous and more volatile, experiencing substantially higher rates of duplication, loss and diversification. PMID:27460588

  4. Molecular Detection, Quantification, and Toxigenicity Profiling of Aeromonas spp. in Source- and Drinking-Water

    PubMed Central

    Robertson, Boakai K; Harden, Carol; Selvaraju, Suresh B; Pradhan, Suman; Yadav, Jagjit S

    2014-01-01

    Aeromonas is ubiquitous in aquatic environments and has been associated with a number of extra-gastrointestinal and gastrointestinal illnesses. This warrants monitoring of raw and processed water sources for pathogenic and toxigenic species of this human pathogen. In this study, a total of 17 different water samples [9 raw and 8 treated samples including 4 basin water (partial sand filtration) and 4 finished water samples] were screened for Aeromonas using selective culturing and a genus-specific real-time quantitative PCR assay. The selective culturing yielded Aeromonas counts ranging 0 – 2 x 103CFU/ml and 15 Aeromonas isolates from both raw and treated water samples. The qPCR analysis indicated presence of a considerable nonculturable population (3.4 x 101 – 2.4 x 104 cells/ml) of Aeromonas in drinking water samples. Virulence potential of the Aeromonas isolates was assessed by multiplex/singleplex PCR-based profiling of the hemolysin and enterotoxin genes viz cytotoxic heat-labile enterotoxin (act), heat-labile cytotonic enterotoxin (alt), heat-stable cytotonic enterotoxin (ast), and aerolysin (aerA) genes. The water isolates yielded five distinct toxigenicity profiles, viz. act, alt, act+alt, aerA+alt, and aerA+alt+act. The alt gene showed the highest frequency of occurrence (40%), followed by the aerA (20%), act (13%), and ast (0%) genes. Taken together, the study demonstrated the occurrence of a considerable population of nonculturable Aeromonads in water and prevalence of toxigenic Aeromonas spp. potentially pathogenic to humans. This emphasizes the importance of routine monitoring of both source and drinking water for this human pathogen and role of the developed molecular approaches in improving the Aeromonas monitoring scheme for water. PMID:24949108

  5. Molecular Detection, Quantification, and Toxigenicity Profiling of Aeromonas spp. in Source- and Drinking-Water.

    PubMed

    Robertson, Boakai K; Harden, Carol; Selvaraju, Suresh B; Pradhan, Suman; Yadav, Jagjit S

    2014-01-01

    Aeromonas is ubiquitous in aquatic environments and has been associated with a number of extra-gastrointestinal and gastrointestinal illnesses. This warrants monitoring of raw and processed water sources for pathogenic and toxigenic species of this human pathogen. In this study, a total of 17 different water samples [9 raw and 8 treated samples including 4 basin water (partial sand filtration) and 4 finished water samples] were screened for Aeromonas using selective culturing and a genus-specific real-time quantitative PCR assay. The selective culturing yielded Aeromonas counts ranging 0 - 2 x 10(3)CFU/ml and 15 Aeromonas isolates from both raw and treated water samples. The qPCR analysis indicated presence of a considerable nonculturable population (3.4 x 10(1) - 2.4 x 10(4) cells/ml) of Aeromonas in drinking water samples. Virulence potential of the Aeromonas isolates was assessed by multiplex/singleplex PCR-based profiling of the hemolysin and enterotoxin genes viz cytotoxic heat-labile enterotoxin (act), heat-labile cytotonic enterotoxin (alt), heat-stable cytotonic enterotoxin (ast), and aerolysin (aerA) genes. The water isolates yielded five distinct toxigenicity profiles, viz. act, alt, act+alt, aerA+alt, and aerA+alt+act. The alt gene showed the highest frequency of occurrence (40%), followed by the aerA (20%), act (13%), and ast (0%) genes. Taken together, the study demonstrated the occurrence of a considerable population of nonculturable Aeromonads in water and prevalence of toxigenic Aeromonas spp. potentially pathogenic to humans. This emphasizes the importance of routine monitoring of both source and drinking water for this human pathogen and role of the developed molecular approaches in improving the Aeromonas monitoring scheme for water.

  6. Lincomycin-induced over-expression of mature recombinant cholera toxin B subunit and the holotoxin in Escherichia coli.

    PubMed

    Arimitsu, Hideyuki; Tsukamoto, Kentaro; Ochi, Sadayuki; Sasaki, Keiko; Kato, Michio; Taniguchi, Koki; Oguma, Keiji; Tsuji, Takao

    2009-10-01

    Cholera toxin (CT) B subunit (CTB) was overproduced using a novel expression system in Escherichia coli. An expression plasmid was constructed by inserting the gene encoding the full-length CTB and the Shine-Dalgarno (SD) sequence derived from CTB or from the heat-labile enterotoxin B subunit (LTB) of enterotoxigenic E. coli into the lacZalpha gene fragment in the pBluescript SK(+) vector. The E. coli strain MV1184 was transformed with each plasmid and then cultured in CAYE broth containing lincomycin. Recombinant CTB (rCTB) was purified from each cell extract. rCTB was overproduced in both transformants without obvious toxicity and was structurally and biologically identical to that of CT purified from Vibrio cholerae, indicating that the original SD and CTB signal sequences were also sufficient to express rCTB in E. coli. Lincomycin-induced rCTB expression was inhibited by mutating the lac promoter, suggesting that lincomycin affects the lactose operon. Based on these findings, we constructed a plasmid that contained the wild-type CT operon and successfully overproduced CT (rCT) using the same procedure for rCTB. Although rCT had an intact A subunit, the amino-terminal modifications and biological properties of the A and B subunits of rCT were identical to those of CT. These results suggest that this novel rCTB over-expression system would also be useful to generate both wild-type and mutant CT proteins that will facilitate further studies on the characteristics of CT, such as mucosal adjuvant activity. PMID:19410003

  7. The tib adherence locus of enterotoxigenic Escherichia coli is regulated by cyclic AMP receptor protein.

    PubMed

    Espert, Shirley M; Elsinghorst, Eric A; Munson, George P

    2011-03-01

    Enterotoxigenic Escherichia coli (ETEC) is a Gram-negative enteric pathogen that causes profuse watery diarrhea through the elaboration of heat-labile and/or heat-stable toxins. Virulence is also dependent upon the expression of adhesive pili and afimbrial adhesins that allow the pathogen to adhere to the intestinal epithelium or mucosa. Both types of enterotoxins are regulated at the level of transcription by cyclic AMP (cAMP) receptor protein (CRP). To further our understanding of virulence gene regulation, an in silico approach was used to identify putative CRP binding sites in the genome of H10407 (O78:H11), an ETEC strain that was originally isolated from the stool of a Bangledeshi patient with cholera-like symptoms circa 1971. One of the predicted binding sites was located within an intergenic region upstream of tibDBCA. TibA is an autotransporter and afimbrial adhesin that is glycosylated by TibC. Expression of the TibA glycoprotein was abolished in an H10407 crp mutant and restored when crp was provided in trans. TibA-dependent aggregation was also abolished in a cyaA::kan strain and restored by addition of exogenous cAMP to the growth medium. DNase I footprinting confirmed that the predicted site upstream of tibDBCA is bound by CRP. Point mutations within the CRP binding site were found to abolish or significantly impair CRP-dependent activation of the tibDB promoter. Thus, these studies demonstrate that CRP positively regulates the expression of the glycosylated afimbrial adhesin TibA through occupancy of a binding site within tibDBp. PMID:21216994

  8. Gastrointestinal and systemic toxicity of fecal extracts from hamsters with clindamycin-induced colitis.

    PubMed

    Rifkin, G D; Silva, J; Fekety, R

    1978-01-01

    The production of toxic substances by intestinal bacteria is one pathogenic mechanism proposed for antibiotic-associated colitis. We demonstrated the presence of a toxic substance(s) in the feces of hamsters developing clindamycin-induced enterocolitis. Suspensions derived from cecal contents of clindamycin-treated animals induced a hemorrhagic ileocecitis and death within 2 to 4 days after being given orogastrically to hamsters. Intraperitoneal injection of sterile filtrates of these suspensions produced an exudative peritonitis, intraabdominal hemorrhages, and death of 80 to 100% of hamsters within 1 day. These effects were not seen with intraperitoneal injection of clindamycin or endotoxin, only small amounts of which were present in the filtrate. Incubation of the filtrate in vitro with polyvalent clostridial antitoxin neuralized its toxicity. In vitro incubation of the filtrate with normal equine serum did not reduce its in vivo toxicity. The toxic substance(s) contained in the filtrate was heat-labile and produced morphological changes in Y-1 adrenal cell cultures characteristic of heat-labile enterotoxins. Cecal filtrates obtained from saline-treated animals produced none of these effects. These preliminary studies suggest that enterotoxin-like substances, possibly produced by clostridia, may play an important role in the pathogenesis of clindamycin-induced colitis in the hamster.

  9. PROMOTIONS: PROper MOTION Software

    NASA Astrophysics Data System (ADS)

    Caleb Wherry, John; Sahai, R.

    2009-05-01

    We report on the development of a software tool (PROMOTIONS) to streamline the process of measuring proper motions of material in expanding nebulae. Our tool makes use of IDL's widget programming capabilities to design a unique GUI that is used to compare images of the objects from two epochs. The software allows us to first orient and register the images to a common frame of reference and pixel scale, using field stars in each of the images. We then cross-correlate specific morphological features in order to determine their proper motions, which consist of the proper motion of the nebula as a whole (PM-neb), and expansion motions of the features relative to the center. If the central star is not visible (quite common in bipolar nebulae with dense dusty waists), point-symmetric expansion is assumed and we use the average motion of high-quality symmetric pairs of features on opposite sides of the nebular center to compute PM-neb. This is then subtracted out to determine the individual movements of these and additional features relative to the nebular center. PROMOTIONS should find wide applicability in measuring proper motions in astrophysical objects such as the expanding outflows/jets commonly seen around young and dying stars. We present first results from using PROMOTIONS to successfully measure proper motions in several pre-planetary nebulae (transition objects between the red giant and planetary nebula phases), using images taken 7-10 years apart with the WFPC2 and ACS instruments on board HST. The authors are grateful to NASA's Undergradute Scholars Research Program (USRP) for supporting this research.

  10. Promoting healthy sleep.

    PubMed

    Price, Bob

    2016-03-01

    Nurses are accustomed to helping others with their sleep problems and dealing with issues such as pain that may delay or interrupt sleep. However, they may be less familiar with what constitutes a healthy night's sleep. This article examines what is known about the process and purpose of sleep, and examines the ways in which factors that promote wakefulness and sleep combine to help establish a normal circadian rhythm. Theories relating to the function of sleep are discussed and research is considered that suggests that sleep deficit may lead to metabolic risks, including heart disease, obesity, type 2 diabetes mellitus and several types of cancer.

  11. Promoting healthy sleep.

    PubMed

    Price, Bob

    2016-03-01

    Nurses are accustomed to helping others with their sleep problems and dealing with issues such as pain that may delay or interrupt sleep. However, they may be less familiar with what constitutes a healthy night's sleep. This article examines what is known about the process and purpose of sleep, and examines the ways in which factors that promote wakefulness and sleep combine to help establish a normal circadian rhythm. Theories relating to the function of sleep are discussed and research is considered that suggests that sleep deficit may lead to metabolic risks, including heart disease, obesity, type 2 diabetes mellitus and several types of cancer. PMID:26959472

  12. [Evaluation of health promotion programmes].

    PubMed

    Berkowska, M; Sito, A

    2000-01-01

    The paper contains a review of definitions of evaluation and discusses the need to evaluate health promotion programmes. The classification of types of evaluation is presented. It is aimed to create a common language of communication between evaluation and the common understanding of terms. The relation between evaluation of health promotion programmes and quality assurance, best practice and evidence based health promotion are discussed.

  13. Health promotion and prevention strategies.

    PubMed

    Bradbury-Golas, Kathleen

    2013-09-01

    Opiate dependency is a medical disorder that requires treatment intervention. Primary health care not only entails treatment of illness but also involves disease prevention and health promotion. Based on Pender's revised Health Promotion Model, a descriptive study comparing the health promoting behaviors/practices in abusing and recovering opiate-dependent drug users is analyzed. Using the Health Promoting Lifestyle Profile II, a comparative descriptive, exploratory, nonexperimental design study was conducted to identify key health-promoting behaviors in recovering opiate-dependent drug users. Prevention strategy recommendations are discussed, along with future research recommendations.

  14. Promoting adolescent health.

    PubMed

    Kelly, Kristina Berg

    2007-10-01

    The aim is to discuss why paediatricians should be involved in adolescent health care and provide youth-friendly-health-services. Global epidemiological data on morbidity and mortality demonstrate that much of ill health in the short and long run are connected to adolescent behaviour and in theory available for prevention. Young people seemingly lose their heads and do not consider dangers. Recent research on brain development provides us with an understanding how this may have a biological base. Also psychology has long taught us how adolescents use experimental behaviours as means to satisfy developmental needs and explore identity. Prevention and health promotion are areas of research where much more needs to be done. There is also a lack of venues for publishing even excellent studies in this field.

  15. Tryptophan promotes charitable donating

    PubMed Central

    Steenbergen, Laura; Sellaro, Roberta; Colzato, Lorenza S.

    2014-01-01

    The link between serotonin (5-HT) and one of the most important elements of prosocial behavior, charity, has remained largely uninvestigated. In the present study, we tested whether charitable donating can be promoted by administering the food supplement L-Tryptophan (TRP), the biochemical precursor of 5-HT. Participants were compared with respect to the amount of money they donated when given the opportunity to make a charitable donation. As expected, compared to a neutral placebo, TRP appears to increase the participants’ willingness to donate money to a charity. This result supports the idea that the food we eat may act as a cognitive enhancer modulating the way we think and perceive the world and others. PMID:25566132

  16. 7 CFR 1150.114 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... promotion, and publicity to advance the image and sales of, and demand for, dairy products generally. ... and Orders; Milk), DEPARTMENT OF AGRICULTURE DAIRY PROMOTION PROGRAM Dairy Promotion and...

  17. Network modularity promotes cooperation.

    PubMed

    Marcoux, Marianne; Lusseau, David

    2013-05-01

    Cooperation in animals and humans is widely observed even if evolutionary biology theories predict the evolution of selfish individuals. Previous game theory models have shown that cooperation can evolve when the game takes place in a structured population such as a social network because it limits interactions between individuals. Modularity, the natural division of a network into groups, is a key characteristic of all social networks but the influence of this crucial social feature on the evolution of cooperation has never been investigated. Here, we provide novel pieces of evidence that network modularity promotes the evolution of cooperation in 2-person prisoner's dilemma games. By simulating games on social networks of different structures, we show that modularity shapes interactions between individuals favouring the evolution of cooperation. Modularity provides a simple mechanism for the evolution of cooperation without having to invoke complicated mechanisms such as reputation or punishment, or requiring genetic similarity among individuals. Thus, cooperation can evolve over wider social contexts than previously reported.

  18. Sales promotions and food consumption.

    PubMed

    Hawkes, Corinna

    2009-06-01

    Sales promotions are widely used to market food to adults, children, and youth. Yet, in contrast to advertising, practically no attention has been paid to their impacts on dietary behaviors, or to how they may be used more effectively to promote healthy eating. This review explores the available literature on the subject. The objective is to identify if and what literature exists, examine the nature of this literature, and analyze what can be learned from it about the effects of sales promotions on food consumption. The review finds that while sales promotions lead to significant sales increases over the short-term, this does not necessarily lead to changes in food-consumption patterns. Nevertheless, there is evidence from econometric modeling studies indicating that sales promotions can influence consumption patterns by influencing the purchasing choices of consumers and encouraging them to eat more. These effects depend on the characteristics of the food product, sales promotion, and consumer. The complexity of the effects means that sales promotions aiming to encourage consumption of nutritious foods need to be carefully designed. These conclusions are based on studies that use mainly sales data as a proxy for dietary intake. The nutrition (and economics) research communities should add to this existing body of research to provide evidence on the impact of sales promotions on dietary intake and related behaviors. This would help support the development of a sales promotion environment conducive to healthy eating. PMID:19519674

  19. 7 CFR 1219.22 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions § 1219.22 Promotion. Promotion means any action to advance the image, desirability, or marketability of Hass...

  20. 7 CFR 1219.22 - Promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions § 1219.22 Promotion. Promotion means any action to advance the image, desirability, or marketability of Hass...

  1. 7 CFR 1219.22 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions § 1219.22 Promotion. Promotion means any action to advance the image, desirability, or marketability of Hass...

  2. 7 CFR 1219.22 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions § 1219.22 Promotion. Promotion means any action to advance the image, desirability, or marketability of Hass...

  3. Health-promoting schools: an opportunity for oral health promotion.

    PubMed Central

    Kwan, Stella Y. L.; Petersen, Poul Erik; Pine, Cynthia M.; Borutta, Annerose

    2005-01-01

    Schools provide an important setting for promoting health, as they reach over 1 billion children worldwide and, through them, the school staff, families and the community as a whole. Health promotion messages can be reinforced throughout the most influential stages of children's lives, enabling them to develop lifelong sustainable attitudes and skills. Poor oral health can have a detrimental effect on children's quality of life, their performance at school and their success in later life. This paper examines the global need for promoting oral health through schools. The WHO Global School Health Initiative and the potential for setting up oral health programmes in schools using the health-promoting school framework are discussed. The challenges faced in promoting oral health in schools in both developed and developing countries are highlighted. The importance of using a validated framework and appropriate methodologies for the evaluation of school oral health projects is emphasized. PMID:16211159

  4. Good Teaching Promotes Student Success.

    ERIC Educational Resources Information Center

    Roueche, John E.; Thompson, Carolyn

    1980-01-01

    Enumerates teaching strategies designed to promote high student retention in developmental courses, which: (1) help develop effective working relationships between student and teacher; (2) assure effective classroom management; (3) promote student involvement in the learning process; and (4) help the instructor recognize his/her own professional…

  5. Promoting Creativity in Young Children.

    ERIC Educational Resources Information Center

    Honig, Alice Sterling

    This paper discusses creativity in young children and what teachers can do to support and promote it. Topics addressed in the paper include: (1) teacher interest in promoting creativity; (2) defining creativity; (3) creativity in the socioemotional domain; (4) the relationship between creativity and empathy for others; (4) bibliotherapy; (5)…

  6. Promoting Reading in Developing Countries.

    ERIC Educational Resources Information Center

    Greaney, Vincent, Ed.

    With the intention of illuminating the many obstacles involved with literacy promotion in the developing nations of Africa, Asia, and South America, the authors of the 10 articles in this collection share their knowledge and experience of literacy promotion in the developing world--including the unique challenges faced by those who publish, print,…

  7. Insurance Incentives for Health Promotion.

    ERIC Educational Resources Information Center

    Hosokawa, Michael C.

    1984-01-01

    To reduce the cost of reimbursements, many insurance companies have begun to use insurance incentives as a way to motivate individuals to participate in health promotion activities. Traditional health education, research and demonstration, and policy-premium incentives are methods of health promotion used by life and health insurance companies.…

  8. Development and accuracy of quantitative real-time polymerase chain reaction assays for detection and quantification of enterotoxigenic Escherichia coli (ETEC) heat labile and heat stable toxin genes in travelers' diarrhea samples.

    PubMed

    Youmans, Bonnie P; Ajami, Nadim J; Jiang, Zhi-Dong; Petrosino, Joseph F; DuPont, Herbert L; Highlander, Sarah K

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.

  9. The problem of bacterial diarrhoea.

    PubMed

    Harries, J T

    1976-01-01

    The reported incidence of "pathogenic" bacteria, as judged by serotype, in the stools of children with acute diarrhoea has varied from 4 to 33% over the last twenty years. Techniques such as tissue culture provide a means for detecting enterotoxin-producing strains of bacteria, strains which often do not possess "pathogenic" serotypes. "Pathogenicity" requires redefinition, and the aetiological importance of bacteria in diarrhoea is probably considerably greater than previous reports have indicated. Colonization of the bowel by a pathogen will result in structural and/or mucosal abnormalities, and will depend on a series of complex interactions between the external environment, the pathogen, and the host and its resident bacterial flora. Enteropathogenic bacteria may be broadly classified as (i) invasive (e.g. Shigella, Salmonella and some Escherichia coli) which predominantly affect the distal bowel, or (ii) non-invasive (e.g. Vibrio cholerae and E. coli) which affect the proximal bowel. V. cholerae and E. coli elaborate heat-labile enterotoxins which activate adenylate cyclase and induce small intestinal secretion; the secretory effects of heat-stable E. coli and heat-labile Shigella dysenteriae enterotoxins are not accompanied by cyclase activation. The two major complications of acute diarrhoea are (i) hypernatraemic dehydration with its attendant neurological, renal and vascular lesions, and (ii) protracted diarrhoea which may lead to severe malnutrition. Deconjugation of bile salts and colonization of the small bowel with toxigenic strains of E. coli may be important in the pathophysiology of the protracted diarrhoea syndrome. The control of bacterial diarrhoea requires a corrdinated political, educational, social, public health and scientific attack. Bacterial diarrhoea is a major health problem throughout the world, and carries an appreciable morbidity and mortality. This is particularly the case during infancy, and in those developing parts of the world

  10. Photovoltaic module with adhesion promoter

    DOEpatents

    Xavier, Grace

    2013-10-08

    Photovoltaic modules with adhesion promoters and methods for fabricating photovoltaic modules with adhesion promoters are described. A photovoltaic module includes a solar cell including a first surface and a second surface, the second surface including a plurality of interspaced back-side contacts. A first glass layer is coupled to the first surface by a first encapsulating layer. A second glass layer is coupled to the second surface by a second encapsulating layer. At least a portion of the second encapsulating layer is bonded directly to the plurality of interspaced back-side contacts by an adhesion promoter.

  11. Nutritional Recommendation Should Promote Sustainability.

    ERIC Educational Resources Information Center

    Reber, Robert J.

    1991-01-01

    Any process or event that disrupts the flow of nutrients and energy becomes a nutrition problem. Nutritionists should promote practices that protect the integrity, stability, and beauty of the land community (soil, water, air, all biological species). (Author)

  12. 7 CFR 1260.122 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion. 1260.122 Section 1260.122 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BEEF PROMOTION AND RESEARCH Beef Promotion and Research Order Definitions § 1260.122 Promotion. Promotion means any action, including...

  13. Promoter-associated RNAs and promoter-targeted RNAs.

    PubMed

    Yan, Bing-Xue; Ma, Jin-Xia

    2012-09-01

    The world of RNAs is much more complex than previously thought, and has rapidly emerged as one of the most actively researched topics in the life sciences. Recently, two findings in this field were reported and given special attention: promoter-associated RNAs (paRNAs), a novel class of RNAs with numerous potential functions; and promoter-targeted RNA-induced transcriptional gene regulation, a new regulatory mechanism to control transcription. In this review, we summarize the studies in these two areas, and outline the current understanding with respect to the potential biological functions of paRNAs, and the molecular mechanisms of promoter-targeted RNA-induced transcriptional gene silencing and activation. Additionally, we seek to integrate these two areas, as paRNAs may have potential biological links with promoter-targeted RNA-induced transcriptional gene regulation. Finally, we will discuss the significance of identifying paRNAs and the possible use of promoter-targeted RNAs in gene regulation and therapy.

  14. 7 CFR 1150.114 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... and Orders; Milk), DEPARTMENT OF AGRICULTURE DAIRY PROMOTION PROGRAM Dairy Promotion and Research... promotion, and publicity to advance the image and sales of, and demand for, dairy products generally....

  15. 7 CFR 1150.114 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MILK), DEPARTMENT OF AGRICULTURE DAIRY PROMOTION PROGRAM Dairy Promotion and Research... promotion, and publicity to advance the image and sales of, and demand for, dairy products generally....

  16. Creating and Promoting a Natural History Collection.

    ERIC Educational Resources Information Center

    Belben, Cathy

    2003-01-01

    Discusses the value of developing and promoting a natural history library by school library media specialists. Topics include benefits to students; promoting outdoor education; recommended reading for high school students; using technology; and other aids to promote outdoor education. (LRW)

  17. 7 CFR 1150.114 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MILK), DEPARTMENT OF AGRICULTURE DAIRY PROMOTION PROGRAM Dairy Promotion and Research... promotion, and publicity to advance the image and sales of, and demand for, dairy products generally....

  18. 7 CFR 1215.16 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POPCORN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Popcorn Promotion, Research, and Consumer Information Order Definitions § 1215.16... popcorn....

  19. 7 CFR 1215.16 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POPCORN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Popcorn Promotion, Research, and Consumer Information Order Definitions § 1215.16... popcorn....

  20. 7 CFR 1215.16 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POPCORN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Popcorn Promotion, Research, and Consumer Information Order Definitions § 1215.16... popcorn....

  1. 7 CFR 1215.16 - Promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POPCORN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Popcorn Promotion, Research, and Consumer Information Order Definitions § 1215.16... popcorn....

  2. 7 CFR 1215.16 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POPCORN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Popcorn Promotion, Research, and Consumer Information Order Definitions § 1215.16... popcorn....

  3. DNA signals at isoform promoters

    PubMed Central

    Dai, Zhiming; Xiong, Yuanyan; Dai, Xianhua

    2016-01-01

    Transcriptional heterogeneity is extensive in the genome, and most genes express variable transcript isoforms. However, whether variable transcript isoforms of one gene are regulated by common promoter elements remain to be elucidated. Here, we investigated whether isoform promoters of one gene have separated DNA signals for transcription and translation initiation. We found that TATA box and nucleosome-disfavored DNA sequences are prevalent in distinct transcript isoform promoters of one gene. These DNA signals are conserved among species. Transcript isoform has a RNA-determined unstructured region around its start site. We found that these DNA/RNA features facilitate isoform transcription and translation. These results suggest a DNA-encoded mechanism by which transcript isoform is generated. PMID:27353836

  4. AAHD's Health Promotion and Wellness, Part 2: Health Promotion Programs

    ERIC Educational Resources Information Center

    Exceptional Parent, 2011

    2011-01-01

    This article is part 2 of a 4-part series on "Health Promotion and Wellness" from the American Association on Health and Disability (AAHD). According to the U.S. Census Bureau, more than 54 million people--one in five Americans--have a disability, and these Americans are more likely to report: (1) Being in poorer overall health; (2) Having less…

  5. Promoting certification: the chapter's role.

    PubMed

    Staul, Luann

    2011-01-01

    The mission of the American Association of Critical-Care Nurses focuses on providing nurses with expert knowledge to promote delivery of excellent, safe, quality care to acutely and critically ill patients and their families. Chapters consist of professional leaders in a community who carry on the mission work of the American Association of Critical-Care Nurses at the local level. Chapters can emphasize the value of certification and continuing education, because they offer a wide spectrum of opportunities to meet the learning and developmental needs of nurses as they advance in their professional careers. This article will highlight strategies that can be implemented by local chapters to facilitate and promote certification.

  6. When Promoting Democracy Is Counterproductive

    ERIC Educational Resources Information Center

    Esfandiari, Haleh; Litwak, Robert S.

    2007-01-01

    The United States has begun a $75-million program to promote democracy by supporting Iranian nongovernmental organizations (NGOs). That program, coupled with loose talk about regime change from members of Congress, commentators close to the administration, and individuals within the administration, has fed a sense of vulnerability and paranoia…

  7. Mutual Benefits in Promoting Bequests.

    ERIC Educational Resources Information Center

    Patterson, Charles W., III

    1979-01-01

    Encouragement of wills and bequests is the backbone of a good planned giving program. Methods of promotion, types of will gifts, tax aspects, identification of potential donors, a simple bequest program, program evaluation, and the need for continuity and patience are discussed. (MLW)

  8. Professional Preparation in Health Promotion.

    ERIC Educational Resources Information Center

    Hill, Charles E.; Fisher, Shirley P.

    1992-01-01

    Colleges and universities must develop curricula to prepare health promotion specialists to work with persons of all ages. Program core should include self-care, consumer awareness, nutrition, weight control, stress management, and substance abuse. Health and physical educators should learn to facilitate change of negative health behaviors into…

  9. Promoting Metacognition in Music Classes

    ERIC Educational Resources Information Center

    Benton, Carol W.

    2013-01-01

    Metacognition is a type of thinking in which learners think about their own cognitive processes. Because it transcends disciplines and grade levels, metacognition is useful in many educational settings and can be transferred from the music classroom to other subject areas. Music educators can promote metacognition by designing and implementing…

  10. Health Promotion: A Resource Book.

    ERIC Educational Resources Information Center

    Anderson, Robert, Ed.; Kickbusch, Ilona, Ed.

    Health promotion redirects thinking about health by: reasserting its social and political aspects; ensuring the people the power to define their own health concerns; and placing health more clearly in the context of other aims in life. This compilation of 41 articles in 8 sections attempts to document this process of redirection of thought. The…

  11. Teachers Promoting Student Mathematical Reasoning

    ERIC Educational Resources Information Center

    Mueller, Mary; Yankelewitz, Dina; Maher, Carolyn

    2014-01-01

    During an informal, after-school, math program, a group of middle school students worked collaboratively on open-ended problems. The students co-constructed arguments, provided justifications for their solutions, and engaged in mathematical reasoning. This paper describes the specific teacher moves that promoted this phenomenon. The findings of…

  12. University Festival Promotes STEM Education

    ERIC Educational Resources Information Center

    Quagliata, Andrew B.

    2015-01-01

    STEM education is argued as an essential ingredient in preparing our children for careers of the future. This study describes a university festival that includes the promotion of STEM-related career interests in young people among its goals. A total of 203 participants between the age of 7 and 17 completed both pre-event and post-event surveys. In…

  13. Promoting Intellectual Growth in Adulthood.

    ERIC Educational Resources Information Center

    Kehle, Thomas J.; Bray, Melissa A.; Chafouleas, Sandra M; McLoughlin, Caven S.

    2002-01-01

    Article discusses problems associated with promoting intellectual growth in adulthood. Defines characteristics of intelligent behavior as incorporating individual attainment of Resources, Intimacy, Competence, and Health (RICH). Presents the RICH theory as a way to define and address the goals of intelligent enhancement. (JDM)

  14. Using Data to Promote Equity

    ERIC Educational Resources Information Center

    Shum, Brenda

    2016-01-01

    Data plays a starring role in promoting educational equity, and data-driven decision making begins with good state policies. With the recent passage of the Every Student Succeeds Act (ESSA) and a proposed federal rule to address racial disproportionality in special education, states will shoulder increased responsibility for eliminating…

  15. Promoting SETI in the UK

    NASA Astrophysics Data System (ADS)

    Penny, Alan

    2013-10-01

    MEETING REPORT What does the UK presently do in the search for extraterrestrial intelligence and what are the plans for the future? Alan Penny reports on a meeting of UK academics active in SETI, held as sessions in the recent National Astronomy Meeting in Scotland - and the formation of the UK SETI Research Network to promote UK academic work.

  16. Advertising and Sales Promotion Guide.

    ERIC Educational Resources Information Center

    North Carolina State Dept. of Public Instruction, Raleigh. Div. of Vocational Education.

    This document contains teacher materials for a 4-unit, 1-year marketing education course in advertising and sales promotion offered in grades 11 and 12 in North Carolina. The preface contains a rationale for the development of the course, a course description, course objectives, a list of the instructional units of the course, and a list of the…

  17. Promoting Inclusive Education in Ghana

    ERIC Educational Resources Information Center

    Djietror, Beauty B. K.; Okai, Edward; Kwapong, Olivia A. T. Frimpong

    2011-01-01

    Inclusive education is critical for nation building. The government of Ghana has put in measures for promoting inclusion from basic through to tertiary level of education. Some of these measures include expansion of school facilities, implementation of the Free Compulsory Universal Basic Education (FCUBE); the change of policy on girls who drop…

  18. Promoting physical activity in schools.

    PubMed

    Armstrong, N

    1993-10-01

    Neil Armstrong, director of the Coronary Prevention in Children Project, argues for a comprehensive programme for promoting children's physical activity. The project's survey of adult coronary risk factors in British children revealed a worryingly low level of physical activity among British schoolchildren. Schools are ideally placed to encourage children to take physical exercise, he writes, but parental role models also play an important part.

  19. Developing and promoting managerial talent.

    PubMed

    Adams-Ender, C L

    1990-03-01

    1. Developing and promoting managerial talent through the use of power and influence, mentoring, and empowerment is essential for seizing opportunities to successfully meet nursing challenges. 2. Power is the ability to affect outcomes, whereas influence is the process of modifying behavior. 3. Mentoring has several advantages: reduction of employee turnover; cultivation of talented employees; and maintenance of organizational goals and traditions.

  20. Health promotion for older Americans.

    PubMed

    Heckler, M M

    1985-01-01

    As American lifespans increase, there is greater concern for the quality of those longer lives. The Department of Health and Human Services, through its many component agencies, has inaugurated a major initiative to promote health and fitness among older Americans to improve life quality and to reduce health care costs. The older population is a fertile ground for such an initiative, because studies indicate that the elderly are extremely health-conscious and very willing to adopt habits that will maintain good health. Investigation disclosed six target areas of concentration at which the health promotion initiative could be aimed: fitness-exercise, nutrition, safe and proper use of drugs and alcohol, accident prevention, other preventive services, and smoking cessation. The initiative includes cooperative programs with States; dissemination of printed information; nutritious meals for the elderly; a Food and Drug Administration consumer education program; Centers for Disease Control programs on accident prevention; a special task force to deal with Alzheimer's disease; and, in cooperation with states, a media campaign of health promotion for the elderly. At least three national health and senior citizens organizations are working closely with HHS agencies on the initiative. A separate Department effort involves the encouragement of fast-growing health maintenance organizations to promote health and prevention for their Medicare members and the persuasion of Medicare beneficiaries generally to seek second medical or surgical opinions. State and local government and the private sector, responding to Department initiatives, have also been developing programs for the aging. Their interest and participation ensures that special health promotion and disease prevention efforts directed toward elderly Americans will continue and proliferate.

  1. 7 CFR 1216.23 - Promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order Definitions § 1216.23 Promotion. Promotion... image of peanuts to the public to improve the competitive position of peanuts in the...

  2. 7 CFR 1216.23 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order Definitions § 1216.23 Promotion. Promotion... image of peanuts to the public to improve the competitive position of peanuts in the...

  3. 7 CFR 1210.312 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE WATERMELON RESEARCH AND PROMOTION PLAN Watermelon Research and Promotion Plan Definitions § 1210.312 Promotion. Promotion means any action taken by the Board, pursuant to the Act, to present a favorable image for watermelons to...

  4. 7 CFR 1210.312 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE WATERMELON RESEARCH AND PROMOTION PLAN Watermelon Research and Promotion Plan Definitions § 1210.312 Promotion. Promotion means any action taken by the Board, pursuant to the Act, to present a favorable image for watermelons to...

  5. 7 CFR 1210.312 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE WATERMELON RESEARCH AND PROMOTION PLAN Watermelon Research and Promotion Plan Definitions § 1210.312 Promotion. Promotion means any action taken by the Board, pursuant to the Act, to present a favorable image for watermelons to...

  6. 7 CFR 1210.312 - Promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE WATERMELON RESEARCH AND PROMOTION PLAN Watermelon Research and Promotion Plan Definitions § 1210.312 Promotion. Promotion means any action taken by the Board, pursuant to the Act, to present a favorable image for watermelons to...

  7. 7 CFR 1210.312 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE WATERMELON RESEARCH AND PROMOTION PLAN Watermelon Research and Promotion Plan Definitions § 1210.312 Promotion. Promotion means any action taken by the Board, pursuant to the Act, to present a favorable image for watermelons to...

  8. 7 CFR 1221.23 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SORGHUM PROMOTION, RESEARCH, AND INFORMATION ORDER Sorghum Promotion, Research, and Information Order Definitions § 1221.23 Promotion. Promotion means any action taken to present a favorable image of sorghum to the public and the...

  9. 7 CFR 1221.23 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SORGHUM PROMOTION, RESEARCH, AND INFORMATION ORDER Sorghum Promotion, Research, and Information Order Definitions § 1221.23 Promotion. Promotion means any action taken to present a favorable image of sorghum to the public and the...

  10. 7 CFR 1221.23 - Promotion.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SORGHUM PROMOTION, RESEARCH, AND INFORMATION ORDER Sorghum Promotion, Research, and Information Order Definitions § 1221.23 Promotion. Promotion means any action taken to present a favorable image of sorghum to the public and the...

  11. 7 CFR 1221.23 - Promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SORGHUM PROMOTION, RESEARCH, AND INFORMATION ORDER Sorghum Promotion, Research, and Information Order Definitions § 1221.23 Promotion. Promotion means any action taken to present a favorable image of sorghum to the public and the...

  12. 7 CFR 1221.23 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SORGHUM PROMOTION, RESEARCH, AND INFORMATION ORDER Sorghum Promotion, Research, and Information Order Definitions § 1221.23 Promotion. Promotion means any action taken to present a favorable image of sorghum to the public and the...

  13. 7 CFR 1218.17 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BLUEBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Blueberry Promotion, Research, and Information Order Definitions § 1218.17 Promotion. Promotion means any action taken to present a favorable image of blueberries to the general public and...

  14. 7 CFR 1218.17 - Promotion.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BLUEBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Blueberry Promotion, Research, and Information Order Definitions § 1218.17 Promotion. Promotion means any action taken to present a favorable image of blueberries to the general public and...

  15. 7 CFR 1218.17 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BLUEBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Blueberry Promotion, Research, and Information Order Definitions § 1218.17 Promotion. Promotion means any action taken to present a favorable image of blueberries to the general public and...

  16. 7 CFR 1218.17 - Promotion.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BLUEBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Blueberry Promotion, Research, and Information Order Definitions § 1218.17 Promotion. Promotion means any action taken to present a favorable image of blueberries to the general public and...

  17. 21 CFR 601.94 - Promotional materials.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Promotional materials. 601.94 Section 601.94 Food... Promotional materials. For biological products being considered for approval under this subpart, unless... preapproval review period copies of all promotional materials, including promotional labeling as well...

  18. 21 CFR 601.45 - Promotional materials.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Promotional materials. 601.45 Section 601.45 Food... Promotional materials. For biological products being considered for approval under this subpart, unless... preapproval review period copies of all promotional materials, including promotional labeling as well...

  19. 7 CFR 1216.23 - Promotion.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order Definitions § 1216.23 Promotion. Promotion... image of peanuts to the public to improve the competitive position of peanuts in the...

  20. 7 CFR 1216.23 - Promotion.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order Definitions § 1216.23 Promotion. Promotion... image of peanuts to the public to improve the competitive position of peanuts in the...