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Sample records for coli heat labile

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

    PubMed Central

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

    1979-01-01

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

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

    PubMed Central

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

    1991-01-01

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

  3. 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

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

    PubMed Central

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

    1978-01-01

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

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

    PubMed Central

    Chapman, P A; Daly, C M

    1989-01-01

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

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

    PubMed Central

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

    1983-01-01

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

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

    PubMed

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

    2005-08-01

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

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

    PubMed Central

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

    1980-01-01

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

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

    PubMed Central

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

    1974-01-01

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

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

    PubMed

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

    2015-01-01

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

  11. 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

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

    PubMed

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

    2007-09-05

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

  13. 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.

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

    PubMed Central

    Kunkel, Steven L.; Robertson, Donald C.

    1979-01-01

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

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

    PubMed

    Gilligan, P H; Robertson, D C

    1979-01-01

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

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

    PubMed

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

    2008-01-01

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

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

    PubMed Central

    Otnaess, A B; Orstavik, I

    1981-01-01

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

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

    PubMed

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

    1993-04-05

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

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

    USDA-ARS?s Scientific Manuscript database

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

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

    PubMed

    Arimitsu, Hideyuki; Sasaki, Keiko; Tsuji, Takao

    2017-01-01

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

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

    PubMed

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

    2008-06-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed Central

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

    1988-01-01

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

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

    PubMed Central

    Ola, Thomas O; Williams, Neil A

    2006-01-01

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

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

    PubMed Central

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

    1983-01-01

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

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

    PubMed Central

    Klipstein, Frederick A.; Engert, Richard F.

    1980-01-01

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

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

    PubMed Central

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

    1999-01-01

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

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

    PubMed Central

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

    1982-01-01

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

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

    PubMed Central

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

    1977-01-01

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

  10. 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

  11. 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.

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

    PubMed Central

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

    1983-01-01

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

  13. HEAT LABILE OPSONINS TO PNEUMOCOCCUS

    PubMed Central

    Smith, Mary Ruth; Wood, W. Barry

    1969-01-01

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

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

    PubMed Central

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

    1988-01-01

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

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

    PubMed Central

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

    1982-01-01

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

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

    PubMed Central

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

    1988-01-01

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

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

    PubMed

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

    1982-09-01

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

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

    PubMed Central

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

    1984-01-01

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

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

    PubMed

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

    2014-09-17

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

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2016-04-01

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

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

    PubMed

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

    2012-10-01

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

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

    Lu, Xi; Fu, Enqing; Xie, Yonghong

    2016-01-01

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

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

    PubMed

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

    2016-08-27

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

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

    PubMed Central

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

    1999-01-01

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

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed

    Chatterjee, Arpita; Chowdhury, Rukhsana

    2008-01-01

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

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

    PubMed Central

    2013-01-01

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

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

    PubMed

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

    2013-10-29

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

  13. 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

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

    PubMed

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

    2013-01-01

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

  15. 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

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

    PubMed Central

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

    1999-01-01

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

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

    DTIC Science & Technology

    1981-09-01

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

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

    DTIC Science & Technology

    1980-01-15

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

  19. 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.

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

    PubMed

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

    2006-07-17

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

  1. 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.

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

    PubMed

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

    2014-09-26

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

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

    PubMed

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

    2011-04-15

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

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

    PubMed Central

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

    2011-01-01

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

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

    SciTech Connect

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

    2012-12-01

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

  6. 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.

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

    PubMed Central

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

    2004-01-01

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

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

    PubMed

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

    2004-07-01

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

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

    PubMed

    Nandre, Rahul M; Lee, John Hwa

    2014-01-09

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

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

    PubMed

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

    2010-02-03

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

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

    PubMed

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

    2014-03-01

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

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

    PubMed Central

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

    2015-01-01

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

  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 Central

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

    2015-01-01

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

  14. 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

  15. 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

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  17. 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.

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

    PubMed

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

    2012-11-09

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2014-01-01

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

  1. 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.

  2. Force relaxation, labile heat and parvalbumin content of skeletal muscle fibres of Xenopus laevis.

    PubMed Central

    Lännergren, J; Elzinga, G; Stienen, G J

    1993-01-01

    1. Measurements were made of stable (hb) and labile (ha) maintenance heat rate, slowing of relaxation as a function of tetanus duration, and parvalbumin (PA) content in intact single muscle fibres of types 1 and 2 from Xenopus laevis. The majority of experiments were performed at 20 degrees C. In addition, total and myofibrillar ATPase activity was measured in skinned Xenopus fibres, also of types 1 and 2; these studies were performed at 4 degrees C. 2. In agreement with a previous study hb was significantly higher in type 1 (175 +/- 13 mW (g wet wt)-1; n = 8) than in type 2 fibres (88 +/- 9 mW (g wet wt)-1; n = 7). The value of ha was 236 +/- 22 and 117 +/- 16 mW (g wet wt)-1, respectively (mean +/- S.E.M.). ha decayed with a time constant of 0.27 +/- 0.02 (n = 8) and 0.33 +/- 0.02 s (n = 7). 3. The early relaxation rate of tetanic force, extrapolated to the onset of stimulation (yo + yb; where yo is 'extra' rate of relaxation and yb steady rate) was 85.6 +/- 4.2 s-1 for type 1 fibres (n = 8) and 62.7 +/- 7.3 s-1 for type 2 fibres (n = 7). Relaxation rate at the end of a 1.8 s tetanus (yb) was 29.4 +/- 1.6 and 33.3 +/- 1.5 s-1, respectively; thus, there was more slowing with tetanus duration in type 1 fibres. The time constant for slowing of relaxation with tetanus duration was similar to that for decay of ha. 4. Parvalbumin concentration, [PA], was 0.45 +/- 0.04 mM in type 1 (n = 7) and 0.22 +/- 0.04 mM (n = 7) in type 2 fibres. 5. For individual fibres positive correlations were found between the 'extra' rate of relaxation (yo), labile heat (ha) and [PA]. Significantly more labile heat was liberated than can be accounted for by the enthalpy change of Ca2+ binding to PA. 6. For five fibres (type 1) studied both at 20 and 10 degrees C, the magnitude of slowing of relaxation, expressed as yo/(yo + yb), was 0.58 +/- 0.03 at 20 degrees C and 0.65 +/- 0.03 at 10 degrees C. 7. Both slowing of relaxation and labile heat were depressed in the second of two closely spaced

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

    PubMed

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

    2015-05-14

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

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

    PubMed Central

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

    1983-01-01

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

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

    PubMed

    Chutkan, Halima; Kuehn, Meta J

    2011-09-01

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

  6. 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.

  7. Initial cloning and sequencing of hydHG, an operon homologous to ntrBC and regulating the labile hydrogenase activity in Escherichia coli K-12.

    PubMed Central

    Stoker, K; Reijnders, W N; Oltmann, L F; Stouthamer, A H

    1989-01-01

    To isolate genes from Escherichia coli which regulate the labile hydrogenase activity, a plasmid library was used to transform hydL mutants lacking the labile hydrogenase. A single type of gene, designated hydG, was isolated. This gene also partially restored the hydrogenase activity in hydF mutants (which are defective in all hydrogenase isoenzymes), although the low hydrogenase 1 and 2 levels were not induced. Therefore, hydG apparently regulates, specifically, the labile hydrogenase activity. Restoration of this latter activity in hydF mutants was accompanied by a proportional increase of the H2 uptake activity, suggesting a functional relationship. H2:fumarate oxidoreductase activity was not restored in complemented hydL mutants. These latter strains may therefore lack, in addition to the labile hydrogenase, a second component (provisionally designated component R), possibly an electron carrier coupling H2 oxidation to the anerobic respiratory chain. Sequence analysis showed an open reading frame of 1,314 base pairs for hydG. It was preceded by a ribosome-binding site but apparently lacked a promoter. Minicell experiments revealed a single polypeptide of approximately 50 kilodaltons. Comparison of the predicted amino acid sequence with a protein sequence data base revealed strong homology to NtrC from Klebsiella pneumoniae, a DNA-binding transcriptional activator. The 411 base pairs upstream from pHG40 contained a second open reading frame overlapping hydG by four bases. The deduced amino acid sequence showed considerable homology with the C-terminal part of NtrB. This sequence was therefore assumed to be part of a second gene, encoding the NtrB-like component, and was designated hydH. The labile hydrogenase activity in E. coli is apparently regulated by a multicomponent system analogous to the NtrB-NtrC system. This conclusion is in agreement with the results of Birkmann et al. (A. Birkmann, R. G. Sawers, and A. Böck, Mol. Gen. Genet. 210:535-542, 1987), who

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

    PubMed

    Li, Jie; Kan, Biao; Zhang, Jingyun

    2014-06-01

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

  9. 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.

  10. Toxigenic Escherichia Coli and Childhood Diarrhea

    PubMed Central

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

    1976-01-01

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

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

    PubMed Central

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

    1975-01-01

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

  12. Investigation of ’Escherichia coli’ Enterotoxins

    DTIC Science & Technology

    1978-05-01

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

  13. Genetic determinants of heat resistance in Escherichia coli

    PubMed Central

    Mercer, Ryan G.; Zheng, Jinshui; Garcia-Hernandez, Rigoberto; Ruan, Lifang; Gänzle, Michael G.; McMullen, Lynn M.

    2015-01-01

    Escherichia coli AW1.7 is a heat resistant food isolate and the occurrence of pathogenic strains with comparable heat resistance may pose a risk to food safety. To identify the genetic determinants of heat resistance, 29 strains of E. coli that differed in their of heat resistance were analyzed by comparative genomics. Strains were classified as highly heat resistant strains, exhibiting a D60-value of more than 6 min; moderately heat resistant strains, exhibiting a D60-value of more than 1 min; or as heat sensitive. A ~14 kb genomic island containing 16 predicted open reading frames encoding putative heat shock proteins and proteases was identified only in highly heat resistant strains. The genomic island was termed the locus of heat resistance (LHR). This putative operon is flanked by mobile elements and possesses >99% sequence identity to genomic islands contributing to heat resistance in Cronobacter sakazakii and Klebsiella pneumoniae. An additional 41 LHR sequences with >87% sequence identity were identified in 11 different species of β- and γ-proteobacteria. Cloning of the full length LHR conferred high heat resistance to the heat sensitive E. coli AW1.7ΔpHR1 and DH5α. The presence of the LHR correlates perfectly to heat resistance in several species of Enterobacteriaceae and occurs at a frequency of 2% of all E. coli genomes, including pathogenic strains. This study suggests the LHR has been laterally exchanged among the β- and γ-proteobacteria and is a reliable indicator of high heat resistance in E. coli. PMID:26441869

  14. Turn Up the Heat—Food and Clinical Escherichia coli Isolates Feature Two Transferrable Loci of Heat Resistance

    PubMed Central

    Boll, Erik J.; Marti, Roger; Hasman, Henrik; Overballe-Petersen, Søren; Stegger, Marc; Ng, Kim; Knøchel, Susanne; Krogfelt, Karen A.; Hummerjohann, Joerg; Struve, Carsten

    2017-01-01

    Heat treatment is a widely used process to reduce bacterial loads in the food industry or to decontaminate surfaces, e.g., in hospital settings. However, there are situations where lower temperatures must be employed, for instance in case of food production such as raw milk cheese or for decontamination of medical devices such as thermo-labile flexible endoscopes. A recently identified locus of heat resistance (LHR) has been shown to be present in and confer heat resistance to a variety of Enterobacteriaceae, including Escherichia coli isolates from food production settings and clinical ESBL-producing E. coli isolates. Here, we describe the presence of two distinct LHR variants within a particularly heat resistant E. coli raw milk cheese isolate. We demonstrate for the first time in this species the presence of one of these LHRs on a plasmid, designated pFAM21805, also encoding type 3 fimbriae and three bacteriocins and corresponding self-immunity proteins. The plasmid was highly transferable to other E. coli strains, including Shiga-toxin-producing strains, and conferred LHR-dependent heat resistance as well as type 3 fimbriae-dependent biofilm formation capabilities. Selection for and acquisition of this “survival” plasmid by pathogenic organisms, e.g., in food production environments, may pose great concern and emphasizes the need to screen for the presence of LHR genes in isolates. PMID:28439262

  15. 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.

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

    PubMed

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

    2015-12-01

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

  18. 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.

  19. Influence of Long Time Storage in Mineral Water on RNA Stability of Pseudomonas aeruginosa and Escherichia coli after Heat Inactivation

    PubMed Central

    Cenciarini, Claire; Courtois, Sophie; Raoult, Didier; La Scola, Bernard

    2008-01-01

    Background Research of RNA viability markers was previously studied for many bacterial species. Few and different targets of each species have been checked and motley results can be found in literature. No research has been done about Pseudomonas aeruginosa in this way. Methodology/Principal Findings Disappearance of 48 transcripts was analyzed by two-steps reverse transcription and real time polymerase chain reaction (RT-PCR) after heat-killing of Pseudomonas aeruginosa previously stored in mineral water or not. Differential results were obtained for each target. 16S rRNA, 23S rRNA, groEL, and rpmE were showed as the most persistent transcripts and rplP, rplV, rplE and rpsD were showed as the most labile transcripts after P. aeruginosa death. However, the labile targets appeared more persistent in bacteria previously stored in mineral water than freshly cultivated (non stored). These nine transcripts were also analyzed in Escherichia coli after heat-killing and different to opposite results were obtained, notably for groEL which was the most labile transcript of E. coli. Moreover, opposite results were obtained between mineral water stored and freshly cultivated E. coli. Conclusions and Significance This study highlights four potential viability markers for P. aeruginosa and four highly persistent transcripts. In a near future, these targets could be associated to develop an efficient viability kit. The present study also suggests that it would be difficult to determine universal RNA viability markers for environmental bacteria, since opposite results were obtained depending on the bacterial species and the physiological conditions. PMID:18941615

  20. 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

  1. Large Surface Blebs on Escherichia coli Heated to Inactivating Temperatures

    PubMed Central

    Scheie, Paul; Ehrenspeck, Susan

    1973-01-01

    Large surface blebs were observed with phase-contrast optics on Escherichia coli B/r and Bs-1 heated to temperatures at which colony-forming ability was lost. Characterization of such blebs was consistent with the view that they were formed by a physical process and were bounded by the outer membrane of the cell. A hypothesis for thermal inactivation of E. coli is presented that places membrane damage near the primary lethal event. Images PMID:4196258

  2. Identification of heat resistant Escherichia coli by qPCR for the locus of heat resistance.

    PubMed

    Ma, Angela; Chui, Linda

    2017-02-01

    Three qPCR assays targeting the locus of heat resistance to identify heat resistant clinical Escherichia coli isolates are described. Of 613 isolates, 3 (0.5%) possessed the locus. The assays are a rapid, highly sensitive and specific alternative to screening by heat shock and can be used in food safety surveillance.

  3. Highly labile elements. [in meteorites

    NASA Technical Reports Server (NTRS)

    Lipschutz, Michael E.; Woolum, Dorothy S.

    1988-01-01

    Certain elements of high lability are very responsive to thermal processes, being either highly volatile during primary nebular condensation or highly mobile by postaccretionary metamorphic or shock heating. Data for highly labile elements indicate that different thermal processes were important in the genesis of each of the chondritic groups and a discussion of each is given. Contents of highly labile elements in a given group of contemporary falls differ from those of the same group that fell in Antarctica more than 0.1 Myr ago. This difference is due either to a time-dependent change in meteorite sources or, less likely, orbital variation of the meteorite flux to Earth.

  4. Highly labile elements. [in meteorites

    NASA Technical Reports Server (NTRS)

    Lipschutz, Michael E.; Woolum, Dorothy S.

    1988-01-01

    Certain elements of high lability are very responsive to thermal processes, being either highly volatile during primary nebular condensation or highly mobile by postaccretionary metamorphic or shock heating. Data for highly labile elements indicate that different thermal processes were important in the genesis of each of the chondritic groups and a discussion of each is given. Contents of highly labile elements in a given group of contemporary falls differ from those of the same group that fell in Antarctica more than 0.1 Myr ago. This difference is due either to a time-dependent change in meteorite sources or, less likely, orbital variation of the meteorite flux to Earth.

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

    PubMed

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

    1998-11-01

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

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

    PubMed Central

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

    1983-01-01

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

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed Central

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

    1984-01-01

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

  9. Hysteresis on heating and cooling of E. coli alkaline phosphatase.

    PubMed

    Uto, Innocent S; Brewer, John M

    2008-01-01

    Measurements of [theta](222) of E. coli phosphatase on heating from 20 degrees to 90 degrees and subsequent cooling to 20 degrees shows a gradual increase in [theta](222) on heating, while cooling shows a symmetric transition centered at 45 degrees . Reheating and cooling shows the same phenomenon. Enzyme heated and cooled once is fully active. The activity of the enzyme depends on its storage conditions (buffer and pH for example), but such changes are least to some extent reversible, especially by heating in different solvents. We conclude the enzyme exists in several forms which are in slow equilibrium with each other, so that the enzyme responds slowly when heated and hence is not at equilibrium during heating/cooling experiments.

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

    PubMed

    Yamamoto, T; Nakazawa, M

    1997-01-01

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

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

    PubMed

    Yamaguchi, H

    1993-02-01

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

  12. 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.

  13. Consensus sequence for Escherichia coli heat shock gene promoters.

    PubMed Central

    Cowing, D W; Bardwell, J C; Craig, E A; Woolford, C; Hendrix, R W; Gross, C A

    1985-01-01

    We have identified promoters for the Escherichia coli heat shock operons dnaK and groE and the gene encoding heat shock protein C62.5. Transcription from each promoter is heat-inducible in vivo, and each is recognized in vitro by RNA polymerase containing sigma 32, the sigma factor encoded by rpoH (htpR) but not by RNA polymerase containing sigma 70. We compared the sequences of the heat shock promoters and propose a consensus promoter sequence, having T-N-t-C-N-C-c-C-T-T-G-A-A in the -35 region and C-C-C-C-A-T-t-T-a in the -10 region. These sequences differ from the consensus sequence recognized by holoenzyme containing sigma 70, the major sigma in E. coli. We suggest that the accumulated consensus sequences of promoters recognized by alternate forms of holoenzyme are compatible with a model in which sigma recognizes only the -10 region of the promoter. Images PMID:3887408

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

    PubMed Central

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

    1988-01-01

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

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

    PubMed

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

    2010-08-27

    DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 degrees C or 65 degrees C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis. Copyright 2010 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2001-08-01

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

  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 Central

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

    2013-01-01

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

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

    PubMed

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

    2017-10-04

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    1974-01-01

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

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

    PubMed

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

    2016-03-01

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

  2. Evaluation of sodium benzoate and licorice (Glycyrrhiza glabra) root extract as heat-sensitizing additives against Escherichia coli O157:H7 in mildly heated young coconut liquid endosperm.

    PubMed

    Gabriel, A A; Salazar, S K P

    2014-08-01

    This study evaluated the use of sodium benzoate (SB) and licorice root extract (LRE) as heat-sensitizing additives against Escherichia coli O157:H7 in mildly heated young coconut liquid endosperm. Consumer acceptance scoring showed that maximum permissible supplementation (MPS) levels for SB and LRE were at 300 and 250 ppm, respectively. The MPS values were considered in the generation of a 2-factor rotatable central composite design for the tested SB and LRE concentration combinations. Liquid endosperm with various SB and LRE supplementation combinations was inoculated with E. coli O157:H7 and heated to 55°C. The susceptibility of the cells towards heating was expressed in terms of the decimal reduction time (D55 ). Response surface analysis showed that only the individual linear effect of benzoate significantly influenced D55 value, where increasing supplementation level resulted in increasing susceptibility. The results reported could serve as baseline information in further investigating other additives that could be used as heat-sensitizing agents against pathogens in heat-labile food systems. Fruit juice products have been linked to outbreaks of microbial infection, where unpasteurized products were proven vectors of diseases. Processors often opt not to apply heat process to juice products as the preservation technique often compromises the sensorial quality. This work evaluated two common additives for their heat-sensitizing effects against E. coli O157:H7 in coconut liquid endosperm, the results of which may serve as baseline information to small- and medium-scale processors, and researchers in the establishment of mild heat process schedule for the test commodity and other similar products. © 2014 The Society for Applied Microbiology.

  3. Enhancement of enterohemorrhagic Escherichia coli O157:H7 stress tolerance via pre-heating.

    PubMed

    Nakano, Masanori; Itoh, Youko; Yamada, Yoshiaki; Nakamura, Kogenta; Sumitomo, Makoto; Nitta, Masakazu

    2012-03-01

    Enterohemorrhagic Escherichia coli O157:H7 infection causes several hundred cases of food poisoning every year in Japan. In severe cases, this type of food poisoning can be fatal. In the present study, we examined the induction of HSP70 in E. coli O157:H7 cells at various temperatures and the thermotolerance of E. coli O157:H7 cells alone and in contaminated food following pre-heating. We evaluated the possibility that thermotolerance by E. coli O157:H7 increases the likelihood of food poisoning. E. coli O157:H7 cells were heated at 43-51 °C, and the survival rate was examined. The temperature of highest induction of HSP70 was used as the pre-heating temperature. We measured the thermotolerance of E. coli O157:H7 cells following pre-heating as the survival after heating at 53 °C (lethal temperature). Additionally, we evaluated the thermotolerance of E. coli O157:H7 cells in ground beef following pre-heating. Heating at 47 °C for 30 min caused the highest induction of HSP70 and this temperature was selected as the pre-heating temperature. The survival rate was significantly higher for 0-90 min compared to that in cultures incubated at 53 °C without pre-heating indicating thermotolerance. Additionally, in ground beef, thermotolerance in E. coli O157:H7 cells was induced by pre-heating. We showed that E. coli O157:H7 cells acquired thermotolerance after pre-heating, which significantly increased survival after a lethal temperature, and increased the likelihood of food poisoning.

  4. Analysis of Heat-Labile Sites Generated by Reactions of Depleted Uranium and Ascorbate in Plasmid DNA

    PubMed Central

    Wilson, Janice; Young, Ashley; Civitello, Edgar R.

    2013-01-01

    The goal of this study was to characterize how depleted uranium (DU) causes DNA damage. Procedures were developed to assess the ability of organic and inorganic DNA adducts to convert to single strand breaks (SSB) in pBR322 plasmid DNA in the presence of heat or piperidine. DNA adducts formed by methyl methanesulfonate (MMS), cis-platin (cis-Pt), and chromic chloride were compared to those formed by reaction of uranyl acetate (UA) and ascorbate (Asc). Uranyl ion in the presence of Asc produced U-DNA adducts that converted to SSB upon heating. Piperidine, which acted on DNA methylated by MMS to convert methyl-DNA adducts to SSB, served in the opposite fashion with U-DNA adducts by decreasing SSB. The observation that piperidine also decreased the gel shift for metal-DNA adducts formed by monofunctional cis-Pt and chromic chloride was interpreted to suggest that piperidine served to remove U-DNA adducts. Radical scavengers did not affect formation of U-induced SSB, suggesting that SSB arose from the presence of U-DNA adducts and not from free radicals. A model is proposed to predict how U-DNA adducts may serve as initial lesions that convert to SSB or AP sites. Results suggest that DU can act as a chemical genotoxin that does not require radiation for its mode of action. Characterizing the DNA lesions formed by DU is necessary to assess the relative importance of different DNA lesions in the formation of DU-induced mutations. Understanding mechanisms of formation of DU-induced mutations may contribute to identification of biomarkers of DU exposures in humans. PMID:24218036

  5. Some Like It Hot: Heat Resistance of Escherichia coli in Food

    PubMed Central

    Li, Hui; Gänzle, Michael

    2016-01-01

    Heat treatment and cooking are common interventions for reducing the numbers of vegetative cells and eliminating pathogenic microorganisms in food. Current cooking method requires the internal temperature of beef patties to reach 71°C. However, some pathogenic Escherichia coli such as the beef isolate E. coli AW 1.7 are extremely heat resistant, questioning its inactivation by current heat interventions in beef processing. To optimize the conditions of heat treatment for effective decontaminations of pathogenic E. coli strains, sufficient estimations, and explanations are necessary on mechanisms of heat resistance of target strains. The heat resistance of E. coli depends on the variability of strains and properties of food formulations including salt and water activity. Heat induces alterations of E. coli cells including membrane, cytoplasm, ribosome and DNA, particularly on proteins including protein misfolding and aggregations. Resistant systems of E. coli act against these alterations, mainly through gene regulations of heat response including EvgA, heat shock proteins, σE and σS, to re-fold of misfolded proteins, and achieve antagonism to heat stress. Heat resistance can also be increased by expression of key proteins of membrane and stabilization of membrane fluidity. In addition to the contributions of the outer membrane porin NmpC and overcome of osmotic stress from compatible solutes, the new identified genomic island locus of heat resistant performs a critical role to these highly heat resistant strains. This review aims to provide an overview of current knowledge on heat resistance of E. coli, to better understand its related mechanisms and explore more effective applications of heat interventions in food industry. PMID:27857712

  6. Occurrence of Hybrid Escherichia coli Strains Carrying Shiga Toxin and Heat-Stable Toxin in Livestock of Bangladesh

    PubMed Central

    Johura, Fatema-Tuz; Parveen, Rozina; Islam, Atiqul; Sadique, Abdus; Rahim, Md Niaz; Monira, Shirajum; Khan, Anisur R.; Ahsan, Sunjukta; Ohnishi, Makoto; Watanabe, Haruo; Chakraborty, Subhra; George, Christine M.; Cravioto, Alejandro; Navarro, Armando; Hasan, Badrul; Alam, Munirul

    2017-01-01

    Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) are important causes of diarrhea in humans and animals worldwide. Although ruminant animals are the main source of STEC, diarrhea due to this pathotype is very low in Bangladesh where ETEC remains the predominant group associated with childhood diarrhea. In the present study, E. coli strains (n = 35) isolated from Bangladesh livestock (goats, sheep, and cattle) and poultry (chicken and ducks) were analyzed for the presence of major virulence factors, such as Shiga toxins (STX-1 and STX-2), heat-labile toxin, and heat-stable toxins (STa and STb). Multiplex polymerase chain reaction results revealed 23 (66%) E. coli strains to be virulent possessing either sta (n = 5), stx (stx1, n = 8; stx2, n = 2), or both (n = 8) genes in varying combinations. Thirty-four percent (8/23) of strains from livestock were hybrid type that carried both stx (either stx1 or stx2) and ETEC-specific enterotoxin gene sta. Serotyping results revealed that the ETEC strains belonged to five serotypes, namely O36:H5, O174:H−, O152:H8, O109:H51, and O8:H21, while the STEC-producing strains belonged to serotypes O76:H19 (n = 3), O43:H2 (n = 2), O87:H16 (n = 2), OR:H2 (n = 1), O110:H16 (n = 1), and O152:H8 (n = 1). The STEC–ETEC hybrid strains belonged to serotypes O76:H19 (n = 3), O43:H2 (n = 2), O87:H16, OR:H2, and O152:H8. Forty percent (2/5) of the ETEC and 20% (2/10) of the STEC strains were multidrug resistant with the highest drug resistance (50%) being found in the hybrid strains. Molecular fingerprinting determined by pulsed-field gel electrophoresis and cluster analyses by dendrogram revealed that, genetically, STEC–ETEC hybrid strains were highly heterogeneous. Multidrug-resistant E. coli STEC–ETEC hybrid strains in domesticated animals pose a public health threat for humans in Bangladesh. PMID:28119905

  7. Occurrence of Hybrid Escherichia coli Strains Carrying Shiga Toxin and Heat-Stable Toxin in Livestock of Bangladesh.

    PubMed

    Johura, Fatema-Tuz; Parveen, Rozina; Islam, Atiqul; Sadique, Abdus; Rahim, Md Niaz; Monira, Shirajum; Khan, Anisur R; Ahsan, Sunjukta; Ohnishi, Makoto; Watanabe, Haruo; Chakraborty, Subhra; George, Christine M; Cravioto, Alejandro; Navarro, Armando; Hasan, Badrul; Alam, Munirul

    2016-01-01

    Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) are important causes of diarrhea in humans and animals worldwide. Although ruminant animals are the main source of STEC, diarrhea due to this pathotype is very low in Bangladesh where ETEC remains the predominant group associated with childhood diarrhea. In the present study, E. coli strains (n = 35) isolated from Bangladesh livestock (goats, sheep, and cattle) and poultry (chicken and ducks) were analyzed for the presence of major virulence factors, such as Shiga toxins (STX-1 and STX-2), heat-labile toxin, and heat-stable toxins (STa and STb). Multiplex polymerase chain reaction results revealed 23 (66%) E. coli strains to be virulent possessing either sta (n = 5), stx (stx1, n = 8; stx2, n = 2), or both (n = 8) genes in varying combinations. Thirty-four percent (8/23) of strains from livestock were hybrid type that carried both stx (either stx1 or stx2) and ETEC-specific enterotoxin gene sta. Serotyping results revealed that the ETEC strains belonged to five serotypes, namely O36:H5, O174:H-, O152:H8, O109:H51, and O8:H21, while the STEC-producing strains belonged to serotypes O76:H19 (n = 3), O43:H2 (n = 2), O87:H16 (n = 2), OR:H2 (n = 1), O110:H16 (n = 1), and O152:H8 (n = 1). The STEC-ETEC hybrid strains belonged to serotypes O76:H19 (n = 3), O43:H2 (n = 2), O87:H16, OR:H2, and O152:H8. Forty percent (2/5) of the ETEC and 20% (2/10) of the STEC strains were multidrug resistant with the highest drug resistance (50%) being found in the hybrid strains. Molecular fingerprinting determined by pulsed-field gel electrophoresis and cluster analyses by dendrogram revealed that, genetically, STEC-ETEC hybrid strains were highly heterogeneous. Multidrug-resistant E. coli STEC-ETEC hybrid strains in domesticated animals pose a public health threat for humans in Bangladesh.

  8. Influence of cyclopropane fatty acids on heat, high pressure, acid and oxidative resistance in Escherichia coli.

    PubMed

    Chen, Yuan Yao; Gänzle, Michael G

    2016-04-02

    Heat and high pressure resistant strains of Escherichia coli are a challenge to food safety. This study investigated effects of cyclopropane fatty acids (CFAs) on stress tolerance in the heat- and pressure-resistant strain E. coli AW1.7 and the sensitive strain E. coli MG1655. The role of CFAs was explored by disruption of cfa coding for CFA synthase with an in-frame, unmarked deletion method. Both wild-type strains consumed all the unsaturated fatty acids (C16:1 and C18:1) that were mostly converted to CFAs and a low proportion to saturated fatty acid (C16:0). Moreover, E. coli AW1.7 contained a higher proportion of membrane C19:0 cyclopropane fatty acid than E. coli MG1655 (P<0.05). The Δcfa mutant strains did not produce CFAs, and the corresponding substrates C16:1 and C18:1 accumulated in membrane lipids. The deletion of cfa did not alter resistance to H2O2 but increased the lethality of heat, high pressure and acid treatments in E. coli AW1.7, and E. coli MG1655. E. coli AW1.7 and its Δcfa mutant were more resistant to pressure and heat but less resistant to acid stress than E. coli MG1655. Heat resistance of wild-type strains and their Δcfa mutant was also assessed in beef patties grilled to an internal temperature of 71 °C. After treatment, cell counts of wild type strains were higher than those of the Δcfa mutant strains. In conclusion, CFA synthesis in E. coli increases heat, high pressure and acid resistance, and increases heat resistance in food. This knowledge on mechanisms of stress resistance will facilitate the design of intervention methods for improved pathogen control in food production. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Heat-treated Escherichia coli as a high-capacity biosorbent for tungsten anions.

    PubMed

    Ogi, Takashi; Makino, Takahiko; Iskandar, Ferry; Tanabe, Eishi; Okuyama, Kikuo

    2016-10-01

    Adsorption performance in the biosorption of tungsten using Escherichia coli cells can be significantly improved by using cell suspensions that have been heat-treated at ⩽100°C. In the case of E. coli cells suspension heated at 100°C, the aqueous tungsten ions concentration rapidly decreased from 0.8mmol/L to practically zero within 1h. This biosorption time is much shorter than that of non-heat treated E. coli cells (7h). Furthermore, the adsorption saturation amount for cells heat-treated at 100°C was significantly increased up to 1.62mmol-W/g-E. coli compared to the unheated E. coli cells case (0.62mmol-W/g-E. coli). Determination of the surface potential and surface structure along with quantitative analyses of free amino acids of heat-treated E. coli cells were also carried out and revealed that heated cells have a high zeta potential and express a higher concentration of amino acids on the cell surface.

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

    PubMed Central

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

    1978-01-01

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

  11. High Heating Rates Affect Greatly the Inactivation Rate of Escherichia coli.

    PubMed

    Huertas, Juan-Pablo; Aznar, Arantxa; Esnoz, Arturo; Fernández, Pablo S; Iguaz, Asunción; Periago, Paula M; Palop, Alfredo

    2016-01-01

    Heat resistance of microorganisms can be affected by different influencing factors. Although, the effect of heating rates has been scarcely explored by the scientific community, recent researches have unraveled its important effect on the thermal resistance of different species of vegetative bacteria. Typically heating rates described in the literature ranged from 1 to 20°C/min but the impact of much higher heating rates is unclear. The aim of this research was to explore the effect of different heating rates, such as those currently achieved in the heat exchangers used in the food industry, on the heat resistance of Escherichia coli. A pilot plant tubular heat exchanger and a thermoresistometer Mastia were used for this purpose. Results showed that fast heating rates had a deep impact on the thermal resistance of E. coli. Heating rates between 20 and 50°C/min were achieved in the heat exchanger, which were much slower than those around 20°C/s achieved in the thermoresistometer. In all cases, these high heating rates led to higher inactivation than expected: in the heat exchanger, for all the experiments performed, when the observed inactivation had reached about seven log cycles, the predictions estimated about 1 log cycle of inactivation; in the thermoresistometer these differences between observed and predicted values were even more than 10 times higher, from 4.07 log cycles observed to 0.34 predicted at a flow rate of 70 mL/min and a maximum heating rate of 14.7°C/s. A quantification of the impact of the heating rates on the level of inactivation achieved was established. These results point out the important effect that the heating rate has on the thermal resistance of E. coli, with high heating rates resulting in an additional sensitization to heat and therefore an effective food safety strategy in terms of food processing.

  12. High Heating Rates Affect Greatly the Inactivation Rate of Escherichia coli

    PubMed Central

    Huertas, Juan-Pablo; Aznar, Arantxa; Esnoz, Arturo; Fernández, Pablo S.; Iguaz, Asunción; Periago, Paula M.; Palop, Alfredo

    2016-01-01

    Heat resistance of microorganisms can be affected by different influencing factors. Although, the effect of heating rates has been scarcely explored by the scientific community, recent researches have unraveled its important effect on the thermal resistance of different species of vegetative bacteria. Typically heating rates described in the literature ranged from 1 to 20°C/min but the impact of much higher heating rates is unclear. The aim of this research was to explore the effect of different heating rates, such as those currently achieved in the heat exchangers used in the food industry, on the heat resistance of Escherichia coli. A pilot plant tubular heat exchanger and a thermoresistometer Mastia were used for this purpose. Results showed that fast heating rates had a deep impact on the thermal resistance of E. coli. Heating rates between 20 and 50°C/min were achieved in the heat exchanger, which were much slower than those around 20°C/s achieved in the thermoresistometer. In all cases, these high heating rates led to higher inactivation than expected: in the heat exchanger, for all the experiments performed, when the observed inactivation had reached about seven log cycles, the predictions estimated about 1 log cycle of inactivation; in the thermoresistometer these differences between observed and predicted values were even more than 10 times higher, from 4.07 log cycles observed to 0.34 predicted at a flow rate of 70 mL/min and a maximum heating rate of 14.7°C/s. A quantification of the impact of the heating rates on the level of inactivation achieved was established. These results point out the important effect that the heating rate has on the thermal resistance of E. coli, with high heating rates resulting in an additional sensitization to heat and therefore an effective food safety strategy in terms of food processing. PMID:27563300

  13. The locus of heat resistance (LHR) mediates heat resistance in Salmonella enterica, Escherichia coli and Enterobacter cloacae.

    PubMed

    Mercer, Ryan G; Walker, Brian D; Yang, Xianqin; McMullen, Lynn M; Gänzle, Michael G

    2017-06-01

    Enterobacteriaceae comprise food spoilage organisms as well as food-borne pathogens including Escherichia coli. Heat resistance in E. coli was attributed to a genomic island called the locus of heat resistance (LHR). This genomic island is also present in several other genera of Enterobacteriaceae, but its function in the enteric pathogens Salmonella enterica and Enterobacter cloacae is unknown. This study aimed to determine the frequency of the LHR in food isolates of E. coli, and its influence on heat resistance in S. enterica and Enterobacter spp. Cell counts of LHR-positive strains of E. coli, S. enterica and E. cloacae were reduced by less than 1, 1, and 4 log (cfu/mL), respectively, after exposure to 60 °C for 5 min, while cell counts of LHR-negative strains of the same species were reduced by more than 7 log (cfu/mL). Introducing an exogenous copy of the LHR into heat-sensitive enteropathogenic E. coli and S. enterica increased heat resistance to a level that was comparable to LHR-positive wild type strains. Cell counts of LHR-positive S. enterica were reduced by less than 1 log(cfu/mL) after heating to 60 °C for 5 min. Survival of LHR-positive strains was improved by increasing the NaCl concentration from 0 to 4%. Cell counts of LHR-positive strains of E. coli and S. enterica were reduced by less than 2 log (cfu/g) in ground beef patties cooked to an internal core temperature of 71 °C. This study indicates that LHR-positive Enterobacteriaceae pose a risk to food safety.

  14. Heat Adaptation Alters Escherichia coli O157:H7 Membrane Lipid Composition and Verotoxin Production

    PubMed Central

    Yuk, Hyun-Gyun; Marshall, Douglas L.

    2003-01-01

    The influence of heat adaptation (growth at 42 and 45°C) on changes in membrane lipid composition and verotoxin concentration of Escherichia coli O157:H7 (ATCC 43895), an rpoS mutant of ATCC 43895 (FRIK 816-3), a verotoxin mutant E. coli O157:H7 (B6-914), and nonpathogenic E. coli (ATCC 25922) was investigated. D values (57°C) of heat-adapted cells were up to 3.9 min longer than those of control cells for all four strains. Heat adaptation increased the amounts of palmitic acid (16:0) and cis-vaccenic acid (18:1ω7c) in membrane lipids of ATCC 43895 and the rpoS mutant, whereas there was a reduction and no change in the amount of cis-vaccenic acid in nonpathogenic and verotoxin mutant E. coli, respectively. The ratio of palmitic to cis-vaccenic acids decreased in ATCC 43895 and in the rpoS mutant, whereas the ratio increased in nonpathogenic E. coli and was not different in the verotoxin mutant with elevated growth temperature. Total verotoxin concentration decreased due to a reduction in intracellular verotoxin amount in heat-adapted ATCC 43895 and rpoS mutant strains. However, extracellular verotoxin concentration increased in heat-adapted cells. The rpoS gene did not influence membrane lipid composition changes although it did affect heat resistance. Results suggest that increased membrane fluidity may have caused increased verotoxin secretion. PMID:12957893

  15. Immunogenicity and Efficacy of a Heat-Killed Whole Cell/B Subunit Cholera Vaccine

    DTIC Science & Technology

    1993-05-15

    occurring enterotoxigenic E . coli infection in U.S. adults in Mexico with a comparison between the two study groups of the following endpoints: rates of...enterotoxigenic E . coli due to heat-labile (LT) and heat-stable (ST) enterotoxin producing strains during a 5-week period, timing of protection in...relationship to vaccine administration, and relationship between enterotoxigenic E . coli diarrhea attack rates and serum IgG as well as fecal IgA

  16. Variation in heat and pressure resistance of verotoxigenic and nontoxigenic Escherichia coli.

    PubMed

    Liu, Yang; Gill, Alex; McMullen, Lynn; Gänzle, Michael G

    2015-01-01

    This study evaluated the heat and pressure resistance of 112 strains of Escherichia coli, including 102 strains of verotoxigenic E. coli (VTEC) representing 23 serotypes and four phylogenetic groups. In an initial screening, the heat and pressure resistance of 100 strains, including 94 VTEC strains, were tested in phosphate-buffered saline (PBS). Treatment at 60°C for 5 min reduced cell counts by 2.0 to 5.5 log CFU/ml; treatment at 600 MPa for 3 min at 25°C reduced the cell counts by 1.1 to 5.5 log CFU/ml. Heat or pressure resistance did not correlate to the phylogenetic group or the serotype. A smaller group of E. coli strains was evaluated for heat and pressure resistance in Luria-Bertani (LB) broth. Generally, the levels of heat resistance of E. coli strains in LB and PBS were similar; however, the levels of pressure resistance observed for treatments in LB broth or PBS were variable. The cell counts of pressure-resistant strains of VTEC were reduced by less than 1.5 log CFU/ml after treatment at 600 MPa for 3 min. E. coli strains were also treated with 600 MPa for 3 min in ground beef or inoculated into beef patties and grilled to 63 or 71°C. The cell counts of the VTEC E. coli O26:H11 strain 05-6544 were reduced by 2 log CFU/g by pressure treatment in ground beef. The cell counts of the heat-resistant E. coli strain AW1.7 were reduced by 1.4 and 3.4 log CFU/g in beef patties grilled to internal temperatures of 63 and 71°C, respectively. The cell counts of E. coli 05-6544 were reduced by less than 3 and 6 log CFU/g in beef patties grilled to internal temperatures of 63 and 71°C, respectively. To study whether the composition of the beef patties influenced heat resistance, E. coli strains AW1.7, AW1.7 Δ pHR1, MG1655, and LMM1030 were mixed into beef patties containing 15 or 35% fat and 0 or 2% NaCl, and the patties were grilled to an internal temperature of 63°C. The highest heat resistance of E. coli was observed in patties containing 15% fat and 2% NaCl.

  17. Molecular responses of Escherichia coli caused by heat stress and recombinant protein production during temperature induction.

    PubMed

    Valdez-Cruz, Norma A; Ramírez, Octavio T; Trujillo-Roldán, Mauricio A

    2011-01-01

    In a recent review, we discussed the extensively used temperature-inducible expression system, based on the pL and/or pR phage lambda promoters that are finely regulated by the thermo-labile cI857 repressor. In this system, an increase in temperature induces the heterologous protein production and activates the heat shock response, as well as the stringent and SOS responses. The same responses are activated just by the overproduction of recombinant protein. All such responses result in a metabolic burden to the cells, a decrease in the specific growth rate, and alterations in the central carbon metabolism. Altogether, these effects can alter the quantity and quality of the produced foreign protein. Here, we compare and discuss the transcription of selected genes, and the concomitant synthesis of heat-shock proteins (hsp) soon after thermal induction, in relation to the responses that occur in other expression systems that also trigger the heat-shock response.

  18. Travelers' diarrhea and toxigenic Escherichia coli.

    PubMed

    Gorbach, S L; Kean, B H; Evans, D G; Evans, D J; Bessudo, D

    1975-05-01

    In a group of 133 United States students studied for 18 days after arriving in Mexico, diarrhea developed in 38 (29 per cent). Diarrhea rarely began before the fourth day, and the mean onset was 13 days after arrival. Symptoms lasted an average of 3.4 days but persisted in 21 per cent of sick students. Heat-labile enterotoxin-producing Escheria coli was found in the stools of 72 per cent of sick and 15 per cent of healthy students. None had heat-labile Esch. coli when they entered Mexico. The incubation period was short, generally 24 to 48 hours, and the carrier state was five days or less in 82 per cent of students surveyed. Entamoeba histolytica was found in 6 per cent of cases of diarrhea, but not salmonella, shigella or penetrating Esch. coli. These studies suggest that approximately 70 per cent of travelers' diarrhea in Mexico is associated with heat-labile toxigenic strains of Esch. coli.

  19. The recovery of heat-stressed Escherichia coli in lake water microcosms.

    PubMed

    Lim, C H; Flint, K P

    1995-12-01

    Escherichia coli was heat stressed at 55 degrees, 60 degrees or 65 degrees C in sterile flasks of lake water. After 6 h at these temperatures the viable count on nutrient agar had dropped below the limits of detection (1 colony in 100 ml). The flasks were transferred to a 15 degrees C incubator and left for 7 d. Recovery of the stressed E. coli was shown to occur within 48 h at this temperature. Recovery also occurred in microcosms amended with 5% (v/v) synthetic sewage. The stressed E. coli multiplied in the amended but not in the unamended microcosms.

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

    PubMed Central

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

    1984-01-01

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

  1. Oxidative stress in E. coli cells upon exposure to heat treatments.

    PubMed

    Marcén, María; Ruiz, Virginia; Serrano, Mª Jesús; Condón, Santiago; Mañas, Pilar

    2017-01-16

    Heat treatments are widely used by the food industry to inactivate microorganisms, however their mode of action on microbial cells is not fully known. In the last years, it has been proposed that the generation of oxidative species could be an important factor contributing to cell death by heat and by other stresses; however, investigations in this field are scarce. The present work studies the generation of reactive oxygen species (ROS) upon heat treatment in E. coli, through the use of cell staining with specific fluorochromes. Results obtained demonstrate that ROS are detected in E. coli cells when they are subjected to heat exposure, and the amount of fluorescence increases with temperature and time, as does the cellular inactivation. The addition of glutathione or tiron, a potent antioxidant and a superoxide quencher, respectively, to the heating medium protected E. coli against heat inactivation and concurrently reduced the detection of ROS, especially in the case of glutathione. Finally, recovery of heated cells under conditions that relief oxidative stress produced an increase in cell survival. Data presented in this work support the view that ROS generation and subsequent control in bacterial cells could be an essential factor determining inactivation and survival upon exposure to heat, and it could be a potential target to increase the efficacy of current treatments. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Enterotoxigenic Escherichia coli

    PubMed Central

    Fleckenstein, James M; Munson, George M; Rasko, David A

    2013-01-01

    The enterotoxigenic Escherichia coli are a pervasive cause of serious diarrheal illness in developing countries. Presently, there is no vaccine to prevent these infections, and many features of the basic pathogenesis of these organisms remain poorly understood. Until very recently most pathogenesis studies had focused almost exclusively on a small subset of known “classical” virulence genes, namely fimbrial colonization factors and the heat-labile (LT) and heat stable (ST) enterotoxins. However, recent investigations of pathogen-host interactions reveal a surprisingly complex and intricately orchestrated engagement involving the interplay of classical and “novel” virulence genes, as well as participation of genes highly conserved in the E. coli species. These studies may inform further rational approaches to vaccine development for these important pathogens. PMID:23892244

  3. Escherichia coli Heat Shock Protein DnaK: Production and Consequences in Terms of Monitoring Cooking

    PubMed Central

    Seyer, Karine; Lessard, Martin; Piette, Gabriel; Lacroix, Monique; Saucier, Linda

    2003-01-01

    Through use of commercially available DnaK proteins and anti-DnaK monoclonal antibodies, a competitive enzyme-linked immunosorbent assay was developed to quantify this heat shock protein in Escherichia coli ATCC 25922 subjected to various heating regimens. For a given process lethality (F7010 of 1, 3, and 5 min), the intracellular concentration of DnaK in E. coli varied with the heating temperature (50 or 55°C). In fact, the highest DnaK concentrations were found after treatments at the lower temperature (50°C) applied for a longer time. Residual DnaK after heating was found to be necessary for cell recovery, and additional DnaK was produced during the recovery process. Overall, higher intracellular concentrations of DnaK tended to enhance cell resistance to a subsequent lethal stress. Indeed, E. coli cells that had undergone a sublethal heat shock (105 min at 55°C, F7010 = 3 min) accompanied by a 12-h recovery (containing 76,786 ± 25,230 molecules/cell) resisted better than exponentially growing cells (38,500 ± 6,056 molecules/cell) when later heated to 60°C for 50 min (F7010 = 5 min). Results reported here suggest that using stress protein to determine cell adaptation and survival, rather than cell counts alone, may lead to more efficient heat treatment. PMID:12788720

  4. Impacts of Individual Animal Response to Heat and Handling Stresses on Escherichia coli and E. coli O157:H7 Fecal Shedding by Feedlot Cattle

    USDA-ARS?s Scientific Manuscript database

    The reduction of foodborne pathogens in cattle destined for human consumption will require knowledge of the factors that impact the carriage and shedding of these organisms. The effects of heat and handling stress levels on the fecal shedding of Escherichia coli O157:H7 and generic E. coli by feedl...

  5. Impacts of individual animal response to heat and handling stresses on Escherichia coli and E. coli O157:H7 fecal shedding by feedlot cattle.

    PubMed

    Brown-Brandl, Tami M; Berry, Elaine D; Wells, James E; Arthur, Terrance M; Nienaber, John A

    2009-09-01

    The reduction of foodborne pathogens in cattle destined for human consumption will require knowledge of the factors that impact the carriage and shedding of these organisms. The objective of this work was to investigate the effects of heat and handling stress levels on the fecal shedding of Escherichia coli O157:H7 and generic E. coli by feedlot cattle. In year 1, 128 feedlot heifers were evaluated for heat tolerance five times per week during the 84-day finishing period from May through August. Heat stress measurements included respiration rate, panting score, and visual assessments. In year 2, panting scores were taken for a group of 256 finishing feedlot heifers on days in July and August for which the temperature humidity index (THI) was predicted to be in the "emergency" category (THI > or = 84). For both years, animals were weighed and temperament scored to assess handling stress on a 28-day schedule. At the same time, rectal fecal samples were collected from each animal individually. The presence and concentrations of E. coli O157:H7 and concentrations of generic E. coli in feces were determined. There were no clear trends between the heat stress levels or temperament scores (as an indicator of response to handling) with either fecal generic E. coli concentrations or E. coli O157:H7 concentrations or prevalence in feces, indicating that neither heat nor handling stress contributes to the food safety risk associated with E. coli O157:H7-positive cattle.

  6. Nonnative Disulfide Bond Formation Activates the σ32-Dependent Heat Shock Response in Escherichia coli

    PubMed Central

    Müller, Alexandra; Hoffmann, Jörg H.; Meyer, Helmut E.; Narberhaus, Franz; Jakob, Ursula

    2013-01-01

    Formation of nonnative disulfide bonds in the cytoplasm, so-called disulfide stress, is an integral component of oxidative stress. Quantification of the extent of disulfide bond formation in the cytoplasm of Escherichia coli revealed that disulfide stress is associated with oxidative stress caused by hydrogen peroxide, paraquat, and cadmium. To separate the impact of disulfide bond formation from unrelated effects of these oxidative stressors in subsequent experiments, we worked with two complementary approaches. We triggered disulfide stress either chemically by diamide treatment of cells or genetically in a mutant strain lacking the major disulfide-reducing systems TrxB and Gor. Studying the proteomic response of E. coli exposed to disulfide stress, we found that intracellular disulfide bond formation is a particularly strong inducer of the heat shock response. Real-time quantitative PCR experiments showed that disulfide stress induces the heat shock response in E. coli σ32 dependently. However, unlike heat shock treatment, which induces these genes transiently, transcripts of σ32-dependent genes accumulated over time in disulfide stress-treated cells. Analyzing the stability of σ32, we found that this constant induction can be attributed to an increase of the half-life of σ32 upon disulfide stress. This is concomitant with aggregation of E. coli proteins treated with diamide. We conclude that oxidative stress triggers the heat shock response in E. coli σ32 dependently. The component of oxidative stress responsible for the induction of heat shock genes is disulfide stress. Nonnative disulfide bond formation in the cytoplasm causes protein unfolding. This stabilizes σ32 by preventing its DnaK- and FtsH-dependent degradation. PMID:23585533

  7. A FaeG-FedF-LT192 fusion elicits protective anti-adhesin and antitoxin antibodies against porcine enterotoxigenic Escherichia coli

    USDA-ARS?s Scientific Manuscript database

    Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 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 anti-adhesin (anti-K88 & anti-F18) and antitoxin (anti-LT & anti-ST) 5 immunity would provide ...

  8. Functional Analysis of Genes Comprising the Locus of Heat Resistance in Escherichia coli.

    PubMed

    Mercer, Ryan; Nguyen, Oanh; Ou, Qixing; McMullen, Lynn; Gänzle, Michael G

    2017-10-15

    The locus of heat resistance (LHR) is a 15- to 19-kb genomic island conferring exceptional heat resistance to organisms in the family Enterobacteriaceae, including pathogenic strains of Salmonella enterica and Escherichia coli The complement of LHR-comprising genes that is necessary for heat resistance and the stress-induced or growth-phase-induced expression of LHR-comprising genes are unknown. This study determined the contribution of the seven LHR-comprising genes yfdX1GI, yfdX2, hdeDGI, orf11, trxGI, kefB, and psiEGI by comparing the heat resistances of E. coli strains harboring plasmid-encoded derivatives of the different LHRs in these genes. (Genes carry a subscript "GI" [genomic island] if an ortholog of the same gene is present in genomes of E. coli) LHR-encoded heat shock proteins sHSP20, ClpKGI, and sHSPGI are not sufficient for the heat resistance phenotype; YfdX1, YfdX2, and HdeD are necessary to complement the LHR heat shock proteins and to impart a high level of resistance. Deletion of trxGI, kefB, and psiEGI from plasmid-encoded copies of the LHR did not significantly affect heat resistance. The effect of the growth phase and the NaCl concentration on expression from the putative LHR promoter p2 was determined by quantitative reverse transcription-PCR and by a plasmid-encoded p2:GFP promoter fusion. The expression levels of exponential- and stationary-phase E. coli cells were not significantly different, but the addition of 1% NaCl significantly increased LHR expression. Remarkably, LHR expression in E. coli was dependent on a chromosomal copy of evgA In conclusion, this study improved our understanding of the genes required for exceptional heat resistance in E. coli and factors that increase their expression in food.IMPORTANCE The locus of heat resistance (LHR) is a genomic island conferring exceptional heat resistance to several foodborne pathogens. The exceptional level of heat resistance provided by the LHR questions the control of pathogens by

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

    DTIC Science & Technology

    2004-12-01

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

  10. Expression of Rice Mature Carbonic Anhydrase Gene Increase E. coli Tolerance to Heat Stress.

    PubMed

    Tianpei, Xiuzi; Mao, Zhinang; Zhu, Yingguo; Li, Shaoqing

    2015-05-01

    Carbonic anhydrate is a zinc-containing metalloenzyme and involved in plant abiotic stress tolerance. In this study, we found that heat stress could induce rice mature carbonic anhydrate gene over-expression in rice plants. An Escherichia coli heterologous expression system was performed to identify the function of rice mature carbonic anhydrate in vitro. By sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mature OsCA fusion protein was identified and proved to be soluble. The results of spot, survival rate, and growth curve assay demonstrated that the expression of the mature OsCA could enhance the thermo-tolerance of the induced mature OsCA recombinants in comparison with controls under heat stress. Meanwhile, compared with controls, the levels of reactive oxygen species in induced mature OsCA recombinants were apparently low under heat stress, and correspondingly, activities of the critical antioxidant enzymes including superoxide dismutase, catalase, and peroxidase in the induced mature OsCA recombinants were significantly increased. Additionally, relative to controls, the activity of the lactate dehydrogenase decreased in the induced mature OsCA recombinants under heat stress. Based on these results, we suggest that mature OsCA protein could confer the E. coli recombinants' tolerance to heat stress by a synergistic fashion of increasing the antioxidant enzymes' activities to reduce the oxidative damage and maintaining the lactate dehydrogenase (LDH) activity of E. coli.

  11. Biofouling on polymeric heat exchanger surfaces with E. coli and native biofilms.

    PubMed

    Pohl, S; Madzgalla, M; Manz, W; Bart, H J

    2015-01-01

    The biofouling affinity of different polymeric surfaces (polypropylene, polysulfone, polyethylene terephthalate, and polyether ether ketone) in comparison to stainless steel (SS) was studied for the model bacterium Escherichia coli K12 DSM 498 and native biofilms originating from Rhine water. The biofilm mass deposited on the polymer surfaces was minimized by several magnitudes compared to SS. The cell count and the accumulated biomass of E. coli on the polymer surfaces showed an opposing linear trend. The promising low biofilm formation on the polymers is attributed to the combination of inherent surface properties (roughness, surface energy and hydrophobicity) when compared to SS. The fouling characteristics of E. coli biofilms show good conformity with the more complex native biofilms investigated. The results can be utilized for the development of new polymer heat exchangers when using untreated river water as coolant or for other processes needing antifouling materials.

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

    PubMed 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

  13. 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.

  14. The effect of heat stress on the antibacterial resistance and plasmid profile in Escherichia coli isolates.

    PubMed

    Ahmadi, M; Ayremlou, N; Sale, H Dastmalchi

    2007-12-01

    In order to study the effect of heat stress on the antibacterial resistance and plasmid profile in Escherichia coli, thirty E. coli were isolated from sheep liver. Antibiotic susceptibility test were done by antibiotic disc diffusion method using filter paper disc on two 24 h cultures of each isolate which grown at 37 and 43 degrees C simultaneously in BHI Broth (Merck VM460193 531). The isolates which grown at 43 degrees C were under heat stress during their growth. Ten commonly used antibiotics, viz., ampicillin, erythromycin, neomycin, trimethoprim-sulfamethoxazol, lincospectine, tetracycline, gentamycin, flumequine, vancomycin and Tiamulin (Padtan Teb). The resistance level of all E. coli isolates against 10 antibacterial drugs compared statistically in 37 and 43 degrees C using MINITAB Version 14 program. Plasmid DNAs were extracted from each of the E. coli isolates which were grown at 37 and 43 degrees C overnight using alkali lysis method. In this study *lambdaDNA (EcoR1+Hind III digested) was used as marker DNA. According to the results of this study, the resistance rate of E. coli isolates have decreased against trimethoprim-sulfamethazol, lincospectine, tiamaulin, tetracyclin and gentamycin at 43 degrees C but only the difference between the resistance rate against gentamycin in 37 degrees C (83.3%) and 43 degrees C (60%) was significant Characterization of Plasmid DNAs by agarose gel electrophoresis showed that each of the thirty drug resistant E. coli harbored a single plasmid. There was no difference among the plasmid profiles of the thirty isolates in 37 and 43 degrees C. As the plasmid profile did not change in 43 degrees C (heat stress) so the resistance differences against antibacterial drugs were not significant except for gentamycine that its resistance may is chromosomal. According to the results of this study, In conclusion it can be said that heat stress could not be effective on antibacterial resistance and plasmid profile if the duration of

  15. Starvation-induced cross protection against heat or H2O2 challenge in Escherichia coli.

    PubMed

    Jenkins, D E; Schultz, J E; Matin, A

    1988-09-01

    Glucose- or nitrogen-starved cultures of Escherichia coli exhibited enhanced resistance to heat (57 degrees C) or H2O2 (15 mM) challenge, compared with their exponentially growing counterparts. The degree of resistance increased with the time for which the cells were starved prior to the challenge, with 4 h of starvation providing the maximal protection. Protein synthesis during starvation was essential for these cross protections, since chloramphenicol addition at the onset of starvation prevented the development of thermal or oxidative resistance. Starved cultures also demonstrated stronger thermal and oxidative resistance than did growing cultures adapted to heat, H2O2, or ethanol prior to the heat or H2O2 challenge. Two-dimensional gel electrophoresis of 35S-pulse-labeled proteins showed that subsets of the 30 glucose starvation proteins were also synthesized during heat or H2O2 adaptation; three proteins were common to all three stresses. Most of the common proteins were among the previously identified Pex proteins (J.E. Schultz, G. I. Latter, and A. Matin, J. Bacteriol. 170:3903-3909, 1988), which are independent of cyclic AMP positive control for their induction during starvation. Induction of starvation proteins dependent on cyclic AMP was not important in these cross protections, since a delta cya strain of E. coli K-12 exhibited the same degree of resistance to heat or H2O2 as the wild-type parent did during both growth and starvation.

  16. Increase in UV mutagenesis by heat stress on UV-irradiated E. coli cells.

    PubMed

    Saha, Swati; Basu, Tarakdas

    2012-06-01

    When leu- auxotrophs of Escherichia coli, after UV irradiation, were grown at temperatures between 30 and 47°C, the frequency of UV-induced mutation from leu- to leu+ revertant increased as the UV dose and the temperature increased. For cells exposed to a UV dose of 45 J/m2, the mutation frequency at 47°C was 1.9 times that at 30°C; for a dose of 90 J/m2, it was 3.25 times; and for 135 J/m2, it was 4.8 times. Similar enhancement of reversion frequency was observed when the irradiated cells were grown at 30°C in the presence of a heat shock inducer, ethanol (8% v/v). Heat shock-mediated enhancement of UV mutagenesis did not occur in an E. coli mutant sigma 32 (heat shock regulator protein), but sigma 32 overexpression in the mutant strain (transformed with a sigma 32-bearing plasmid) increased the UV-induced mutation frequency. These results suggest that heat stress alone has no mutagenic property, but when applied to UV-damaged cells, it enhances the UV-induced frequency of cell mutation.

  17. Heat resistance of viable but non-culturable Escherichia coli cells determined by differential scanning calorimetry.

    PubMed

    Castro-Rosas, Javier; Gómez-Aldapa, Carlos Alberto; Villagómez Ibarra, José Roberto; Santos-López, Eva María; Rangel-Vargas, Esmeralda

    2017-10-16

    Several reports have suggested that the viable but non-culturable (VBNC) state is a resistant form of bacterial cells that allows them to remain in a dormant form in the environment. Nevertheless, studies on the resistance of VBNC bacterial cells to ecological factors are limited, mainly because techniques that allow this type of evaluation are lacking. Differential scanning calorimetry (DSC) has been used to study the thermal resistance of culturable bacteria but has never been used to study VBNC cells. In this work, the heat resistance of Escherichia coli cells in the VBNC state was studied using the DSC technique. The VBNC state was induced in E. coli ATCC 25922 by suspending bacterial cells in artificial sea water, followed by storage at 3 ± 2°C for 110 days. Periodically, the behaviour of E. coli cells was monitored by plate counts, direct viable counts and DSC. The entire bacterial population entered the VBNC state after 110 days of storage. The results obtained with DSC suggest that the VBNC state does not confer thermal resistance to E. coli cells in the temperature range analysed here. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Heat inactivation of Escherichia coli O157:H7 in apple juice exposed to chlorine.

    PubMed

    Folsom, J P; Frank, J F

    2000-08-01

    Exposure of Escherichia coli O157:H7 to chlorine before heat treatment results in increased production of heat shock proteins. Current heating regimens for pasteurizing apple cider do not account for chlorine exposure in the wash water. This research determined the effect of sublethal chlorine treatment on thermal inactivation of E. coli O157:H7. D58-values were calculated for stationary-phase cells exposed to 0.6 mg/liter of total available chlorine and unchlorinated cells in commercial shelf-stable apple juice (pH 3.6). D58-values for unchlorinated and chlorine-exposed cells in buffer were 5.45 and 1.65 min, respectively (P < 0.01). Death curves of chlorine-exposed and unchlorinated cells in apple juice were not completely linear. Unchlorinated cells heated in apple juice exhibit a 3-min delay before onset of linear inactivation. Chlorine treatment eliminated this shoulder, indicating an overall loss of thermotolerance. The linear portion of each curve represented a small fraction of the total population. D58-values calculated from these populations are 0.77 min for unexposed cells and 1.19 min for chlorine-exposed cells (P = 0.05). This indicates that a subpopulation of chorine-treated cells is possibly more resistant to heat because of chlorine treatment. The effect of chlorine treatment, however, is insignificant when compared with the effect of losing the shoulder. This is illustrated by the time required to kill the initial 90% of the cell population. This is observed to be 3.14 min for unchlorinated versus 0.3 min for chlorine-exposed cells (P < 0.001). These observations indicate that current heat treatments need not be adjusted for the effect of chlorine treatment.

  19. Destruction of the outer membrane permeability barrier of Escherichia coli by heat treatment.

    PubMed

    Tsuchido, T; Katsui, N; Takeuchi, A; Takano, M; Shibasaki, I

    1985-08-01

    Heat treatment of a wild-type Escherichia coli strain at 55 degrees C in 50 mM Tris-hydrochloride buffer with or without 10 mM magnesium sulfate or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 8.0 caused an increase in cell surface hydrophobicity. By determining the location of n-hexadecane droplets attached to cells by phase-contrast microscopy, the septal and polar regions of heated cells appeared to become the most frequently hydrophobic. Some of the lipopolysaccharide molecules in the outer membrane were released from heated cells, and the cells became susceptible to the hydrolytic action of added phospholipase C. Heat-treated cells also became permeable to the hydrophobic dye crystal violet, which was added externally. The release of part of the outer membrane by heat treatment appeared to bring about the disorganization of the outer membrane structure and, as a consequence, to result in the partial disruption of the permeability barrier function of the outer membrane. Tris was found to enhance damage to the outer membrane by heat.

  20. Characterization of heat-shock proteins in Escherichia coli strains under thermal stress in vitro.

    PubMed

    Urban-Chmiel, Renata; Dec, Marta; Puchalski, Andrzej; Wernicki, Andrzej

    2013-12-01

    The aim of this study was to evaluate the effect of heat stress in in vitro conditions on the induction of heat-shock protein (Hsp)70 by Escherichia coli cells, and to determine the localization of Hsps in cell fractions. The material consisted of wild strains of E. coli isolated from the digestive tract of calves, suspended in an exponential-phase culture and subjected to 41.5 °C for 2 h. Individual fractions were analysed by SDS-PAGE and two-dimensional electrophoresis. Western blotting with mouse anti-Hsp70 and anti-Hsp60 mAbs was used to identify the proteins. Electrophoretic analysis of the heat-treated cells detected Hsp70 in all three fractions, cytoplasmic, periplasmic and membrane, which was confirmed by Western blotting. The proteins obtained had diverse localizations in the pH gradient in two-dimensional electrophoresis, which may indicate changes in their conformation and physical properties leading to stabilization and protection of intracellular structures in stress conditions. The presence of these Hsps in different cell fractions indicates a very strong protective adaptation in the bacteria in unfavourable conditions, which is critical for the organism infected by them.

  1. Role of the heat shock response in stability of mRNA in Escherichia coli K-12.

    PubMed Central

    Henry, M D; Yancey, S D; Kushner, S R

    1992-01-01

    The heat shock response in Escherichia coli involves extensive induction of the heat shock proteins, with the concomitant suppression of the synthesis of the non-heat shock proteins. While the induction of the heat shock proteins has been shown to occur primarily at the transcriptional level, the suppression of non-heat shock proteins is poorly understood. We have investigated the possibility that an increased decay of non-heat shock mRNAs is a means of decreasing the synthesis of non-heat shock proteins during the heat shock response. Heat shock response-defective strains were compared with wild-type controls by several criteria to evaluate both mRNA stability and the induction of enzymes known to be involved in mRNA turnover. Our results indicate that increased mRNA decay is not a mechanism used to regulate the synthesis of non-heat shock proteins. PMID:1732210

  2. Role of the 3'-untranslated region of human endothelin-1 in vascular endothelial cells. Contribution to transcript lability and the cellular heat shock response.

    PubMed

    Mawji, Imtiaz A; Robb, G Brett; Tai, Sharon C; Marsden, Philip A

    2004-03-05

    Endothelin-1 (ET-1) is a potent vasoconstrictor peptide expressed in the vascular endothelium. Stringent control over ET-1 expression is achieved through a highly regulated promoter and rapid mRNA turnover. Since little is known about mechanisms governing ET-1 post-transcriptional regulation, and changes in ET-1 mRNA stability are implicated in disease processes, we characterized these pathways using a variety of functional approaches. We expressed human ET-1 and luciferase transcripts with or without a wild type ET-1 3'-untranslated region (3'-UTR) and found that the 3'-UTR had potent mRNA destabilizing activity. Deletion analysis localized this activity to two domains of the 3'-UTR we have termed destabilizing elements 1 and 2 (DE1 and DE2). Mutational studies revealed that DE1 functions as an AU-rich element (ARE) dependent on a 100-nucleotide region. This activity was further localized to a 10-nucleotide region at position 978-987 of the 3'-UTR. Depletion of AUF1 by RNA interference up-regulated ET-1 in endothelial cells suggesting AUF1-dependent regulation. Since AUF1 functions through the ubiquitin-proteasome pathway, we disrupted this pathway with heat shock and proteasome inhibitor in endothelial cells and observed stabilization of endogenous ET-1 mRNA. Chimeric transcripts bearing wild type ET-1 3'-UTRs were also stabilized in response to proteasome inhibition whereas DE1 mutants failed to respond. Taken together, these findings suggest a complex model of ARE-mediated mRNA turnover dependent on two 3'-UTR domains, DE1 and DE2. Furthermore, DE1 functions as an ARE directing mRNA half-life through the proteasome. Finally, this data provides evidence for a novel pathway of ET-1 mRNA stabilization by heat shock.

  3. Centrifugal field effects on the sedimentation coefficient of the Escherichia coli nucleoid after heat treatment.

    PubMed

    Pellón, J R; Gómez, R F

    1981-12-01

    The effects of the centrifugal field on the sedimentation coefficient of the heated (50 degrees C, 30 min) Escherichia coli nucleoid were investigated. Form 3,000 r.p.m. the sedimentation coefficients of the heated nucleoids were highly dependent on rotor speed. At 3,000 r.p.m. thier sedimentation coefficient was about 4,000 S while at 7,000 r.p.m. if was about 1,500-1,700 S. At 7,000 r.p.m. and over, nucleoid aggregations occurred and it was difficult to differentiate speed dependence from nucleoid aggregation. Factors likely to cause speed dependence and/or nucleoid aggregation are indicated. The practical importance of these findings is pointed out.

  4. Virulence gene profiling of enteroaggregative Escherichia coli heat-stable enterotoxin 1-harboring E. coli (EAST1EC) derived from sporadic diarrheal patients.

    PubMed

    Konno, Takayuki; Yatsuyanagi, Jun; Saito, Shioko

    2012-04-01

    Between 2007 and 2009, a total of 2168 Escherichia coli strains derived from diarrheal patients, defined as putative diarrheagenic E. coli (DEC), were collected from medical institutions in Akita prefecture, Japan. Thirty five of the strains lacked typical pathogenic determinants of DEC other than astA, which encodes enteroaggregative E. coli (EAggEC) heat-stable enterotoxin 1 (EAST1). These E. coli strains are referred to as EAST1EC. Several studies have suggested a role of EAST1 in diarrhea; however, the correlation between diarrhea and the presence of astA remains inconclusive. To investigate whether EAST1EC strains derived from diarrheal patients shared pathogenic factors other than EAST1, virulence gene profiling of 12 virulence genes - iha, lpfA, ldaG, pilS, pic, pet, irp2, daa, aah, aid, cdtB and hlyA - was carried out. PCR analysis revealed that four of the 35 EAST1EC strains harbored only astA, 24 harbored genes associated with adhesins and intestinal colonization, three strains harbored the gene for α-hemolysin, and 24 strains harbored the gene for a siderophore. These results indicated that some EAST1EC strains harbor various virulence genes associated with distinct E. coli pathotypes, primarily enterohemorrhagic E. coli and EAggEC, which may represent additional pathogenic determinants of EAST1EC.

  5. The different ecological niches of enterotoxigenic E scherichia coli

    PubMed Central

    Gonzales‐Siles, Lucia

    2015-01-01

    Summary Enterotoxigenic E scherichia coli (ETEC) is a water and food‐borne pathogen that infects the small intestine of the human gut and causes diarrhoea. Enterotoxigenic E. coli adheres to the epithelium by means of colonization factors and secretes two enterotoxins, the heat labile toxin and/or the heat stable toxin that both deregulate ion channels and cause secretory diarrhoea. Enterotoxigenic E. coli as all E. coli, is a versatile organism able to survive and grow in different environments. During transmission and infection, ETEC is exposed to various environmental cues that have an impact on survivability and virulence. The ability to cope with exposure to different stressful habitats is probably shaping the pool of virulent ETEC strains that cause both endemic and epidemic infections. This review will focus on the ecology of ETEC in its different habitats and interactions with other organisms as well as abiotic factors. PMID:26522129

  6. Solute Transport Proteins and the Outer Membrane Protein NmpC Contribute to Heat Resistance of Escherichia coli AW1.7▿

    PubMed Central

    Ruan, Lifang; Pleitner, Aaron; Gänzle, Michael G.; McMullen, Lynn M.

    2011-01-01

    This study aimed to elucidate determinants of heat resistance in Escherichia coli by comparing the composition of membrane lipids, as well as gene expression, in heat-resistant E. coli AW1.7 and heat-sensitive E. coli GGG10 with or without heat shock. The survival of E. coli AW1.7 at late exponential phase was 100-fold higher than that of E. coli GGG10 after incubation at 60°C for 15 min. The cytoplasmic membrane of E. coli AW1.7 contained a higher proportion of saturated and cyclopropane fatty acids than that of E. coli GGG10. Microarray hybridization of cDNA libraries obtained from exponentially growing or heat-shocked cultures was performed to compare gene expression in these two strains. Expression of selected genes from different functional groups was quantified by quantitative PCR. DnaK and 30S and 50S ribosomal subunits were overexpressed in E. coli GGG10 relative to E. coli AW1.7 upon heat shock at 50°C, indicating improved ribosome stability. The outer membrane porin NmpC and several transport proteins were overexpressed in exponentially growing E. coli AW1.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of membrane properties confirmed that NmpC is present in the outer membrane of E. coli AW1.7 but not in that of E. coli GGG10. Expression of NmpC in E. coli GGG10 increased survival at 60°C 50- to 1,000-fold. In conclusion, the outer membrane porin NmpC contributes to heat resistance in E. coli AW1.7, but the heat resistance of this strain is dependent on additional factors, which likely include the composition of membrane lipids, as well as solute transport proteins. PMID:21398480

  7. Heat production in chemically skinned smooth muscle of guinea-pig taenia coli.

    PubMed Central

    Lönnbro, P; Hellstrand, P

    1991-01-01

    1. The rate of heat production of chemically skinned guinea-pig taenia coli smooth muscle at 25 degrees C was measured using microcalorimetric techniques. 2. Muscle strips were mounted isometrically and incubated in solutions containing MgATP (3.2 mM) and phosphocreatine (PCr, 12 mM), pH 6.9. Activation was obtained by the injection of Ca2+ into the sample compartment of the calorimeter. 3. The heat production rate of the resting preparation (pCa 9) was 0.40 +/- 0.03 mW g-1 wet weight (n = 23). During maximal activation (pCa 4.8) the heat rate increased to 1.12 +/- 0.07 mW g-1 (mean +/- S.E.M., n = 15). With stepwise increase in [Ca2+] from pCa 9 to 4.8 the energetic cost of force maintenance tended to increase at higher [Ca2+]. 4. After activation by Ca2+, the heat production rate reached its maximum while force was still increasing. 5. Changing ionic strength from 90 to 150 mM had no effect on either basal or activated heat rate. Oligomycin, amphotericin B and the adenylate kinase inhibitor Ap5A had no effect on the basal heat rate. 6. Exchanging ATP in the incubation medium for inosine triphosphate (ITP) reduced the force and heat production after injection of Ca2+. The basal heat production was not lowered when ATP was exchanged for ITP. 7. The observed enthalpy change for PCr splitting at 25 degrees C (pH 6.9, ionic strength 90 mM) was -28 +/- 3 kJ mol-1 (mean +/- S.E.M., n = 9). After correction for the phosphate equilibrium, buffer reactions, and Mg2+ binding to PCr and HPO42-, the net enthalpy change is calculated to be -39 +/- 3 kJ mol-1. 8. Heat production in the skinned smooth muscle consists of one basal component present in relaxed muscle, and one component associated with contraction. The nature of the basal heat production is unclear but does not seem to involve turnover of phosphate on the myosin light chains. The increase in the energetic tension cost with increasing activation by Ca2+ has implications for the understanding of the contractile

  8. Survival of Escherichia coli O157:H7 in ground beef after sublethal heat shock and subsequent isothermal cooking.

    PubMed

    Wiegand, K M; Ingham, S C; Ingham, B H

    2009-08-01

    Heat shock of Escherichia coli O157:H7 in broth media reportedly leads to enhanced survival during subsequent heating in broth medium or ground beef. Survival of E. coli O157:H7 during slow cooking thus may be enhanced by prior exposure to sublethal heat shock conditions, thereby jeopardizing the safety of slow-cooked products such as beef roasts. This study examined the effect of heat shocking E. coli O157:H7-inoculated lean (6 to 9% fat) ground beef on the survival of the pathogen in the same ground beef during a subsequent 4-h, 54.4 degrees C cooking process. Six different combinations of heat shock temperature (47.2, 48.3, or 49.4 degrees C) and time (5 or 30 min) were applied to a five-strain cocktail of microaerophilically grown cells in 25 g of prewarmed ground beef, which was followed by cooking at 54.4 degrees C. Temperature during a 30-min heat shock treatment did not significantly affect E. coli O157:H7 survival during subsequent isothermal cooking (P > 0.05). Survival after a 5-min heat shock was higher when the heat shock temperature was 48.3 or 49.4 degrees C (P < 0.05) than when it was 47.2 degrees C. The D-values at 54.4 degrees C (130 degrees F) (D54.4-value) of the process significantly increased only when cells were exposed to a heat shock combination of 5 min at 49.4 degrees C. Mean (n = 3 trials) reductions in E. coli O157:H7 during the 4-h, 54.4 degrees C isothermal cooking process ranged from 4.3 to 7.5 log CFU/g. Heating E. coli O157:H7-contaminated beef at the high end of the sublethal temperature range for 5 min could increase survival of E. coli O157:H7 during subsequent slow-cooking processes.

  9. Effects of disruption of heat shock genes on susceptibility of Escherichia coli to fluoroquinolones

    PubMed Central

    Yamaguchi, Yuko; Tomoyasu, Toshifumi; Takaya, Akiko; Morioka, Mizue; Yamamoto, Tomoko

    2003-01-01

    Background It is well known that expression of certain bacterial genes responds rapidly to such stimuli as exposure to toxic chemicals and physical agents. It is generally believed that the proteins encoded in these genes are important for successful survival of the organism under the hostile conditions. Analogously, the proteins induced in bacterial cells exposed to antibiotics are believed to affect the organisms' susceptibility to these agents. Results We demonstrated that Escherichia coli cells exposed to levofloxacin (LVFX), a fluoroquinolone (FQ), induce the syntheses of heat shock proteins and RecA. To examine whether the heat shock proteins affect the bactericidal action of FQs, we constructed E. coli strains with mutations in various heat shock genes and tested their susceptibility to FQs. Mutations in dnaK, groEL, and lon increased this susceptibility; the lon mutant exhibited the greatest effects. The increased susceptibility of the lon mutant was corroborated by experiments in which the gene encoding the cell division inhibitor, SulA, was subsequently disrupted. SulA is induced by the SOS response and degraded by the Lon protease. The findings suggest that the hypersusceptibility of the lon mutant to FQs could be due to abnormally high levels of SulA protein resulting from the depletion of Lon and the continuous induction of the SOS response in the presence of FQs. Conclusion The present results show that the bactericidal action of FQs is moderately affected by the DnaK and GroEL chaperones and strongly affected by the Lon protease. FQs have contributed successfully to the treatment of various bacterial infections, but their widespread use and often misuse, coupled with emerging resistance, have gradually compromised their utility. Our results suggest that agents capable of inhibiting the Lon protease have potential for combination therapy with FQs. PMID:12911840

  10. Synergistic effect of heat and solar UV on DNA damage and water disinfection of E. coli and bacteriophage MS2.

    PubMed

    Theitler, Dana Jennifer; Nasser, Abid; Gerchman, Yoram; Kribus, Abraham; Mamane, Hadas

    2012-12-01

    The response of a representative virus and indicator bacteria to heating, solar irradiation, or their combination, was investigated in a controlled solar simulator and under real sun conditions. Heating showed higher inactivation of Escherichia coli compared to the bacteriophage MS2. Heating combined with natural or simulated solar irradiation demonstrated a synergistic effect on the inactivation of E. coli, with up to 3-log difference for 50 °C and natural sun insolation of 2,000 kJ m(-2) (compared to the sum of the separate treatments). Similar synergistic effect was also evident when solar-UV induced DNA damage to E. coli was assessed using the endonuclease sensitive site assay (ESS). MS2 was found to be highly resistant to irradiation and heat, with a slightly synergistic effect observed only at 59 °C and natural sun insolation of 5,580 kJ m(-2). Heat treatment also hindered light-dependent recovery of E. coli making the treatment much more effective.

  11. Genome-wide transcriptional responses of Escherichia coli K-12 to continuous osmotic and heat stresses.

    PubMed

    Gunasekera, Thusitha S; Csonka, Laszlo N; Paliy, Oleg

    2008-05-01

    Osmotic stress is known to increase the thermotolerance and oxidative-stress resistance of bacteria by a mechanism that is not adequately understood. We probed the cross-regulation of continuous osmotic and heat stress responses by characterizing the effects of external osmolarity (0.3 M versus 0.0 M NaCl) and temperature (43 degrees C versus 30 degrees C) on the transcriptome of Escherichia coli K-12. Our most important discovery was that a number of genes in the SoxRS and OxyR oxidative-stress regulons were up-regulated by high osmolarity, high temperature, or a combination of both stresses. This result can explain the previously noted cross-protection of osmotic stress against oxidative and heat stresses. Most of the genes shown in previous studies to be induced during the early phase of adaptation to hyperosmotic shock were found to be also overexpressed under continuous osmotic stress. However, there was a poorer overlap between the heat shock genes that are induced transiently after high temperature shifts and the genes that we found to be chronically up-regulated at 43 degrees C. Supplementation of the high-osmolarity medium with the osmoprotectant glycine betaine, which reduces the cytoplasmic K(+) pool, did not lead to a universal reduction in the expression of osmotically induced genes. This finding does not support the hypothesis that K(+) is the central osmoregulatory signal in Enterobacteriaceae.

  12. [Effect of aggregate state, tonicity, and level of nutrients in media on recovery of Escherichia coli bacteria from heat damage].

    PubMed

    Morozov, I I

    1996-01-01

    The influence of aggregate condition, nutrient composition and tonicity of incubation medium on recovery from damages induced by heat at 52 degrees C Escherichia coli B/r and Escherichia coli BS-1 cells was studied. The cells were shown to be recovered from heat damages only in liquid and isotonic medium. The observed increase in a number of viable organisms was not greatly depended on source of energy, plastic material and macroelements of incubation medium. The recovered cells showed increase of osmotic tolerance to hyper- and hypotonic shock similar to intact cells. No increase in survivors was observed when heated cells were incubated on solid and/or anisotonic medium. The additional cell killing and high osmosensitivity may be seen in this case. The role of the system of osmotic homeostasis, including membrane state, in the modification of cell viability and osmoresistance of heated bacterial cells was discussed on the base of the data obtained.

  13. 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.

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

    PubMed

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

    2014-01-01

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

  15. 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.

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

    PubMed

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

    2010-09-01

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

  17. Bactericidal Effect of Selected Antidiarrhoeal Medicinal Plants on Intracellular Heat-Stable Enterotoxin-Producing Escherichia coli

    PubMed Central

    Birdi, Tannaz J.; Brijesh, S.; Daswani, Poonam G.

    2014-01-01

    Diarrhoeal diseases due to enterotoxigenic Escherichia coli continue to be a cause of global concern. Medicinal plants have been gaining popularity as promising antidiarrhoeal agents. In the present study, four antidiarrhoeal plants, viz. Aegle marmelos, Cyperus rotundus, Psidium guajava and Zingiber officinale were screened against a heat-stable toxin-producing enterotoxigenic E. coli strain. Decoctions of these plants were studied for their effect on intracellular killing of the bacterial strain using murine monocytic cell line, J774. [3H] thymidine release assay was used to evaluate the apoptotic/necrotic effect. All plants at concentrations <1% enhanced intracellular killing of the bacteria by J774 cells. However, at higher concentrations, the decoctions induced apoptosis in J774 cells. The study demonstrates that these plants could control diarrhoea caused by heat-stable toxin-producing enterotoxigenic E. coli through their immunomodulatory effect. PMID:25035535

  18. Survival and heat resistance of Salmonella enterica and Escherichia coli O157:H7 in peanut butter.

    PubMed

    He, Yingshu; Guo, Dongjing; Yang, Jingyun; Tortorello, Mary Lou; Zhang, Wei

    2011-12-01

    Significant differences (P < 0.05) were found between the survival rates of Salmonella enterica and Escherichia coli O157:H7 in peanut butter with different formulations and water activity. High carbohydrate content in peanut butter and low incubation temperature resulted in higher levels of bacterial survival during storage but lower levels of bacterial resistance to heat treatment.

  19. Induction of DnaK and GroEL heat shock proteins by fluoroquinolones in Escherichia coli.

    PubMed Central

    Mizushima, T; Matsuo, M; Sekimizu, K

    1997-01-01

    Various fluoroquinolones (norfloxacin, enoxacin, ofloxacin, levofloxacin, and sparfloxacin) induce DnaK and GroEL heat shock proteins in Escherichia coli. The induction is transient, consistent with the kinetics of cellular DNA relaxation. The concentrations of fluoroquinolones required for induction are similar to those required for DNA relaxation and much higher than those required for cell death. PMID:8980780

  20. Survival and Heat Resistance of Salmonella enterica and Escherichia coli O157:H7 in Peanut Butter ▿ †

    PubMed Central

    He, Yingshu; Guo, Dongjing; Yang, Jingyun; Tortorello, Mary Lou; Zhang, Wei

    2011-01-01

    Significant differences (P < 0.05) were found between the survival rates of Salmonella enterica and Escherichia coli O157:H7 in peanut butter with different formulations and water activity. High carbohydrate content in peanut butter and low incubation temperature resulted in higher levels of bacterial survival during storage but lower levels of bacterial resistance to heat treatment. PMID:21965404

  1. Detection of Escherichia coli enterotoxins in stools.

    PubMed Central

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

    1980-01-01

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

  2. Expression of stress and virulence genes in Escherichia coli O157:H7 heat shocked in fresh dairy compost.

    PubMed

    Singh, Randhir; Jiang, Xiuping

    2015-01-01

    The purpose of this study was to determine the gene expression of Escherichia coli O157:H7 heat shocked in dairy compost. A two-step real-time PCR assay was used to evaluate the expression of stress and virulence genes in E. coli O157:H7 heat shocked in compost at 47.5°C for 10 min. Heat-shocked E. coli O157:H7 in compost was isolated by using an immunomagnetic bead separation method, followed by total RNA extraction, which was then converted to cDNA by using a commercial kit. E. coli O157:H7 heat shocked in broth served as the media control. In compost, heat shock genes (clpB, dnaK, and groEL) and the alternative sigma factor (rpoH) of E. coli O157:H7 were upregulated (P < 0.05), whereas the expression of trehalose synthesis genes did not change. Virulence genes, such as stx1 and fliC, were upregulated, while genes stx2, eaeA, and hlyA were downregulated. In the toxin-antitoxin (TA) system, toxin genes, mazF, hipA, and yafQ were upregulated, whereas among antitoxin genes, only dinJ was upregulated (P < 0.05). In tryptic soy broth, all heat shock genes (rpoH, clpB, dnaK, and groEL) were upregulated (P < 0.05), and most virulence genes (stx1, stx2, hlyA, and fliC) and TA genes (mazF-mazE, hipA-hipB, and yafQ-dinJ and toxin gene chpS) were down-regulated. Our results revealed various gene expression patterns when E. coli O157:H7 inoculated in compost was exposed to a sublethal temperature. Clearly, induction of the heat shock response is one of the important protective mechanisms that prolongs the survival of pathogens during the composting process. In addition, other possible mechanisms (such as the TA system) operating along with heat shock response may be responsible for the extended survival of pathogens in compost.

  3. Hypochlorous acid activates the heat shock and soxRS systems of Escherichia coli.

    PubMed

    Dukan, S; Dadon, S; Smulski, D R; Belkin, S

    1996-11-01

    A series of plasmids, containing fusions of different stress promoters to lux reporter genes, was used in an attempt to monitor the defense circuits activated upon exposure of Escherichia coli to sublethal doses of free chlorine. A significant level of activation was exhibited by promoters of three heat shock genes (grpE, dnaK, and lon), in an rpoH-dependent manner. The promoter of micF, a gene under the control of the soxRS regulon, was also strongly induced, but not in a soxR mutant. This induction was not affected by sodA and sodB mutations, implying that it did not involve oxygen radical activity. Free-chlorine activation of both heat shock and soxRS regulons required an exposure of less then I s in duration. The oxyR or the SOS regulons were apparently not induced by free chlorine (as judged by lack of activation of katG and recA, respectively), and neither was the universal stress (uspA) protein.

  4. ANIMAL ENTEROTOXIGENIC ESCHERICHIA COLI

    PubMed Central

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

    2016-01-01

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

  5. Improvements in the passive immune hemolysis test for assaying enterotoxigenic Escherichia coli.

    PubMed Central

    de Castro, A F; Serafim, M B; Gomes, J A; Gatti, M S

    1980-01-01

    The sensitivity of the passive immune hemolysis test for the detection of heat-labile enterotoxin from Escherichia coli was increased when the test was carried out with Veronal-buffered saline plus Ca2+ and Mg2+ as diluent, instead of phosphate-buffered saline. The performance of the test was further improved by using stationary cultures to which mitomycin C had been added at the end of the lag phase. PMID:6792218

  6. Distribution and Polymorphism of the Flagellin Genes from Isolates of Campylobacter coli and Campylobacter jejuni

    DTIC Science & Technology

    1993-05-01

    American Society for Microbioloc% Distribution and Polymorphism of the Flagellin Genes from Isolates of Campylobacter coli and Campylobacter jejuni RICHARD...in Campylobacter jejuni . serogroups both the flaA and flaB genes are extremely Mol. M;crobiol. 5:1151-1158. z homologous. Within most LIO heat-labile...irllwn hungatei. J1. Bacteriol. 123:-28 proteins of Campylobacter jejuni 81116. Infect. Immun. 59: 42. Thomashow, L S., and S. C. Rittenberg. 198

  7. The effect of catalase on recovery of heat-injured DNA-repair mutants of Escherichia coli.

    PubMed

    Mackey, B M; Seymour, D A

    1987-06-01

    The apparent sensitivity of Escherichia coli K12 to mild heat was increased by recA (def), recB and polA, but not by uvrA, uvrB or recF mutations. However, addition of catalase to the rich plating medium used to assess viability restored counts of heat-injured recA, recB and polA strains to wild-type levels. E. coli p3478 polA was sensitized by heat to a concentration of hydrogen peroxide similar to that measured in autoclaved recovery medium. The apparent heat sensitivity of DNA-repair mutants is thus due to heat-induced sensitivity to the low levels of peroxide present in rich recovery media. It is proposed that DNA damage in heated cells could occur indirectly by an oxidative mechanism. The increased peroxide sensitivity of heat-injured cells was not due to a decrease in total catalase activity but may be related specifically to inactivation of the inducible catalase/peroxidase (HPI).

  8. Viability of Clostridium perfringens, Escherichia coli, and Listeria monocytogenes surviving mild heat or aqueous ozone treatment on beef followed by heat, alkali, or salt stress.

    PubMed

    Novak, John S; Yuan, James T C

    2003-03-01

    The threat of pathogen survival following ozone treatment of meat necessitates careful evaluation of the microorganisms surviving under such circumstances. The objective of this study was to determine whether sublethal aqueous ozone treatment (3 ppm of O3 for 5 min) of microorganisms on beef surfaces would result in increased or decreased survival with respect to subsequent heat, alkali, or NaCl stress. A mild heat treatment (55 degrees C for 30 min) was used for comparison. Reductions in three-strain cocktails of Clostridium perfringens, Escherichia coli O157:H7, and Listeria monocytogenes on beef following the heat treatment were 0.14, 0.77, and 1.47 log10 CFU/g, respectively, whereas reductions following ozone treatment were 1.28, 0.85, and 1.09 log10 CFU/g, respectively. C. perfringens cells exhibited elevated heat resistance at 60 degrees C (D60 [time at 60 degrees C required to reduce the viable cell population by 1 log10 units or 90%] = 17.76 min) following heat treatment of beef (55 degrees C for 30 min) but exhibited reduced viability at 60 degrees C following ozone treatment (D60 = 7.64 min) compared with the viability of untreated control cells (D60 = 13.84 min). The D60-values for L. monocytogenes and E. coli O157:H7 following heat and ozone exposures were not significantly different (P > 0.05). C. perfringens cells that survived ozone treatment did not exhibit increased resistance to pH (pH 6 to 12) relative to non-ozone-treated cells when grown at 37 degrees C for 24 h. The heat treatment also resulted in decreased numbers of surviving cells above and below neutral pH values for both E. coli O157:H7 and L. monocytogenes relative to those of non-heat-treated cells grown at 37 degrees C for 24 h. There were significant differences (P < 0.05) in C. perfringens reductions with increasing NaCl concentrations. The effects of NaCl were less apparent for E. coli and L. monocytogenes survivors. It is concluded that pathogens surviving ozone treatment of beef

  9. 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

  10. 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

  11. Enterotoxigenic Escherichia coli diarrhea of travelers: a prospective study of American Peace Corps volunteers.

    PubMed

    Sack, D A; Kaminsky, D C; Sack, R B; Wamola, I A; Orskov, F; Orskov, I; Slack, R C; Arthur, R R; Kapikian, A Z

    1977-08-01

    Travelers' diarrhea was studied prospectively in a group of 39 American Peace Corps Volunteers (PCVs) during their first five weeks in Kenya. Twenty-seven developed diarrheal disease and 12 remained well. Multiple episodes were documented in 11 of the symptomatic volunteers. Enterotoxigenic Escherichia coli of many serotypes producing heat-labile and/or heat-stable enterotoxin were isolated from 17 of the 27 volunteers with diarrhea and from 1 of the 12 well volunteers. The enterotoxigenic E. coli were more likely to be antibiotic sensitive than the non-enterotoxigenic E. coli. A serum antibody rise to the heat-labile toxin (LT) was detected in six symptomatic volunteers, five of whom had a positive culture for LT-producing E. coli, and from one asymptomatic, culture negative volunteer. Salmonella cubana was isolated from two volunteers, and three volunteers had serologic evidence of infection with human reovirus-like (rotavirus) agent. This study confirms the role of enterotoxigenic E. coli as a major cause of travelers' diarrhea and suggests that the disease is similar in widely separated geographic areas.

  12. An Escherichia coli mutant resistant to phleomycin, bleomycin, and heat inactivation is defective in ubiquinone synthesis.

    PubMed Central

    Collis, C M; Grigg, G W

    1989-01-01

    A mutant of Escherichia coli, selected for resistance to the antibiotic and antitumor agent phleomycin, has been characterized, and the phleomycin resistance determinant has been identified. The mutant is equally resistant to bleomycins. The resistance to phleomycin is strongly dependent on the nature of the C-terminal amine of the drug, with the greatest resistance being shown to phleomycins and bleomycins with the most basic terminal amines. The mutation also confers resistance to the lethal effects of heating at 52 degrees C. Other characteristics of the phleomycin-resistant strain include a slow growth rate, an inability to grow on succinate as the sole carbon source (Suc- phenotype), cross resistance to aminoglycoside antibiotics, and a slight sensitivity to hydrogen peroxide, methyl methanesulfonate, and gamma-irradiation. Some of these characteristics, together with mapping data, suggested that the phleomycin resistance and Suc- determinant probably lies within the ubiF gene coding for an enzyme effecting a step in the biosynthesis of ubiquinone. The phenotypes of known mutants defective in this and other steps of the ubiquinone pathway were found to be closely similar to those of the original phleomycin-resistant strain. PMID:2475481

  13. A Portable, Shock-Proof, Surface-Heated Droplet PCR System for Escherichia coli Detection

    PubMed Central

    Angus, Scott V.; Cho, Soohee; Harshman, Dustin K.; Song, Jae-Young; Yoon, Jeong-Yeol

    2015-01-01

    A novel polymerase chain reaction (PCR) device was developed that uses wire-guided droplet manipulation (WDM) to guide a droplet over three different heating chambers. After PCR amplification, end-point detection is achieved using a smartphone-based fluorescence microscope. The device was tested for identification of the 16S rRNA gene V3 hypervariable region from Escherichia coli genomic DNA. The lower limit of detection was 103 genome copies per sample. The device is portable with smartphone-based end-point detection and provides the assay results quickly (15 min for a 30-cycle amplification) and accurately. The system is also shock and vibration resistant, due to the multiple points of contact between the droplet and the thermocouple and the Teflon film on the heater surfaces. The thermocouple also provides realtime droplet temperature feedback to ensure it reaches the set temperature before moving to the next chamber/step in PCR. The device is equipped to use either silicone oil or coconut oil. Coconut oil provides additional portability and ease of transportation by eliminating spilling because its high melting temperature means it is solid at room temperature. PMID:26164008

  14. Comparative sequence analysis of enteroaggregative Escherichia coli heat-stable enterotoxin 1 identified in Korean and Japanese Escherichia coli strains.

    PubMed

    Seo, Dong Joo; Choi, SunKeum; Jeon, Su Been; Jeong, Suntak; Park, Hyunkyung; Lee, Bog-Hieu; Kim, Geun-Bae; Yang, Soo-Jin; Nishikawa, Yoshikazu; Choi, Changsun

    2017-02-21

    The aim of this study was to compare the sequence of the astA gene found in 8 Korean and 11 Japanese Escherichia coli isolates. Conventional PCR was used to amplify the astA gene from the chromosomal and plasmid DNA preparation samples of each isolate using commercial DNA extraction kits. Cloning of the PCR products, sequence analysis, and pulse field gel electrophoresis (PFGE) were sequentially performed. An identical copy of astA in each isolate were found for 8 Korean and 8 Japanese E. coli strains isolated from bovine, porcine, and healthy human carriers. Among these, 1 Korean and 4 Japanese isolates carried a stop mutation at residue 16. Three Japanese outbreak strains (V199, V638, and 96-127-23) carried multiple clones of astA gene with multiple amino acids changes at residues 11, 16, 20, 23, 30, 33, and 34. Compared with the non-diarrheal isolates, clonal diversity and sequence variations of the astA gene in outbreak isolates may be associated with virulence potential of EAST1.

  15. A systematic review and meta-analysis of the epidemiology of pathogenic Escherichia coli of calves and the role of calves as reservoirs for human pathogenic E. coli.

    PubMed

    Kolenda, Rafał; Burdukiewicz, Michał; Schierack, Peter

    2015-01-01

    Escherichia coli bacteria are the most common causes of diarrhea and septicemia in calves. Moreover, calves form a major reservoir for transmission of pathogenic E. coli to humans. Systematic reviews and meta-analyses of publications on E. coli as calf pathogens and the role of calves as reservoir have not been done so far. We reviewed studies between 1951 and 2013 reporting the presence of virulence associated factors (VAFs) in calf E. coli and extracted the following information: year(s) and country of sampling, animal number, health status, isolate number, VAF prevalence, serotypes, diagnostic methods, and biological assays. The prevalence of VAFs or E. coli pathotypes was compared between healthy and diarrheic animals and was analyzed for time courses. Together, 106 papers with 25,982 E. coli isolates from 27 countries tested for VAFs were included. F5, F17, and F41 fimbriae and heat-stable enterotoxin (ST) - VAFs of enterotoxigenic E. coli (ETEC) were significantly associated with calf diarrhea. On the contrary, ETEC VAF F4 fimbriae and heat-labile enterotoxin as well as enteropathogenic (EPEC), Shiga toxin-producing (STEC), and enterohemorrhagic E. coli (EHEC) were not associated with diarrhea. The prevalence increased overtime for ST-positive isolates, but decreased for F5- and STEC-positive isolates. Our study provides useful information about the history of scientific investigations performed in this domain so far, and helps to define etiological agents of calf disease, and to evaluate calves as reservoir hosts for human pathogenic E. coli.

  16. Degradation of sigma 32, the heat shock regulator in Escherichia coli, is governed by HflB.

    PubMed Central

    Herman, C; Thévenet, D; D'Ari, R; Bouloc, P

    1995-01-01

    The heat shock response in Escherichia coli is governed by the concentration of the highly unstable sigma factor sigma 32. The essential protein HflB (FtsH), known to control proteolysis of the phage lambda cII protein, also governs sigma 32 degradation: an HflB-depleted strain accumulated sigma 32 and induced the heat shock response, and the half-life of sigma 32 increased by a factor up to 12 in mutants with reduced HflB function and decreased by a factor of 1.8 in a strain overexpressing HflB. The hflB gene is in the ftsJ-hflB operon, one promoter of which is positively regulated by heat shock and sigma 32. The lambda cIII protein, which stabilizes sigma 32 and lambda cII, appears to inhibit the HflB-governed protease. The E. coli HflB protein controls the stability of two master regulators, lambda cII and sigma 32, responsible for the lysis-lysogeny decision of phage lambda and the heat shock response of the host. Images Fig. 2 Fig. 4 Fig. 5 PMID:7724592

  17. Effect of incubation media on the recovery of Escherichia coli K12 heated at 52 degrees C.

    PubMed

    Ahmad, M; Srivastava, B S; Agarwala, S C

    1978-07-01

    The exposure of exponentially grown Escherichia coli K12 to 52 degrees C for 30 min in Tris/Mg2+ buffer resulted in a considerable loss of viability when plated on tryptone agar. When such heated bacteria were held at 37 degrees C for 2 h in tryptone broth before plating on tryptone agar, there was a significant increase in viability. Thus, heat damage was repaired in tryptone broth but not on tryptone agar. Recovery was greater in tryptone broth than in synthetic medium. In tryptone broth, recA or polA mutants also recovered but a lex mutant did not. As a result of heating, the sensitivity of bacteria to ultraviolet radiation (u.v.), to mitomycin C and to plating on high salt medium was enhanced. After incubation for 2 h in tryptone broth at 37 degrees C, the bacteria regained their resistance to u.v. and mitomycin C and tolerance to high salt medium. Recovery of viability required RNA and protein synthesis, whereas recovery of u.v. resistance did not require protein synthesis. Heating for 30 min inhibited the release of acid-soluble material from DNA in all strains of E. coli used.

  18. Comparative genomics and transcriptomics of Escherichia coli isolates carrying virulence factors of both enteropathogenic and enterotoxigenic E. coli.

    PubMed

    Hazen, Tracy H; Michalski, Jane; Luo, Qingwei; Shetty, Amol C; Daugherty, Sean C; Fleckenstein, James M; Rasko, David A

    2017-06-14

    Escherichia coli that are capable of causing human disease are often classified into pathogenic variants (pathovars) based on their virulence gene content. However, disease-associated hybrid E. coli, containing unique combinations of multiple canonical virulence factors have also been described. Such was the case of the E. coli O104:H4 outbreak in 2011, which caused significant morbidity and mortality. Among the pathovars of diarrheagenic E. coli that cause significant human disease are the enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC). In the current study we use comparative genomics, transcriptomics, and functional studies to characterize isolates that contain virulence factors of both EPEC and ETEC. Based on phylogenomic analysis, these hybrid isolates are more genomically-related to EPEC, but appear to have acquired ETEC virulence genes. Global transcriptional analysis using RNA sequencing, demonstrated that the EPEC and ETEC virulence genes of these hybrid isolates were differentially-expressed under virulence-inducing laboratory conditions, similar to reference isolates. Immunoblot assays further verified that the virulence gene products were produced and that the T3SS effector EspB of EPEC, and heat-labile toxin of ETEC were secreted. These findings document the existence and virulence potential of an E. coli pathovar hybrid that blurs the distinction between E. coli pathovars.

  19. Distribution of colonization factor antigens among enterotoxigenic Escherichia coli strains isolated from patients with diarrhea in Nepal, Indonesia, Peru, and Thailand.

    PubMed Central

    Nirdnoy, W; Serichantalergs, O; Cravioto, A; LeBron, C; Wolf, M; Hoge, C W; Svennerholm, A M; Taylor, D N; Echeverria, P

    1997-01-01

    Samples (1,318) of enterotoxigenic Escherichia coli (ETEC) isolated in 1994-1995 from children with diarrhea from Nepal, Indonesia, Peru, and Thailand were examined for colonization factor antigen (CFA) and coli surface (CS) antigens. Fifty-five percent of 361 heat-labile and heat-stable (LT-ST), 14% of 620 LT-only, and 48% of 337 ST-only ETEC had CFA/CS antigens. LT-ST ETEC strains were predominantly in the CFA II group, and ST only strains were in the CFA IV group. Additional studies are needed to identify ETEC strains that do not have CFA/CS antigens. PMID:9003636

  20. Suitability of selective plating media for recovering heat- or freeze-stressed Escherichia coli O157:H7 from tryptic soy broth and ground beef.

    PubMed

    Rocelle, M; Clavero, S; Beuchat, L R

    1995-09-01

    The efficacy of tryptic soy agar (TSA), modified sorbitol MacConkey agar (MSMA), modified eosin methylene blue (MEMB) agar, and modified SD-39 (MSD) agar in recovering a five-strain mixture of enterohemorrhagic Escherichia coli O157:H7 and five non-O157 strains of E. coli heated in tryptic soy broth at 52, 54, or 56 degrees C for 10, 20, and 30 min was determined. Nonselective TSA supported the highest recovery of heated cells. Significantly (P < or = 0.05) lower recovery of heat-stressed cells was observed on MSMA than on TSA, MEMB agar, or MSD agar. The suitability of MEMB agar or MSD agar for recovery of E. coli O157:H7 from heated or frozen (-20 degrees C) low- or high-fat ground beef was determined. Recovery of E. coli O157:H7 from heated ground beef was significantly (P < or = 0.05) higher on TSA than on MEMB agar, which in turn supported higher recovery than MSD agar did; MSMA was inferior. Recovery from frozen ground beef was also higher on MEMB and MSD agars than on MSMA. Higher populations were generally recovered from high-fat beef than from low-fat beef, but the relative performance of the recovery media was the same. The inability of MSMA to recover stressed cells of E. coli O157:H7 underscores the need to develop a better selective medium for enumerating E. coli O157:H7.

  1. Suitability of selective plating media for recovering heat- or freeze-stressed Escherichia coli O157:H7 from tryptic soy broth and ground beef.

    PubMed Central

    Rocelle, M; Clavero, S; Beuchat, L R

    1995-01-01

    The efficacy of tryptic soy agar (TSA), modified sorbitol MacConkey agar (MSMA), modified eosin methylene blue (MEMB) agar, and modified SD-39 (MSD) agar in recovering a five-strain mixture of enterohemorrhagic Escherichia coli O157:H7 and five non-O157 strains of E. coli heated in tryptic soy broth at 52, 54, or 56 degrees C for 10, 20, and 30 min was determined. Nonselective TSA supported the highest recovery of heated cells. Significantly (P < or = 0.05) lower recovery of heat-stressed cells was observed on MSMA than on TSA, MEMB agar, or MSD agar. The suitability of MEMB agar or MSD agar for recovery of E. coli O157:H7 from heated or frozen (-20 degrees C) low- or high-fat ground beef was determined. Recovery of E. coli O157:H7 from heated ground beef was significantly (P < or = 0.05) higher on TSA than on MEMB agar, which in turn supported higher recovery than MSD agar did; MSMA was inferior. Recovery from frozen ground beef was also higher on MEMB and MSD agars than on MSMA. Higher populations were generally recovered from high-fat beef than from low-fat beef, but the relative performance of the recovery media was the same. The inability of MSMA to recover stressed cells of E. coli O157:H7 underscores the need to develop a better selective medium for enumerating E. coli O157:H7. PMID:7574637

  2. Stress-Induced Evolution of Heat Resistance and Resuscitation Speed in Escherichia coli O157:H7 ATCC 43888

    PubMed Central

    Gayán, Elisa; Cambré, Alexander; Michiels, Chris W.

    2016-01-01

    ABSTRACT The development of resistance in foodborne pathogens to food preservation techniques is an issue of increasing concern, especially in minimally processed foods where safety relies on hurdle technology. In this context, mild heat can be used in combination with so-called nonthermal processes, such as high hydrostatic pressure (HHP), at lower individual intensities to better retain the quality of the food. However, mild stresses may increase the risk of (cross-)resistance development in the surviving population, which in turn might compromise food safety. In this investigation, we examined the evolution of Escherichia coli O157:H7 strain ATCC 43888 after recurrent exposure to progressively intensifying mild heat shocks (from 54.0°C to 60.0°C in 0.5°C increments) with intermittent resuscitation and growth of survivors. As such, mutant strains were obtained after 10 cycles of selection with ca. 106-fold higher heat resistance than that for the parental strain at 58.0°C, although this resistance did not extend to temperatures exceeding 60.0°C. Moreover, these mutant strains typically displayed cross-resistance against HHP shock and displayed signs of enhanced RpoS and RpoH activity. Interestingly, additional cycles of selection maintaining the intensity of the heat shock constant (58.5°C) selected for mutant strains in which resuscitation speed, rather than resistance, appeared to be increased. Therefore, it seems that resistance and resuscitation speed are rapidly evolvable traits in E. coli ATCC 43888 that can compromise food safety. IMPORTANCE In this investigation, we demonstrated that Escherichia coli O157:H7 ATCC 43888 rapidly acquires resistance to mild heat exposure, with this resistance yielding cross-protection to high hydrostatic pressure treatment. In addition, mutants of E. coli ATCC 43888 in which resuscitation speed, rather than resistance, appeared to be improved were selected. As such, both resistance and resuscitation speed seem to be

  3. Studies on the Chick-lethal Toxin of Escherichia coli

    PubMed Central

    Truscott, R. B.

    1973-01-01

    A toxin which is lethal for two week old chicks has been recovered from strains of Escherichia coli O78:K80 of bovine and avian origin and from avian isolates of serogroups O2, O45 and O109. The toxin is heat-labile, antigenic, high in protein, inactivated by pronase, trypsin, amylase, and pancreatic lipase. The toxin may be precipitated by ammonium sulfate or TCA treatment from the supernatant obtained by repeated centrifugation of sonicated cells. Considerable purification has been obtained by column chromatography using Sepharose 6B. PMID:4270809

  4. Extreme Heat Resistance of Food Borne Pathogens Campylobacter jejuni, Escherichia coli, and Salmonella typhimurium on Chicken Breast Fillet during Cooking

    PubMed Central

    de Jong, Aarieke E. I.; van Asselt, Esther D.; Zwietering, Marcel H.; Nauta, Maarten J.; de Jonge, Rob

    2012-01-01

    The aim of this research was to determine the decimal reduction times of bacteria present on chicken fillet in boiling water. The experiments were conducted with Campylobacter jejuni, Salmonella, and Escherichia coli. Whole chicken breast fillets were inoculated with the pathogens, stored overnight (4°C), and subsequently cooked. The surface temperature reached 70°C within 30 sec and 85°C within one minute. Extremely high decimal reduction times of 1.90, 1.97, and 2.20 min were obtained for C. jejuni, E. coli, and S. typhimurium, respectively. Chicken meat and refrigerated storage before cooking enlarged the heat resistance of the food borne pathogens. Additionally, a high challenge temperature or fast heating rate contributed to the level of heat resistance. The data were used to assess the probability of illness (campylobacteriosis) due to consumption of chicken fillet as a function of cooking time. The data revealed that cooking time may be far more critical than previously assumed. PMID:22389647

  5. Assembly of lipopolysaccharide in Escherichia coli requires the essential LapB heat shock protein.

    PubMed

    Klein, Gracjana; Kobylak, Natalia; Lindner, Buko; Stupak, Anna; Raina, Satish

    2014-05-23

    Here, we describe two new heat shock proteins involved in the assembly of LPS in Escherichia coli, LapA and LapB (lipopolysaccharide assembly protein A and B). lapB mutants were identified based on an increased envelope stress response. Envelope stress-responsive pathways control key steps in LPS biogenesis and respond to defects in the LPS assembly. Accordingly, the LPS content in ΔlapB or Δ(lapA lapB) mutants was elevated, with an enrichment of LPS derivatives with truncations in the core region, some of which were pentaacylated and exhibited carbon chain polymorphism. Further, the levels of LpxC, the enzyme that catalyzes the first committed step of lipid A synthesis, were highly elevated in the Δ(lapA lapB) mutant. Δ(lapA lapB) mutant accumulated extragenic suppressors that mapped either to lpxC, waaC, and gmhA, or to the waaQ operon (LPS biosynthesis) and lpp (Braun's lipoprotein). Increased synthesis of either FabZ (3-R-hydroxymyristoyl acyl carrier protein dehydratase), slrA (novel RpoE-regulated non-coding sRNA), lipoprotein YceK, toxin HicA, or MurA (UDP-N-acetylglucosamine 1-carboxyvinyltransferase) suppressed some of the Δ(lapA lapB) defects. LapB contains six tetratricopeptide repeats and, at the C-terminal end, a rubredoxin-like domain that was found to be essential for its activity. In pull-down experiments, LapA and LapB co-purified with LPS, Lpt proteins, FtsH (protease), DnaK, and DnaJ (chaperones). A specific interaction was also observed between WaaC and LapB. Our data suggest that LapB coordinates assembly of proteins involved in LPS synthesis at the plasma membrane and regulates turnover of LpxC, thereby ensuring balanced biosynthesis of LPS and phospholipids consistent with its essentiality. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Delineation and comparison of ganglioside-binding epitopes for the toxins of Vibrio cholerae, Escherichia coli, and Clostridium tetani: evidence for overlapping epitopes.

    PubMed

    Angström, J; Teneberg, S; Karlsson, K A

    1994-12-06

    Binding studies of various glycolipids, mainly belonging to the ganglio series, to the toxins isolated from Vibrio cholerae, Escherichia coli, and Clostridium tetani have been performed, using the microtiter well assay. By using the found binding preferences in conjunction with minimum-energy conformations obtained from molecular modeling of the various ligands, binding epitopes on the natural receptor glycolipids for the toxins have been defined. The binding preferences for the cholera toxin and the heat-labile E. coli toxin are very similar, with the ganglioside GM1 being the most efficient ligand. The tetanus toxin binds strongly to gangliosides of the G1b series, with GT1b as the most efficient ligand. It is found that the binding epitope on GM1 for the cholera and heat-labile toxins to a large extent overlaps with the epitope on GQ1b for the tetanus toxin.

  7. Induction of the heat shock regulon of Escherichia coli markedly increases production of bacterial viruses at high temperatures

    SciTech Connect

    Wiberg, J.S.; Mowrey-Mckee, M.F.; Stevens, E.J.

    1988-01-01

    Production of bacteriophages T2, T4, and T6 at 42.8 to 44/sup 0/C was increased from 8- to 260-fold by adapting the Escherichia coli host (grown at 30/sup 0/C) to growth at the high temperature for 8 min before infection; this increase was abolished if the host htpR (rpoH) gene was inactive. Others have shown that the htpR protein increases or activates the synthesis of at least 17 E. coli heat shock proteins upon raising the growth temperature above a certain level. At 43.8 to 44/sup 0/C in T4-infected, unadapted cells, the rates of RNA, DNA, and protein synthesis were about 100, 70 and 70%, respectively, of those in T4-infected, adapted cells. Production of the major processed capsid protein, gp23, was reduced significantly more than that of most other T4 proteins in unadapted cells relative to adapted cells. Only 4.6% of the T4 DNA made in unadapted cells was resistant to micrococcal nuclease, versus 50% in adapted cells. Thus, defective maturation of T4 heads appears to explain the failure of phage production in unadapted cells. Overproduction of the heat shock protein GroEL from plasmids restored T4 production in unadapted cells to about 50% of that seen in adapted cells. T4-infected, adapted E. coli B at around 44/sup 0/C exhibited a partial tryptophan deficiency. Production of bacteriophage T7 at 44/sup 0/C was increased two- to fourfold by adapting the host to 44/sup 0/C before infection; evidence against involvement of the htpR (rpoH) gene is presented. This work and recent work with bacteriophage delta appear to represent the first demonstrations for any virus that expression of the heat shock regulon of a host is necessary for virus production at high temperature.

  8. Inactivation of Escherichia coli in a tropical fruit smoothie by a combination of heat and pulsed electric fields.

    PubMed

    Walkling-Ribeiro, M; Noci, F; Cronin, D A; Lyng, J G; Morgan, D J

    2008-10-01

    Moderate heat in combination with pulsed electric fields (PEF) was investigated as a potential alternative to thermal pasteurization of a tropical fruit smoothie based on pineapple, banana, and coconut milk, inoculated with Escherichia coli K12. The smoothie was heated from 25 degrees C to either 45 or 55 degrees C over 60 s and subsequently cooled to 10 degrees C. PEF was applied at electric field strengths of 24 and 34 kV/cm with specific energy inputs of 350, 500, and 650 kJ/L. Both processing technologies were combined using heat (45 or 55 degrees C) and the most effective set of PEF conditions. Bacterial inactivation was estimated on standard and NaCl-supplemented tryptone soy agar (TSA) to enumerate sublethally injured cells. By increasing the temperature from 45 to 55 degrees C, a higher reduction in E. coli numbers (1 compared with 1.7 log(10) colony forming units {CFU} per milliliter, P < 0.05) was achieved. Similarly, as the field strength was increased during stand-alone PEF treatment from 24 to 34 kV/cm, a greater number of E. coli cells were inactivated (2.8 compared with 4.2 log(10) CFU/mL, P < 0.05). An increase in heating temperature from 45 to 55 degrees C during a combined heat/PEF hurdle approach induced a higher inactivation (5.1 compared with 6.9 log(10) CFU/mL, respectively [P < 0.05]) with the latter value comparable to the bacterial reduction of 6.3 log(10) CFU/mL (P> or = 0.05) achieved by thermal pasteurization (72 degrees C, 15 s). A reversed hurdle processing sequence did not affect bacterial inactivation (P> or = 0.05). No differences were observed (P> or = 0.05) between the bacterial counts estimated on nonselective and selective TSA, suggesting that sublethal cell injury did not occur during single PEF treatments or combined heat/PEF treatments.

  9. Inactivation of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes in orange and tomato juice using ohmic heating.

    PubMed

    Sagong, Hun-Gu; Park, Sang-Hyun; Choi, Young-Jin; Ryu, Sangryeol; Kang, Dong-Hyun

    2011-06-01

    The effects of ohmic heating on reduction of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes in orange and tomato juice were investigated. Orange and tomato juice inoculated with E. coli O157:H7, Salmonella Typhimurium, and L. monocytogenes were subjected to ohmic heating with selected parameters including electric field strength from 10 to 20 V/cm and treatment times from 0 to 540 s. The number of pathogens was reduced by increasing the electric field strength from 10 to 20 V/cm as well as increasing treatment time. The population of E. coli O157:H7 was reduced more than 5 log after 120, 210, and 540 s of treatment in orange juice with 20, 15, and 10 V/cm electric field strengths, respectively. In tomato juice, levels of E. coli O157:H7 were reduced more than 5 log after 90, 180, and 480 s with the same electric field strengths. Similar phenomena were observed for Salmonella Typhimurium and L. monocytogenes, but E. coli O157:H7 was the most resistant to ohmic heating treatment. These results show that ohmic heating is potentially useful for inactivation of E. coli O157:H7, Salmonella Typhimurium, and L. monocytogenes and that the effect of inactivation depends on applied electric field strength, treatment time, pathogen species, and type of juice.

  10. Serologic responses to somatic O and colonization-factor antigens of enterotoxigenic Escherichia coli in travelers.

    PubMed

    Deetz, T R; Evans, D J; Evans, D G; DuPont, H L

    1979-07-01

    To improve the retrospective diagnoses of enterotoxigenic Escherichia coli (ETEC) as a cause of travelers' diarrhea, as well as to determine the presence of colonization-factor antigens in these infections, a study of serologic responses to antigens of ETEC was done. Paired sera from 60 United States students in Cholula, Puebla, Mexico, were analyzed for rises in titer of antibody to heat-labile toxin, eight somatic antigen O serogroups associated with ETEC, and two colonization-factor antigens, CFA/I and CFA/II. Only 9% had a response to O antigens, while 20% had responses to the colonization-factor antigens. Response to the colonization-factor antigens correlated significantly with response to the heat-labile toxin and with culture evidence of ETEC infection. Serologic studies confirmed that colonization-factor antigen has a role in naturally acquired cases of travelers' diarrhea and that it can be used as an additional determinant of infection with ETEC.

  11. Severely Heat Injured Survivors of E. coli O157:H7 ATCC 43888 Display Variable and Heterogeneous Stress Resistance Behavior

    PubMed Central

    Gayán, Elisa; Govers, Sander K.; Michiels, Chris W.; Aertsen, Abram

    2016-01-01

    Although minimal food processing strategies aim to eliminate foodborne pathogens and spoilage microorganisms through a combination of mild preservation techniques, little is actually known on the resistance behavior of the small fraction of microorganisms surviving an inimical treatment. In this study, the conduct of severely heat stressed survivors of E. coli O157:H7 ATCC 43888, as an indicator for the low infectious dose foodborne enterohemorrhagic strains, was examined throughout their resuscitation and outgrowth. Despite the fact that these survivors were initially sublethally injured, they were only marginally more sensitive to a subsequent heat treatment and actually much more resistant to a subsequent high hydrostatic pressure (HHP) shock in comparison with unstressed control cells. Throughout further resuscitation, however, their initial HHP resistance rapidly faded out, while their heat resistance increased and surpassed the initial heat resistance of unstressed control cells. Results also indicated that the population eventually emerging from the severely heat stressed survivors heterogeneously consisted of both growing and non-growing cells. Together, these observations provide deeper insights into the particular behavior and heterogeneity of stressed foodborne pathogens in the context of food preservation. PMID:27917163

  12. Heat treatment of unclarified Escherichia coli homogenate improved the recovery efficiency of recombinant hepatitis B core antigen.

    PubMed

    Ng, Michelle Y T; Tan, Wen Siang; Abdullah, Norhafizah; Ling, Tau Chuan; Tey, Beng Ti

    2006-10-01

    Heat precipitation procedure has been regularly incorporated as a selective purification step in various thermostable proteins expressed in different hosts. This method is efficient in precipitation of most of the host proteins and also deactivates various host proteases that can be harmful to the desired gene products. In this study, introduction of heat treatment procedure in the purification of hepatitis B core antigen (HBcAg) produced in Escherichia coli has been investigated. Thermal treatment of the cell homogenate at 60 degrees C for 30 min prior to subsequent clarification steps has resulted in 1.4 times and 18% higher in purity and recovery yield, respectively, compared to the non-heat-treated cell homogenate. In direct capture of HBcAg by using anion-exchangers from unclarified feedstock, pre-conditioning the feedstock by heat treatment at 60 degrees C for 45 min has increased the recovery yield of HBcAg by 2.9-fold and 42% in purity compared to that treated for 10 min. Enzyme-linked immunosorbent assay (ELISA) analysis showed that the antigenicity of the core particles was not affected by the heat treatment process.

  13. Towards the quantification of the effect of acid treatment on the heat tolerance of Escherichia coli K12 at lethal temperatures.

    PubMed

    Velliou, E G; Van Derlinden, E; Cappuyns, A M; Nikolaidou, E; Geeraerd, A H; Devlieghere, F; Van Impe, J F

    2011-06-01

    The aim of this work is to investigate the effect of acid treatment -before and during heat inactivation- on the heat resistance of Escherichia coli K12 MG1655 cells at lethal temperatures. E. coli cells were grown in Brain Heart Infusion broth until they reached the stationary phase (≈10(9) cfu/mL). Approximately 30 min before thermal inactivation the early stationary phase cells were added in Brain Heart Infusion broth with a specific pH value, achieved with addition of either acetic (50% (v/v)), lactic (50% (v/v)) or hydrochloric acid (30% (v/v)), and inactivation experiments took place at 54 °C and 58 °C. The inactivation dynamics are analysed using the inactivation model of Geeraerd et al. (2000). This enables to define the induced thermotolerance of E. coli as a prolongation of the shoulder and/or a reduction of the inactivation rate. Generally, addition of acids increased the heat resistance of E. coli. The induced resistance depends on the type of acid and on the quantity added, i.e. different levels of acidification lead to a different level of heat resistance. This work provides additional knowledge on the reaction of bacterial cultures to heat after acid treatment -before and during heat treatment- and, therefore, it contributes to an improved understanding of the effect of acid exposure on the bacterial heat resistance.

  14. Repair of radiation-induced damage to the cell division mechanism of Escherichia coli.

    PubMed

    Adler, H I; Fisher, W D; Hardigree, A A; Stapleton, G E

    1966-02-01

    Adler, Howard I. (Oak Ridge National Laboratory, Oak Ridge, Tenn.), William D. Fisher, Alice A. Hardigree, and George E. Stapleton. Repair of radiation-induced damage to the cell division mechanism of Escherichia coli. J. Bacteriol. 91:737-742. 1966.-Microscopic observations of irradiated populations of filamentous Escherichia coli cells indicated that filaments can be induced to divide by a substance donated by neighboring cells. We have made this observation the basis for a quantitative technique in which filaments are incubated in the presence of nongrowing donor cells. The presence of "donor" organisms promotes division and subsequent colony formation in filaments. "Donor" bacteria do not affect nonfilamentous cells. An extract of "donor" cells retains the division-promoting activity. The extract has been partially fractionated, and consists of a heat-stable and a heat-labile component. The heat-stable component is inactive in promoting cell division, but enhances the activity of the heat-labile component. The division-promoting system is discussed as a radiation repair mechanism and as a normal component of the cell division system in E. coli.

  15. Genetic probes for enterotoxigenic Escherichia coli isolated from childhood diarrhea in the Central African Republic.

    PubMed Central

    Georges, M C; Wachsmuth, I K; Birkness, K A; Moseley, S L; Georges, A J

    1983-01-01

    Escherichia coli strains were isolated from 778 children with diarrhea and 151 well children in the Central African Republic over a period of 1 year. These 929 strains were assayed for heat-labile and heat-stable enterotoxin production and were hybridized (probed) with structural genes for these enterotoxins. Twenty-four isolates from diarrheal patients and one isolate from a well child were found to be toxigenic by assay and probe. Minor discrepancies were encountered with both assays and probes during initial screening procedures, but the two methodologies were ultimately comparable. Images PMID:6350346

  16. Diarrhoeagenic Escherichia coli and salmonellae in calves and lambs in Kashmir absence, prevalence and antibiogram.

    PubMed

    Wani, S A; Hussain, I; Beg, S A; Rather, M A; Kabli, Z A; Mir, M A; Nishikawa, Y

    2013-12-01

    Polymerase chain reaction assays and culture were used to investigate 728 faecal samples from 404 calves (286 diarrhoeic, 118 healthy) and 324 lambs (230 diarrhoeic, 94 healthy) in Kashmir, India, for the presence of enterotoxigenic Escherichia coli (ETEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC) and salmonellae. Antimicrobial sensitivity patterns were also investigated. In total, 23 ETEC isolates were obtained from the diarrhoeic calves and 12 from diarrhoeic lambs. Most (74%) of the isolates from calves harboured the gene encoding heat-labile enterotoxin I, whereas 75% of the isolates from lambs possessed only the gene encoding for heat-stable enterotoxin a. The ETEC isolates belonged to 20 serogroups, among which serogroups O15 (five isolates) and O8 (four isolates) were the most frequent. Salmonella Typhimurium or S. Enteritidis was identified in three samples from diarrhoeic lambs. The ETEC isolates and the salmonellae showed multidrug resistance. No EAEC or DAEC was detected in any of the samples.

  17. Inactivation of Escherichia coli O157:H7 in orange juice using a combination of high pressure and mild heat.

    PubMed

    Linton, M; McClements, J M; Patterson, M F

    1999-03-01

    The effect of high pressure on the survival of a pressure-resistant strain of Escherichia coli O157:H7 (NCTC 12079) in orange juice was investigated over the pH range 3.4 to 5.0. The pH of commercial, sterile orange juice was adjusted to 3.4, 3.6, 3.9, 4.5, or 5.0. The juice was then inoculated with 10(8) CFU ml(-1) of E. coli O157:H7. The inoculated orange juice was subjected to pressure treatments of 400, 500, or 550 MPa at 20 degrees C or 30 degrees C to determine the conditions that would give a 6-log10 inactivation of E. coli O157:H7. A pressure treatment of 550 MPa for 5 min at 20 degrees C produced this level of kill at pH 3.4, 3.6, 3.9, and 4.5 but not at pH 5.0. Combining pressure treatment with mild heat (30 degrees C) did result in a 6-log10 inactivation at pH 5.0. Thus, the processing conditions (temperature and time) must be considered when pressure-treating orange juice to ensure microbiological safety.

  18. Efficacy of disinfectants and heat against Escherichia coli O157:H7.

    PubMed

    Oie, S; Kamiya, A; Tomita, M; Katayama, A; Iwasaki, A; Miyamura, S

    1999-01-01

    The bactericidal activity of disinfectants and hot water against ten Escherichia coli O157:H7 strains, which were isolated from faeces of patients with enterohaemorrhagic E. coli infection, were evaluated and showed different DNA patterns. After exposure to 0.1% benzalkonium chloride, 0.1% chlorhexidine gluconate containing a nonionic surfactant, and 80% (v/v) ethanol, 99.99% of viable bacterial cells were killed at 20 degrees C within 15 s irrespective of the presence or absence of 0.1% albumin. On the other hand, after exposure to hot water, 99.99% of the bacterial cells were killed within 15 s at 70 degrees C. These results suggest that benzalkonium chloride, chlorhexidine gluconate containing a nonionic surfactant, ethanol, and hot water at 70 degrees C or more are effective for disinfection of E. coli O157:H7 in hospitals.

  19. Stress-Induced Evolution of Heat Resistance and Resuscitation Speed in Escherichia coli O157:H7 ATCC 43888.

    PubMed

    Gayán, Elisa; Cambré, Alexander; Michiels, Chris W; Aertsen, Abram

    2016-11-15

    The development of resistance in foodborne pathogens to food preservation techniques is an issue of increasing concern, especially in minimally processed foods where safety relies on hurdle technology. In this context, mild heat can be used in combination with so-called nonthermal processes, such as high hydrostatic pressure (HHP), at lower individual intensities to better retain the quality of the food. However, mild stresses may increase the risk of (cross-)resistance development in the surviving population, which in turn might compromise food safety. In this investigation, we examined the evolution of Escherichia coli O157:H7 strain ATCC 43888 after recurrent exposure to progressively intensifying mild heat shocks (from 54.0°C to 60.0°C in 0.5°C increments) with intermittent resuscitation and growth of survivors. As such, mutant strains were obtained after 10 cycles of selection with ca. 10(6)-fold higher heat resistance than that for the parental strain at 58.0°C, although this resistance did not extend to temperatures exceeding 60.0°C. Moreover, these mutant strains typically displayed cross-resistance against HHP shock and displayed signs of enhanced RpoS and RpoH activity. Interestingly, additional cycles of selection maintaining the intensity of the heat shock constant (58.5°C) selected for mutant strains in which resuscitation speed, rather than resistance, appeared to be increased. Therefore, it seems that resistance and resuscitation speed are rapidly evolvable traits in E. coli ATCC 43888 that can compromise food safety.

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

    PubMed Central

    2014-01-01

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

  1. Effects of age, ambient temperature, and heat-stable Escherichia coli enterotoxin on intestinal transit in infant mice.

    PubMed

    Moon, H W; Fung, P Y; Isaacson, R E; Booth, G D

    1979-07-01

    Some interrelationships among age, ambient temperature, intestinal transit, and enterotoxigenic Escherichia coli infection were studied in an infant mouse model. The transit of dye in the small intestine was accelerated during the response to heat-stable E. coli enterotoxin. Transit in the small intestine of normal mice accelerated with increased age (from less than 17 h to 8 days old) and accelerated with increased ambient temperature (from 25 to 37 degrees C). Transit was more rapid in the jejunum than in the ileum throughout the range of experimental conditions studied. E. coli strains that do not produce any of the pili known facilitate intestinal colonization were cleared from the small intestine more rapidly at 37 degrees C than at 25 degrees C. This clearance was thought to be due to accelerated transit at the higher temperature. In contrast, a strain of E. coli that produces K99 (pili previously shown to facilitate intestinal colonization in other species) was not cleared from the small intestine and colonized more intensively at 37 degrees C than at 25 degrees C. Intensified colonization by this strain was thought to be due to increased production of K99 at the higher temperature. It was suggested that sluggish intestinal transit may also be characteristic of the neonates of other species and be one of the factors predisposing them to intestinal colonization by enteropathogens. It was speculated that this predisposition may be enhanced if the neonates are chilled. However, the effect of ambient temperature on intestinal transit in homeothermic neonates such as pigs, calves, and humans may be different from that in mice because neonatal mice are poikilothermic.

  2. Co-expression of a heat shock transcription factor to improve conformational quality of recombinant protein in Escherichia coli.

    PubMed

    Hsu, Shao-Yen; Lin, Yu-Sheng; Li, Shu-Jyuan; Lee, Wen-Chien

    2014-09-01

    A co-expression system was established in Escherichia coli for enhancing the cellular expression of heat shock transcription factor, sigma 32 (σ(32)). A Shine-Dalgarno sequence and the rpoH gene of E. coli, which encodes σ(32), were cloned into a bacterial plasmid containing a gene fusion encoding a doubly tagged N-acetyl-d-neuraminic acid aldolase (GST-Neu5Ac aldolase-5R). After the IPTG induction, a substantially higher level of sigma 32 was observed up to 3 h in the co-expression cells, but an enhancement in the solubility of target protein was manifest only in the first hour. Nevertheless, the co-expression of sigma 32 led to higher level of Neu5Ac aldolase enzymatic activity in both the soluble and insoluble (inclusion body) fractions. The Neu5Ac aldolase activity of the supernatant from the lysate of cells co-expressing GST-Neu5Ac aldolase-5R and recombinant σ(32) was 3.4-fold higher at 3 h postinduction than that in cells overexpressing GST-Neu5Ac aldolase-5R in the absence of recombinantly expressed σ(32). The results of acrylamide quenching indicated that the conformational quality of the fusion protein was improved by the co-expression of recombinant σ(32). Thus, the increased level of intracellular σ(32) might have created favorable conditions for the proper folding of recombinant proteins through the cooperative effects of chaperones/heat shock proteins expressed by the E. coli host, which resulted in smaller inclusion bodies, improved conformational quality and a higher specific activity of the overexpressed GST-Neu5Ac aldolase-5R protein. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  3. Influence of growth temperature on inactivation and injury of Escherichia coli O157:H7 by heat, acid, and freezing.

    PubMed

    Semanchek, J J; Golden, D A

    1998-04-01

    The influence of growth temperature on heat-, lactic acid-, and freeze-induced inactivation and injury of Escherichia coli O157:H7 in 0.1% peptone water was investigated. Three strains of E. coli O157:H7 isolated respectively from salami, apple cider, and ground beef were evaluated. Growth of strains at 10 degrees C compared with growth at 37 degrees C had a significant impact on reducing (P < 0.01) D values obtained for heating (DH value), acid exposure (DA value), with the exception of the cider strain stored in lactic acid solutions. When strains were cultivated at 10 and 37 degrees C and heated at 54 and 56 degrees C, the salami strain possessed the highest (P < 0.01) DH values (5.9 to 59.7 min). When grown at 10 degrees C, the beef strain had the lowest (P < 0.01) DH values after heating at 52, 54, and 56 degrees C (11.2, 4.1 and 2.5 min, respectively). The salami strain grown at 10 degrees C had the highest (P < 0.01) DA values in all concentrations of lactic acid. When grown at 37 degrees C, the salami strain had the highest (P < 0.01) DA values after storage in 0.1 and 0.25% lactic acid, while DA values for the salami and beef strains did not differ (P > 0.05) when stored in 0.5% lactic acid. Portions of strain populations were sublethally injured by heat and lactic acid treatments, as evidenced by the inability of injured organisms to form colonies on tryptone soy agar containing 2% NaCl. Strains cultured at 10 degrees C were more susceptible to sublethal heat injury than the strains cultured at 37 degrees C. Storage of test strains at -20 degrees C for 7 months resulted in a 4- to 6-log CFU/ml reduction in viable population, but induced only minimal sublethal injury. After 5 months at -20 degrees C, strains cultured at 10 degrees C were more sensitive to freeze inactivation than strains cultured at 37 degrees C. When grown at 10 and cultured at 37 degrees C. When grown at 10 and 37 degrees C and stored at -20 degrees C for 7 months, the cider strain

  4. Evaluation of antisera used for detecting enterotoxigenic Escherichia coli in Sao Paulo.

    PubMed Central

    Guth, B E; Trabulsi, L R

    1985-01-01

    The usefulness of antisera in detecting enterotoxigenic Escherichia coli (ETEC) strains in Sao Paulo was evaluated. Polyvalent antisera detected 49% of ETEC isolates and were more effective in identifying E. coli that produced heat-labile and heat-stable enterotoxins and in strains that produced only heat-stable enterotoxin. ETEC strains not detected by the antisera belonged to different serogroups not isolated in Sao Paulo before; 34% of these strains had undetermined O antigens, and most of them produced only heat-labile toxin. A variation of serogroups over time was especially observed among strains that produced heat-stable toxin. The importance of H-antigen determinations in the effectiveness of ETEC diagnosis by serological methods became evident, as non-ETEC strains were also detected by polyvalent antisera, but their serotypes were different from those of ETEC strains. Although antisera can be used to identify O:H types of ETEC strains with accuracy, serotyping cannot be recommended for routine diagnosis. However, such a procedure may be useful for studying outbreaks of ETEC diarrhea if the involved serotypes are already known. PMID:3908475

  5. Induction of the heat shock regulon of Escherichia coli markedly increases production of bacterial viruses at high temperatures.

    PubMed Central

    Wiberg, J S; Mowrey-McKee, M F; Stevens, E J

    1988-01-01

    Production of bacteriophages T2, T4, and T6 at 42.8 to 44 degrees C was increased from 8- to 260-fold by adapting the Escherichia coli host (grown at 30 degrees C) to growth at the high temperature for 8 min before infection; this increase was abolished if the host htpR (rpoH) gene was inactive. Others have shown that the htpR protein increases or activates the synthesis of at least 17 E. coli heat shock proteins upon raising the growth temperature above a certain level. At 43.8 to 44 degrees C in T4-infected, unadapted cells, the rates of RNA, DNA, and protein synthesis were about 100, 70, and 70%, respectively, of those in T4-infected, adapted cells. Production of the major processed capsid protein, gp23, was reduced significantly more than that of most other T4 proteins in unadapted cells relative to adapted cells. Only 4.6% of the T4 DNA made in unadapted cells was resistant to micrococcal nuclease, versus 50% in adapted cells. Thus, defective maturation of T4 heads appears to explain the failure of phage production in unadapted cells. Overproduction of the heat shock protein GroEL from plasmids restored T4 production in unadapted cells to about 50% of that seen in adapted cells. T4-infected, adapted E. coli B at around 44 degrees C exhibited a partial tryptophan deficiency; this correlated with reduced uptake of uracil that is probably caused by partial induction of stringency. Production of bacteriophage T7 at 44 degrees C was increased two- to fourfold by adapting the host to 44 degrees C before infection; evidence against involvement of the htpR (rpoH) gene is presented. This work and recent work with bacteriophage lambda (C. Waghorne and C.R. Fuerst, Virology 141:51-64, 1985) appear to represent the first demonstrations for any virus that expression of the heat shock regulon of a host is necessary for virus production at high temperature. Images PMID:2446014

  6. Suppression of rpoH (htpR) mutations of Escherichia coli: heat shock response in suhA revertants.

    PubMed

    Tobe, T; Kusukawa, N; Yura, T

    1987-09-01

    Temperature-resistant pseudorevertants were isolated from rpoH (htpR) mutants of Escherichia coli K-12 that cannot grow at a high temperature owing to a deficiency in sigma 32 required for the induction of heat shock proteins. Among them was a class of revertants carrying a suppressor mutation, designated suhA, that suppressed all the nonsense and missense rpoH mutations tested. suhA is located at 77 min, about 1 min away from rpoH, on the genetic map. In contrast to the rpoH mutants, the suhA revertants that contained both rpoH (nonsense) and suhA mutations were fully or partially proficient in the induction of heat shock proteins upon exposure to a high temperature. Under these conditions, transcription from two heat shock promoters as determined by operon fusion was transiently activated. In one of the rpoH(Am) suhA revertants studied in detail, an increase in temperature caused the synthesis of significant amounts of sigma 32, accompanied by increased stability and accumulation of rpoH mRNAs. On the other hand, the same mutation (suhA6) only weakly suppressed the rpoH deletion mutant; however, two of the major heat shock genes, dnaK and groE, were apparently induced in the absence of sigma 32. Thus, suhA6 seems to bring about the induction of heat shock genes by at least two mechanisms, one increasing the level of sigma 32 synthesis, and the other activating some transcription factor other than sigma 32.

  7. Influence of dimethyl dicarbonate on the resistance of Escherichia coli to a combined UV-Heat treatment in apple juice

    PubMed Central

    Gouma, Maria; Gayán, Elisa; Raso, Javier; Condón, Santiago; Álvarez, Ignacio

    2015-01-01

    Commercial apple juice inoculated with Escherichia coli was treated with UV-C, heat (55°C) and dimethyl dicarbonate – DMDC (25, 50, and 75 mg/L)-, applied separately and in combination, in order to investigate the possibility of synergistic lethal effects. The inactivation levels resulting from each treatment applied individually for a maximum treatment time of 3.58 min were limited, reaching 1.2, 2.9, and 0.06 log10 reductions for UV, heat, and DMDC (75 mg/L), respectively. However, all the investigated combinations resulted in a synergistic lethal effect, reducing the total treatment time and UV dose, with the synergistic lethal effect being higher when larger concentrations of DMDC were added to the apple juice. The addition of 75 mg/L of DMDC prior to the combined UV-C light treatment at 55°C resulted in 5 log10 reductions after only 1.8 min, reducing the treatment time and UV dose of the combined UV-Heat treatment by 44%. PMID:26042117

  8. An Outbreak of Diarrhea in Mandera, Kenya, Due to Escherichia coli Serogroup O-Nontypable Strain That Had a Coding Gene for Enteroaggregative E. coli Heat-Stable Enterotoxin 1.

    PubMed

    Ochi, Sadayuki; Shah, Mohammad; Odoyo, Erick; Bundi, Martin; Miringu, Gabriel; Guyo, Sora; Wandera, Ernest; Kathiiko, Cyrus; Kariuki, Samuel; Karama, Mohamed; Tsuji, Takao; Ichinose, Yoshio

    2017-02-08

    In an outbreak of gastroenteritis in December 2009, in Mandera, Kenya, Escherichia coli O-nontypable (ONT) strain was isolated from stool specimens of patients (18/24, 75%). The E. coli ONT organisms could not be assigned to any of the recognized diarrheagenic groups of E. coli However, they possessed the enteroaggregative E. coli heat-stable enterotoxin-1 gene. The cell-free culture filtrates of the E. coli ONT strain isolated from the outbreak cases induced considerable amount of fluid accumulation in suckling mouse intestine, indicating production of an enterotoxic factor(s). These results identify E. coli that did not have any diarrheagenic characteristics except astA as the etiological agent of the diarrheal outbreak in Mandera. It is however considered necessary to characterize the fluid accumulation factor(s) to determine whether any novel toxins were responsible for the fluid accumulation. Moreover, it is important to study dissemination of strains producing the enterotoxic factor(s) to assess their public health significance distribution in the environment. © The American Society of Tropical Medicine and Hygiene.

  9. Biofilm Formation Potential of Heat-Resistant Escherichia coli Dairy Isolates and the Complete Genome of Multidrug-Resistant, Heat-Resistant Strain FAM21845

    PubMed Central

    Schmid, Michael; Kulli, Sandra; Schneeberger, Kerstin; Naskova, Javorka; Knøchel, Susanne; Ahrens, Christian H.

    2017-01-01

    ABSTRACT We tested the biofilm formation potential of 30 heat-resistant and 6 heat-sensitive Escherichia coli dairy isolates. Production of curli and cellulose, static biofilm formation on polystyrene (PS) and stainless steel surfaces, biofilm formation under dynamic conditions (Bioflux), and initial adhesion rates (IAR) were evaluated. Biofilm formation varied greatly between strains, media, and assays. Our results highlight the importance of the experimental setup in determining biofilm formation under conditions of interest, as correlation between different assays was often not a given. The heat-resistant, multidrug-resistant (MDR) strain FAM21845 showed the strongest biofilm formation on PS and the highest IAR and was the only strain that formed significant biofilms on stainless steel under conditions relevant to the dairy industry, and it was therefore fully sequenced. Its chromosome is 4.9 Mb long, and it harbors a total of five plasmids (147.2, 54.2, 5.8, 2.5, and 1.9 kb). The strain carries a broad range of genes relevant to antimicrobial resistance and biofilm formation, including some on its two large conjugative plasmids, as demonstrated in plate mating assays. IMPORTANCE In biofilms, cells are embedded in an extracellular matrix that protects them from stresses, such as UV radiation, osmotic shock, desiccation, antibiotics, and predation. Biofilm formation is a major bacterial persistence factor of great concern in the clinic and the food industry. Many tested strains formed strong biofilms, and especially strains such as the heat-resistant, MDR strain FAM21845 may pose a serious issue for food production. Strong biofilm formation combined with diverse resistances (some encoded on conjugative plasmids) may allow for increased persistence, coselection, and possible transfer of these resistance factors. Horizontal gene transfer may conceivably occur in the food production setting or the gastrointestinal tract after consumption. PMID:28550056

  10. Variation in Resistance of Natural Isolates of Escherichia coli O157 to High Hydrostatic Pressure, Mild Heat, and Other Stresses

    PubMed Central

    Benito, Amparo; Ventoura, Georgia; Casadei, Maria; Robinson, Tobin; Mackey, Bernard

    1999-01-01

    Strains of Escherichia coli O157 isolated from patients with clinical cases of food-borne illness and other sources exhibited wide differences in resistance to high hydrostatic pressure. The most pressure-resistant strains were also more resistant to mild heat than other strains. Strain C9490, a representative pressure-resistant strain, was also more resistant to acid, oxidative, and osmotic stresses than the pressure-sensitive strain NCTC 12079. Most of these differences in resistance were observed only in stationary-phase cells, the only exception being acid resistance, where differences were also apparent in the exponential phase. Membrane damage in pressure-treated cells was revealed by increased uptake of the fluorescent dyes ethidium bromide and propidium iodide. When strains were exposed to the same pressure for different lengths of time, the pressure-sensitive strains took up stain sooner than the more resistant strain, which suggested that the differences in resistance may be related to susceptibility to membrane damage. Our results emphasize the importance of including stress-resistant strains of E. coli O157 when the efficacy of a novel or mild food preservation treatment is tested. PMID:10103251

  11. Characterization of heat-stable enterotoxin from a hypertoxigenic Escherichia coli strain that is pathogenic for cattle.

    PubMed Central

    Saeed, A M; Magnuson, N S; Gay, C C; Greenberg, R N

    1986-01-01

    An enterotoxigenic Escherichia coli (ETEC) strain isolated from a calf with clinical scours was found to produce over 17- to 60-fold more heat-stable enterotoxin (STa) than four laboratory-adapted bovine ETEC strains. The purified STa of this strain was identical to those produced by other ETEC strains. A severe form of scours was induced in 5- to 15-day-old colostrum-fed calves and in 1- to 2-week-old piglets by oral administration of the purified STa. This study demonstrates that STa is a mediator of diarrhea in newborn calves and piglets and that under identical growth conditions diverse strains of bovine ETEC may produce variable amounts of homologous STa's. PMID:3525417

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

    PubMed

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

    2007-02-01

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

  13. Quercetin as a shuttle for labile iron.

    PubMed

    Baccan, Mayara Marinovic; Chiarelli-Neto, Orlando; Pereira, Regina Mara Silva; Espósito, Breno Pannia

    2012-02-01

    The antioxidant activity of flavonoids may involve their ability to complex body iron in non-redox-active forms. In this study, it was found that the catechol flavonoids rutin and quercetin are able to suppress redox-active labile plasma iron (LPI) in both buffered solution and in iron-overloaded sera. Both flavonoids are effective in loading the metal into the iron-transport protein transferrin. Iron derivatives of quercetin and rutin are able to permeate cell membranes, however, only free quercetin is able to gain access to the cytosol and decrease intracellular labile iron pools. These results suggest that the antioxidant activity of quercetin may be dependent on its ability to shuttle labile iron from cell compartments followed by its transfer to transferrin.

  14. Labile sulfide and sulfite in phytochelatin complexes

    SciTech Connect

    Eannetta, N.T.; Steffens, J.C. )

    1989-04-01

    Heavy metals such as cadmium induce tomato cell cultures to synthesize the metal binding polypeptides ({gamma}-Glu-Cys){sub 3} and ({gamma}-Glu-Cys){sub 4}-Gly (phytochelatins). Tomato cells selected for growth on normally lethal concentrations of CdCl{sub 2} synthesize higher quantities of these polypeptides. Cd{sup r} cells are not cross-resistant to other heavy metals, and recent work suggests that metal detoxification by these peptides may be Cd-specific. The occurrence of labile sulfur as a component of the metal complex raises questions concerning possible functions of phytochelatins besides that of Cd binding. The presence of acid-labile sulfide ion in phytochelatin complexes has been reported by several groups. We report the additional finding that labile sulfite is also present in these complexes and in higher amounts than sulfide. Sulfide and sulfite are both released from the metal binding complex by acidification or by treatment with EDTA.

  15. Virulence Genes in Expanded-Spectrum-Cephalosporin-Resistant and -Susceptible Escherichia coli Isolates from Treated and Untreated Chickens.

    PubMed

    Baron, S; Delannoy, S; Bougeard, S; Larvor, E; Jouy, E; Balan, O; Fach, P; Kempf, I

    2015-12-14

    This study investigated antimicrobial resistance, screened for the presence of virulence genes involved in intestinal infections, and determined phylogenetic groups of Escherichia coli isolates from untreated poultry and poultry treated with ceftiofur, an expanded-spectrum cephalosporin. Results show that none of the 76 isolates appeared to be Shiga toxin-producing E. coli or enteropathogenic E. coli. All isolates were negative for the major virulence factors/toxins tested (ehxA, cdt, heat-stable enterotoxin [ST], and heat-labile enterotoxin [LT]). The few virulence genes harbored in isolates generally did not correlate with isolate antimicrobial resistance or treatment status. However, some of the virulence genes were significantly associated with certain phylogenetic groups.

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

    PubMed Central

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

    1989-01-01

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

  17. A low cost technique for synthesis of gold nanoparticles using microwave heating and its application in signal amplification for detecting Escherichia Coli O157:H7 bacteria

    NASA Astrophysics Data System (ADS)

    Thanh Ngo, Vo Ke; Giang Nguyen, Dang; Phat Huynh, Trong; Lam, Quang Vinh

    2016-09-01

    In the present work a low cost technique for preparation of gold nanoparticles (AuNPs) using microwave heating was developed. The effect of different elements (precursor reagents, irradiation time, and microwave radiation power) on the final morphology of AuNPs obtained through the microwave assisted technique has been investigated. The characterization of the samples has been carried out by transmission electron microscopy, UV-vis absorption spectroscopy, Fourier transform infrared spectroscopy, and powder x-ray diffraction. The results showed that to some extent the above-mentioned characterizations influenced the size of synthetized nanoparticles and application of microwave heating has many advantages such as low cost, rapid preparation and highly uniform particles. As an application in quartz crystal microbalance (QCM) immunosensor, AuNPs are conjugated with the Escherichia coli (E.coli) O157:H7 antibodies for signal amplification to detect E.coli O157:H7 bacteria residual in QCM system.

  18. [The influence of cooling rates and medium tonicity on Escherichia coli B/r survival after heating with different lethal temperatures].

    PubMed

    Morozov, I I; Morozova, G V; Petin, V G

    2005-01-01

    Cell thermosensitivity of Escherichia coli B/r increases with the cooling rise, especially in hypertonic conditions after heating at 50, 55 and 60 degrees C. A certain relationship is suggested between the observed phenomenon and the osmotic homeostasis system of microorganisms under condition of thermogenic and tonic stress.

  19. Linkage of heat-stable enterotoxin activity and ampicillin resistance in a plasmid isolated from an Escherichia coli strain of human origin.

    PubMed Central

    Stieglitz, H; Fonseca, R; Olarte, J; Kupersztoch-Portnoy, Y M

    1980-01-01

    In an Escherichia coli strain of human origin, ampicillin resistance and heat-stable enterotoxin activity were shown by EcoRI restriction endonuclease and genetic analysis to be in an 80-megadalton plasmid. Images Fig. 1 Fig. 2 PMID:6254890

  20. Comparative Effects of Ohmic and Conventional Heating for Inactivation of Escherichia coli O157:H7, Salmonella enterica Serovar Typhimurium, and Listeria monocytogenes in Skim Milk and Cream.

    PubMed

    Kim, Sang-Soon; Kang, Dong-Hyun

    2015-06-01

    Ohmic heating has proven advantages over conventional thermal processing and novel thermal alternative technologies. In this study, the effect of ohmic and conventional heating for pasteurizing skim milk and cream was examined. All treatment conditions for ohmic and conventional heating were identical except for composition of the heating chamber. In most cases, the reduction of three pathogens did not differ significantly between ohmic heating and conventional heating at fixed treatment temperatures and times. However, temperature can be increased more rapidly with ohmic than with conventional heating treatment, both in skim milk and in cream. Therefore, E. coli O157:H7, Salmonella Typhimurium, and L. monocytogenes were inactivated more effectively by ohmic heating treatment for the same treatment time intervals. Also, the time required for pathogen populations to decrease to below the detection limit was less for ohmic heating than conventional heating. Quality aspects (viscosity, pH, and color) of skim milk and cream suffered less degradation by ohmic than by conventional heating. Although there was little evidence of a nonthermal effect of ohmic heating, the results demonstrate significant advantages in the use of ohmic heating over conventional methods for pasteurizing skim milk and cream.

  1. The intertidal copepod Tigriopus japonicus small heat shock protein 20 gene (Hsp20) enhances thermotolerance of transformed Escherichia coli.

    PubMed

    Seo, Jung Soo; Lee, Young-Mi; Park, Heum Gi; Lee, Jae-Seong

    2006-02-17

    To understand the role of the Tigriopus japonicus Hsp20 gene, we isolated this gene from a whole body cDNA library and found two heat shock factor elements at the 5'-UTR. The transformed bacteria containing Tigriopus Hsp20 showed thermotolerance against heat shock (54 degrees C) with different ranges of time. The Tigriopus Hsp20 gene is comprised of 174 amino acid residues and shows similarity to Caenorhabditis elegans (27% identity), silkworm (24.1% identity), moth (24.1% identity), Mexican tetra (19.5% identity), zebrafish (19.5% identity), and spiny dogfish (17.2% identity) genes, but shows more similarity in the C-terminal region that contains an alpha-crystallin domain. Protein motifs such as an N-glycosylation site (67-70 NKSE) and a casein kinase II phosphorylation site were found in Tigriopus Hsp20. The genomic structure of the Tigriopus Hsp20 gene did not contain introns. To characterize the biochemical characteristics of the Tigriopus Hsp20 protein, we expressed Tigriopus Hsp20 in Escherichia coli and purified the soluble protein via 6x His-tag chromatography. To analyze the gene expression of Tigriopus Hsp20 against environmental stresses (e.g., water temperature and salinity), we performed a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). On exposure to different salinities, significant change in the expression of Tigriopus Hsp20 was not observed. However, upon heat shock (30 degrees C), Tigriopus Hsp20 expression was significantly increased, but in the case of cold shock (4 or 10 degrees C), expression was likely downregulated. These findings provide a better understanding of cellular protection mechanisms against environmental stress such as heat shock.

  2. Effect of milk fat content on the performance of ohmic heating for inactivation of Escherichia coli O157:H7, Salmonella enterica Serovar Typhimurium and Listeria monocytogenes.

    PubMed

    Kim, S-S; Kang, D-H

    2015-08-01

    The effect of milk fat content on ohmic heating compared to conventional heating for inactivation of food-borne pathogens was investigated. Sterile cream was mixed with sterile buffered peptone water and adjusted to 0, 3, 7, 10% (w/v) milk fat content. These samples with varying fat content were subjected to ohmic and conventional heating. The effect of milk fat on temperature increase and electrical conductivity were investigated. Also, the protective effect of milk fat on the inactivation of foodborne pathogens was studied. For conventional heating, temperatures of samples increased with time and were not significantly (P > 0.05) different regardless of fat content. Although the inactivation rate of Escherichia coli O157:H7, Salmonella Typhimurium and L. monocytogens decreased in samples of 10% fat content, a protective effect was not observed for conventional heating. In contrast with conventional heating, ohmic heating was significantly affected by milk fat content. Temperature increased more rapidly with lower fat content for ohmic heating due to higher electrical conductivity. Nonuniform heat generation of nonhomogeneous fat-containing samples was verified using a thermal infrared camera. Also, the protective effect of milk fat on E. coli O157:H7 and Listeria monocytogenes was observed in samples subjected to ohmic heating. These results indicate that food-borne pathogens can survive in nonhomogeneous fat-containing foods subjected to ohmic heating. Therefore, more attention is needed regarding ohmic heating than conventional heating for pasteurizing fat-containing foods. The importance of adequate pasteurization for high milk fat containing foods was identified. © 2015 The Society for Applied Microbiology.

  3. Combination effect of ozone and heat treatments for the inactivation of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes in apple juice.

    PubMed

    Sung, Hye-Jung; Song, Won-Jae; Kim, Kwang-Pyo; Ryu, Sangryeol; Kang, Dong-Hyun

    2014-02-03

    We investigated the combination effect of ozone and heat treatments in apple juice for the inactivation of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes. Apple juices inoculated with the three pathogens were treated with gaseous ozone and heat simultaneously for up to 1 min. Gaseous ozone treatment was progressed at a flow rate of 3.0 l/min with a concentration of 2.0-3.0 g/m³ and heat treatment was performed at temperatures of 25, 45, 50, and 55 °C. Populations of surviving pathogens decreased in all samples as treatment temperature increased from 25 to 55 °C. Heat treatment alone (25, 45, 50 and 55 °C) resulted in 0.20, 0.37, 2.16 and 2.54 log CFU/ml reductions of E. coli O157:H7, respectively, in apple juice. Combination treatment of ozone and heat for 1 min reduced this pathogen by 1.50 and 1.60 log CFU/ml, respectively, at 25 and 45 °C, and below the detection limit (1 log CFU/ml) at 50 and 55 °C. We found a synergistic effect in the inactivation of pathogens in apple juice treated with ozone and heated at 50 °C. The reduction trend of S. Typhimurium and L. monocytogenes in apple juice was similar to that of E. coli O157:H7. There were no significant changes of Hunter color values when apple juices were treated with heat only and the combination of ozone and heat. Residual ozone was measured following ozone treatment. In all ozone treated samples, the concentration of residual ozone was reduced to under acceptable levels (<0.4 mg/l). In conclusion, the combination treatment of ozone and heat was significantly effective in the inactivation of foodborne pathogens while maintaining acceptable apple juice quality.

  4. Effect of cell immobilization on heat-induced sublethal injury of Escherichia coli, Salmonella Typhimurium and Listeria innocua.

    PubMed

    Noriega, Estefanía; Velliou, Eirini; Van Derlinden, Eva; Mertens, Laurence; Van Impe, Jan F M

    2013-12-01

    The occurrence of sublethally injured cells in foods poses major public health concerns and is an essential aspect when assessing the microbial response to food preservation strategies, yet there is limited research dealing with its specific implications for mild heating. All available studies so far have been performed in broths colonized by planktonic cells, although their susceptibility to lethal agents has often been reported to be markedly different to the stress tolerance of cell colonies developed in solid foods. In this work, the effect of planktonic and colony growth, as well as the influence of colony density on sublethal injury induced by mild heating of Escherichia coli, Salmonella Typhimurium and Listeria innocua were assessed in food model systems. Detection of injured survivors relied on their inability to form visible colonies on salt-based selective media, which do not affect the growth of healthy cells. Sublethal injury (SI) increased rapidly with shorter exposure times and afterwards, decreased progressively, suggesting a mechanism of cumulative damage triggering lethal instead of SI. Cell arrangement affected the degree of SI, higher values being generally found for gelified systems, although the effect of colony density depended on the target microorganism. This information is essential for optimizing the design of food safety assurance systems.

  5. Heat Resistance in Liquids of Enterococcus spp., Listeria spp., Escherichia coli, Yersinia enterocolitica, Salmonella spp. and Campylobacter spp

    PubMed Central

    Sörqvist, S

    2003-01-01

    The aim of the work was to collect, evaluate, summarize and compare heat resistance data reported for Campylobacter, Enterococcus, Escherichia, Listeria, Salmonella and Yersinia spp. The work was limited to resistance in liquids with pH values 6–8. Results obtained under similar experimental conditions were sought. Thermal destruction lines for the various bacterial groups studied were constructed using log10 D values and treatment temperatures. There was a good linear relationship between log10 D and temperature with Escherichia coli, listerias and salmonellas. For campylobacters, enterococci and yersinias the relationships were weaker but, nevertheless, present. Using the slopes of the lines and their 95% confidence limits, z values and their 95% confidence limits were calculated. z values were compared with z values obtained from reports. The equations for the lines were also used for calculation of predicted means of D values at various treatment temperatures. 95% confidence limits on predicted means of D values and on predicted individual D values were also calculated. Lines and values are shown in figures and tables. Differences in heat resistance noted between and within the bacterial groups studied are discussed. PMID:14650540

  6. Differences in serological responses and excretion patterns of volunteers challenged with enterotoxigenic Escherichia coli with and without the colonization factor antigen.

    PubMed Central

    Evans, D G; Satterwhite, T K; Evans, D J; DuPont, H L

    1978-01-01

    Double-blind studies were performed to compare the virulence of enterotoxigenic Escherichia coli with and without the fimbriate colonization factor antigen (CFA), using young healthy adults (mean age, 23 years) as volunteers. In the first study one group of volunteers ingested 1 X 10(6) E. coli H-10407, the CFA-positive strain, and another group ingested 1 X 10(6) E. coli H-10407-P, the CFA-negative spontaneous derivative of strain H-10407. The second study was similar except that the test strains were administered at a dose of 1 X 10(8) viable cells. Three parameters of infection were monitored: (i) diarrhea and associated symptoms; (ii) excretion pattern of test strains; and (iii) humoral antibody response to CFA, somatic antigen, and heat-labile enterotoxin. Significant signs of illness occurred only in six of seven volunteers who ingested E. coli H-10407 at a dose of 1 X 10(8). At both doses, E. coli H-10407-P appeared in the stool on day 1 postchallenge and disappeared by day 4. In contrast, strain H-10407 was persistently excreted from the first to the last day of the study. Also, only those volunteers in the H-10407 challenge groups (12 of 13 analyzed) responded with a fourfold antibody titer rise to CFA, somatic antigen, and/or heat-labile enterotoxin. No reversion of H-10407-P to H-10407 was detected. PMID:346488

  7. Lability of Humic-Bound Phosphorus

    USDA-ARS?s Scientific Manuscript database

    Phosphorus (P) has long been known to be present in humic substances from various sources. However, information on the lability of humic-bound P is very limited although such information is critical for understanding the role of humic substances in P cycling and nutrition. In this presentation, we d...

  8. Radio-frequency heating to inactivate Salmonella Typhimurium and Escherichia coli O157:H7 on black and red pepper spice.

    PubMed

    Kim, Sung-Youn; Sagong, Hun-Gu; Choi, Sang Ho; Ryu, Sangryeol; Kang, Dong-Hyun

    2012-02-01

    The efficacy of radio-frequency (RF) heating to inactivate Salmonella Typhimurium and Escherichia coli O157:H7 on black and red pepper spice was investigated. A 27.12 MHz RF heating system consisted of two parallel-plate electrodes was used, with the sample being placed between them. Black peppers (whole and ground) and red peppers (+ 16 mesh, -16 + 25 mesh, and -25 mesh) inoculated with S. Typhimurium and E. coli O157:H7 were treated with RF energy during 50s for black peppers and 40s for red peppers, and color change of samples was evaluated after treatment. RF heating for 50s resulted in 2.80 to 4.29 log CFU/g reductions of S. Typhimurium and E. coli O157:H7 in black peppers and RF heating of red peppers for 40s reduced pathogens by 3.38 log CFU/g to more than 5 log CFU/g (below the detection limit) without affecting the color quality change. The results suggest that RF heating has the potential for novel thermal process to control foodborne pathogens in spice. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Heat resistance of Escherichia coli O157:H7 in a nutrient medium and in ground beef patties as influenced by storage and holding temperatures.

    PubMed

    Jackson, T C; Hardin, M D; Acuff, G R

    1996-03-01

    Stationary-phase cultures of Escherichia coli O157:H7 were inoculated into tryptic soy broth, sealed in vials, and stored at -18 degrees C for 1, 8, and 15 days, or 3 or 15 degrees C for 3, 6, and 9 h. Thermal resistance was determined at 55 degrees C. Each storage treatment was repeated with additional holding at 23 or 30 degrees C for 1, 2, 3, or 4 h prior to heating to simulate potential temperature abuse during handling. Cultures under treatments enabling the growth of E. coli O157:H7 were generally more heat sensitive than those held at temperatures which restricted growth or enabled growth to stationary phase. Cultures stored frozen (-18 degrees C) without holding at elevated temperatures had greater heat resistance than those stored under refrigeration (3 degrees C) or at 15 degrees C. Subsequent holding of frozen cultures at 23 or 30 degrees C resulted in a decrease in heat resistance. To determine whether these responses would be observed under typical commercial preparation procedures, ground beef patties were inoculated with E. coli O157:H7 and stored at 3 or 15 degrees C for 9 h or at -18 degrees C for 8 d and then held at 21 or 30 degrees C for 0 or 4 h. Patties were grilled to an internal temperature of 54.4 degrees C (130 degrees F), 62.8 degrees C (145 degrees F), or 68.3 degrees C (155 degrees F). Cultures were most resistant in frozen patties, while cultures in patties stored at 15 degrees C were the most heat sensitive. Holding patties at 21 or 30 degrees C prior to grilling resulted in increased sensitivity. Storage and holding temperatures similar to those encountered in food service may influence the ability of E. coli O157:H7 to survive heat treatments.

  10. A small heat shock protein enables Escherichia coli to grow at a lethal temperature of 50°C conceivably by maintaining cell envelope integrity.

    PubMed

    Ezemaduka, Anastasia N; Yu, Jiayu; Shi, Xiaodong; Zhang, Kaiming; Yin, Chang-Cheng; Fu, Xinmiao; Chang, Zengyi

    2014-06-01

    It is essential for organisms to adapt to fluctuating growth temperatures. Escherichia coli, a model bacterium commonly used in research and industry, has been reported to grow at a temperature lower than 46.5°C. Here we report that the heterologous expression of the 17-kDa small heat shock protein from the nematode Caenorhabditis elegans, CeHSP17, enables E. coli cells to grow at 50°C, which is their highest growth temperature ever reported. Strikingly, CeHSP17 also rescues the thermal lethality of an E. coli mutant deficient in degP, which encodes a protein quality control factor localized in the periplasmic space. Mechanistically, we show that CeHSP17 is partially localized in the periplasmic space and associated with the inner membrane of E. coli, and it helps to maintain the cell envelope integrity of the E. coli cells at the lethal temperatures. Together, our data indicate that maintaining the cell envelope integrity is crucial for the E. coli cells to grow at high temperatures and also shed new light on the development of thermophilic bacteria for industrial application.

  11. Comparison of enterotoxic activities of heat-stable enterotoxins from class 1 and class 2 Escherichia coli of swine origin.

    PubMed Central

    Whipp, S C; Moon, H W; Argenzio, R A

    1981-01-01

    Pig small intestine develops age-dependent resistance to some (class 2 strains) enterotoxigenic Escherichia coli while remaining susceptible to others (class 1 strains). This study tested the hypothesis that class 1 and class 2 strains produce different subtypes of heat-stable enterotoxin (ST). The dose-response curves of small intestine to crude ST preparations from a class 1 and a class 2 strain were compared in several species. In infant mice, the class 1 ST preparation was less active than the class 2 ST preparation, whereas in rabbits the preparations were equally potent. However, in 1-, 7-, and 14-week-old pigs, the class 1 ST preparation was more active than the class 2 preparation. At low doses, both preparations caused reduced absorption in pigs of all three age groups, and at high doses the class 1 preparation caused secretion in all three age groups. In contrast, at high doses the class 2 preparation caused secretion in 1-week-old pigs but only reduced absorption in older pigs. when class 1 and class 2 ST preparations were fractionated by methanol extraction, in both cases the mouse-negative, pig-positive activity was associated with the methanol-insoluble fraction and mouse-positive, pig-positive activity was associated with the methanol-soluble fraction. The results are consistent with a hypothesis that class 1 and class 2 strains of enterotoxigenic E. coli produce different subtypes of ST and that the response of pig intestine to ST varies with both age and toxin subtype. PMID:7011991

  12. Proteome analysis of the Escherichia coli heat shock response under steady-state conditions

    PubMed Central

    Lüders, Svenja; Fallet, Claas; Franco-Lara, Ezequiel

    2009-01-01

    In this study a proteomic approach was used to investigate the steady-state response of Escherichia coli to temperature up-shifts in a cascade of two continuously operated bioreactors. The first reactor served as cell source with optimal settings for microbial growth, while in the second chemostat the cells were exposed to elevated temperatures. By using this reactor configuration, which has not been reported to be used for the study of bacterial stress responses so far, it is possible to study temperature stress under well-defined, steady-state conditions. Specifically the effect on the cellular adaption to temperature stress using two-dimensional gel electrophoresis was examined and compared at the cultivation temperatures of 37°C and 47.5°C. As expected, the steady-state study with the double bioreactor configuration delivered a different protein spectrum compared to that obtained with standard batch experiments in shaking flasks and bioreactors. Setting a high cut-out spot-to-spot size ratio of 5, proteins involved in defence against oxygen stress, functional cell envelope proteins, chaperones and proteins involved in protein biosynthesis, the energy metabolism and the amino acid biosynthesis were found to be differently expressed at high cultivation temperatures. The results demonstrate the complexity of the stress response in a steady-state culture not reported elsewhere to date. PMID:19772559

  13. Small Heat-Shock Proteins, IbpAB, Protect Non-Pathogenic Escherichia coli from Killing by Macrophage-Derived Reactive Oxygen Species

    PubMed Central

    Goeser, Laura; Fan, Ting-Jia; Tchaptchet, Sandrine; Stasulli, Nikolas; Goldman, William E.; Sartor, R. Balfour; Hansen, Jonathan J.

    2015-01-01

    Many intracellular bacterial pathogens possess virulence factors that prevent detection and killing by macrophages. However, similar virulence factors in non-pathogenic bacteria are less well-characterized and may contribute to the pathogenesis of chronic inflammatory conditions such as Crohn’s disease. We hypothesize that the small heat shock proteins IbpAB, which have previously been shown to reduce oxidative damage to proteins in vitro and be upregulated in luminal non-pathogenic Escherichia strain NC101 during experimental colitis in vivo, protect commensal E. coli from killing by macrophage-derived reactive oxygen species (ROS). Using real-time PCR, we measured ibpAB expression in commensal E. coli NC101 within wild-type (wt) and ROS-deficient (gp91phox-/-) macrophages and in NC101 treated with the ROS generator paraquat. We also quantified survival of NC101 and isogenic mutants in wt and gp91phox-/- macrophages using gentamicin protection assays. Similar assays were performed using a pathogenic E. coli strain O157:H7. We show that non-pathogenic E. coli NC101inside macrophages upregulate ibpAB within 2 hrs of phagocytosis in a ROS-dependent manner and that ibpAB protect E. coli from killing by macrophage-derived ROS. Moreover, we demonstrate that ROS-induced ibpAB expression is mediated by the small E. coli regulatory RNA, oxyS. IbpAB are not upregulated in pathogenic E. coli O157:H7 and do not affect its survival within macrophages. Together, these findings indicate that ibpAB may be novel virulence factors for certain non-pathogenic E. coli strains. PMID:25798870

  14. Overexpression of CsLEA11, a Y3SK2-type dehydrin gene from cucumber (Cucumis sativus), enhances tolerance to heat and cold in Escherichia coli.

    PubMed

    Zhou, Yong; He, Peng; Xu, Yaping; Liu, Qiang; Yang, Yingui; Liu, Shiqiang

    2017-09-29

    As the group II LEA (late embryogenesis abundant) proteins, dehydrins (DHNs) play an important role in plant growth and development, as well as in response to abiotic or biotic stress challenges. In this study, a DHN gene named CsLEA11 was identified and characterized from Cucumis sativus. Sequence analysis of CsLEA11 showed that it is a Y3SK2-type DHN protein rich in hydrophilic amino acids. Expression analyses revealed that the transcription of CsLEA11 could be significantly induced by heat and cold stress. The recombinant plasmid was transformed into Escherichia coli BL21 and isopropy-β-D-thiogalactoside (IPTG) was used to induce recombinant E. coli to express CsLEA11 gene. Overexpression of CsLEA11 in E. coli enhanced cell viability and conferred tolerance to heat and cold stress. Furthermore, CsLEA11 protein could protect the activity of lactate dehydrogenase (LDH) under heat stress. Taken together, our data demonstrate that CsLEA11 might function in tolerance of cucumber to heat and cold stress.

  15. Short communication: Heat-resistant Escherichia coli as potential persistent reservoir of extended-spectrum β-lactamases and Shiga toxin-encoding phages in dairy.

    PubMed

    Marti, Roger; Muniesa, Maite; Schmid, Michael; Ahrens, Christian H; Naskova, Javorka; Hummerjohann, Jörg

    2016-11-01

    Here we report the isolation of heat-resistant Escherichia coli from raw milk cheeses. Detection of the heat-resistance markers clpK and orfI by PCR was followed by phenotypical confirmation of increased heat-resistance. These strains were Shiga toxin-negative and, although several were found to be multidrug resistant, no plasmids encoding extended-spectrum β-lactamases (ESBL) were found in any of the isolates. The aim of this study was to assess the potential of these strains to acquire ESBL plasmids and a modified Shiga toxin-encoding phage. Only 4 ESBL-encoding, heat-sensitive E. coli strains were isolated from 1,251 dairy samples (2/455 raw milk and 2/796 raw milk cheese samples). One incompatibility group FII plasmid (CTX-M-14, 79.0 kb) and 3 incompatibility group I1 plasmids (CTX-M-15, 95.2, 96.1, and 97.8 kb) were fully sequenced and de novo assembled. All 4 plasmids are readily transferred to heat-resistant E. coli isolates in plate matings (9.7×10(-5) to 3.7×10(-1) exconjugants per recipient) and, to a lesser extent, in milk (up to 7.4×10(-5) exconjugants per recipient). Importantly, the plasmids are stably maintained during passaging in liquid media without antimicrobial pressure. The heat-resistant isolate FAM21805 was also shown to be capable of acting as donor of all 4 ESBL plasmids. In addition, 3 of 11 tested ESBL exconjugants of heat-resistant strains were lysogenized by the modified Shiga toxin-encoding phage 933W ∆stx::gfp::cat. The higher fraction of heat-resistant E. coli (93 of 256 isolates) compared with the estimated 2% previously predicted based on genomic prevalence of heat resistance genes seems to indicate a selection advantage in the raw milk cheese production environment. The combination of 2 factors may lead to said advantage: increased survival during thermization of raw milk (heating to subpasteurization temperatures) and increased survival rates during cheese ripening. Should these strains acquire ESBL-encoding plasmids, Shiga

  16. Expanded Monomeric Intermediate upon Cold and Heat Unfolding of Phosphofructokinase-2 from Escherichia coli

    PubMed Central

    Baez, Mauricio; Wilson, Christian A.M.; Ramírez-Sarmiento, César A.; Guixé, Victoria; Babul, Jorge

    2012-01-01

    Folding studies have been focused mainly on small, single-domain proteins or isolated single domains of larger proteins. However, most of the proteins present in biological systems are composed of multiple domains, and to date, the principles that underlie its folding remain elusive. The unfolding of Pfk-2 induced by GdnHCl has been described by highly cooperative three-state equilibrium (N2↔2I↔2U). This is characterized by a strong coupling between the subunits’ tertiary structure and the integrity of the dimer interface because “I” represents an unstructured and expanded monomeric intermediate. Here we report that cold and heat unfolding of Pfk-2 resembles the N2↔2I step of chemically induced unfolding. Moreover, cold unfolding appears to be as cooperative as that induced chemically and even more so than its heat-unfolding counterpart. Because Pfk-2 is a large homodimer of 66 kDa with a complex topology consisting of well-defined domains, these results are somewhat unexpected considering that cold unfolding has been described as a special kind of perturbation that decouples the cooperative unfolding of several proteins. PMID:23200052

  17. Topoisomerase activity during the heat shock response in Escherichia coli K-12.

    PubMed Central

    Camacho-Carranza, R; Membrillo-Hernández, J; Ramírez-Santos, J; Castro-Dorantes, J; Chagoya de Sánchez, V; Gómez-Eichelmann, M C

    1995-01-01

    During the upshift of temperature from 30 to 42, 45, 47, or 50 degrees C, an increase in the level of supercoiling of a reporter plasmid was observed. This increase was present in groE and dnaK mutants but was inhibited in cells treated with chloramphenicol and novobiocin. The intracellular [ATP]/[ADP] ratio increased rapidly after an upshift in temperature from 30 to 47 degrees C and then decreased to reach a level above that observed at 30 degrees C. These results suggest that gyrase and proteins synthesized during heat shock are responsible for the changes seen in plasmid supercoiling. Proteins GroE and DnaK are probably not involved in this phenomenon. PMID:7768879

  18. rpoE, the gene encoding the second heat-shock sigma factor, sigma E, in Escherichia coli.

    PubMed Central

    Rouvière, P E; De Las Peñas, A; Mecsas, J; Lu, C Z; Rudd, K E; Gross, C A

    1995-01-01

    In Escherichia coli, the heat shock response is under the control of two alternative sigma factors: sigma 32 and sigma E. The sigma 32-regulated response is well understood, whereas little is known about that of sigma E, except that it responds to extracytoplasmic immature outer membrane proteins. To further understand this response, we located the rpoE gene at 55.5' and analyzed the role of sigma E. sigma E is required at high temperature, and controls the transcription of at least 10 genes. Some of these might contribute to the integrity of the cell since delta rpoE cells are more sensitive to SDS plus EDTA and crystal violet. sigma E controls its own transcription from a sigma E-dependent promoter, indicating that rpoE transcription plays a role in the regulation of E sigma E activity. Indeed, under steady-state conditions, the transcription from this promoter mirrors the levels of E sigma E activity in the cell. However, it is unlikely that the rapid increase in E sigma E activity following induction can be accounted for solely by increased transcription of rpoE. Based upon homology arguments, we suggest that a gene encoding a negative regulator of sigma E activity is located immediately downstream of rpoE and may function as the target of the E sigma E inducing signal. Images PMID:7889934

  19. Binding of E. coli heat-stable enterotoxin to rat intestinal brush borders and to basolateral membranes

    SciTech Connect

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

    1987-09-01

    We studied the binding of E. coli heat-stable enterotoxin (STa) to rat brush borders (BB) and to basolateral membranes (BLM) using a biologically active monoiodinated radioligand (( /sup 125/I)STa) and highly enriched BB and BLM preparations free of other significant organelle contamination. Binding of (/sup 125/I)STa to BB was specific; time-, temperature-, and pH-dependent; saturable; and partially reversible. Nonlabeled toxin competitively inhibited the binding of radioligand to BB in a dose-related manner. Scatchard analysis revealed a single class of receptors with an apparent affinity constant of 8.7 +/- 1.5 X 10(8) l/mol. Binding was not affected by amino acids, sugars, and lectins. Proteolytic enzymes significantly decreased binding, although several did so by modifying the radioligand. Trypsin inhibited binding without modifying the radioligand thus supporting the proteinaceous nature of the receptor. Since the enrichment in binding activity in the BB over the homogenate was significantly lower than the enrichment in sucrase activity, we concluded that binding activity is probably associated with other membranous domains, but direct examination revealed no binding activity on basolateral membranes.

  20. Reduction of the secretory response to Escherichia coli heat-stable enterotoxin by thiol and disulfide compounds. [Mice

    SciTech Connect

    Greenberg, R.N.; Dunn, J.A.; Guerrant, R.L.

    1983-07-01

    We examined the effects of disulfide and thiol compounds on Escherichia coli heat-stable enterotoxin (ST) and cyclic GMP-induced secretion. Both cystamine and cystine (disulfide compounds) reduced the secretory responses to submaximal doses of ST in suckling mice (at 0.5 mumol per mouse) and reduced ST activation of guanylate cyclase (by 33 to 73% at 1 mM). In higher doses, cystamine completely eradicated a maximally effective ST dose as well. In addition, the sulfhydryl (thiol) compounds cysteamine, cysteine, and acetylcysteine strikingly reduced the secretory response and the guanylate cyclase response to ST. Neither the disulfide nor the thiol compounds tested reduced cyclic GMP-induced secretion. These studies suggest that disulfide and thiol compounds both block ST-induced secretion before its activation of guanylate cyclase. Taken with the work of others, these findings suggest that disulfide compounds may alter the oxidation reduction state of a cell or act directly on the guanylate cyclase enzyme, whereas thiol compounds may inactivate ST itself by breaking its disulfide bridges, or it may alter guanylate cyclase activation by ST. Both families of compounds deserve further consideration among potential antisecretory agents for application in the control of ST-induced diarrhea.

  1. Effect of heat-assisted pulsed electric fields and bacteriophage on enterohemorrhagic Escherichia coli O157:H7.

    PubMed

    Walkling-Ribeiro, Markus; Anany, Hany; Griffiths, Mansel W

    2015-01-01

    Pulsed electric fields (PEF), heat-assisted PEF (H-PEF), and virulent bacteriophage (VP) are non-thermal techniques for pathogen inactivation in liquids that were investigated individually, and in combination (PEF/VP, H-PEF/VP) to control enterohemorrhagic Escherichia coli (EHEC) O157:H7 in Luria-Bertani broth (LBB) and Ringer's solution (RS). Treated cells were subsequently incubated at refrigeration (4°C) and temperature-abuse conditions (12°C) for 5 days. When EHEC cells grown in LBB were subjected to non-thermal processing and subsequently stored at 12°C for 5 days, reductions in count of between 0.1 and 0.6 log cycles were observed and following storage at 4°C the decrease in counts varied between 0.2 and 1.1 log10 . For bacteria cells suspended in RS values ranged from 0.1 to ≥3.9 log cycles at both storage temperatures. The most effective treatments were H-PEF and H-PEF/VP, both producing a >3.4 log cycle reduction of cells suspended in non-nutrient RS. Analysis of EHEC recovery on selective and non-selective media indicated no occurrence of sub-lethal damage for VP, PEF/VP, and H-PEF/VP-treated cells. The findings indicate that combining PEF and lytic phage may represent a suitable alternative to conventional fluid decontamination following further process optimization.

  2. Effect of Escherichia coli heat-stable enterotoxin on cell volume and intracellular inorganic ions of rat intestinal cells.

    PubMed

    Gilles-Baillien, M; Gerday, C; Kaeckenbeeck, A; Flamee, P A; Duchesne, P Y

    1987-11-01

    1.--Electron micrographs of rat jejunum mucosa incubated for 1 h in the presence of Escheria coli heat-stable enterotoxin (STa) in the lumen shows alterations of villous cells as well as of crypt cells. The brush border of mature enterocytes is partially desintegrated and covered with a thick mucus. Crypts are occupied on half of their height by cells very similar to Paneth cells, loaded with numerous large dark inclusions. 2.--Cell volume and intracellular inorganic ion concentrations have been estimated in mucosal scrapings of jejunum sacs, incubated in vitro for 1 or 3 h. The quick action (1 h of incubation) of STa is a swelling of the intestinal calls accompanied by an increase in Na+, Cl- and Ca2+ intracellular concentrations and a decrease in the K+ and Mg2+ ones. The delayed action (3 h of incubation) is an increase of extracellular space and a decrease in cell volume; and at the same time the intracellular concentration of Na+, Cl-, K+, Ca2+ and Mg2+ is augmented. 3.--After 3 h of incubation intestinal cells from the other levels of intestine (duodenum, ileum and colon) show the same variations in cell volume and intracellular inorganic ion concentrations under the influence of STa, as those recorded in the jejunum. 4.--The present work favours the hypothesis that all intestinal cells, villous or cryptic, are involved in the alteration of fluid ion transport ending in diarrhea.

  3. Carvacrol facilitates heat-induced inactivation of Escherichia coli O157:H7 and inhibits formation of heterocyclic amines in grilled ground beef patties.

    PubMed

    Friedman, Mendel; Zhu, Libin; Feinstein, Yelena; Ravishankar, Sadhana

    2009-03-11

    Heating meat at high temperature and/or for a long time to kill foodborne pathogens increases the formation of potentially carcinogenic heterocyclic amines. To overcome this problem, 1% carvacrol, the main ingredient of oregano oil widely used in salad dressings, was added to ground beef, which was mixed well and then inoculated with Escherichia coli O157:H7. Beef patties were then prepared and heat-treated on a preheated electrical skillet to reach an internal temperature of 65, 70, or 80 degrees C at the cold spot. Samples were enumerated for surviving E. coli O157:H7 population by plating on appropriate media. Heterocyclic amines (MeIQ, MeIQx, and PhIP) were extracted from ground beef using solid phase extraction and analyzed by mass spectrometry. Multiple reaction monitoring (MRM) scan type in positive mode was used to monitor the amines of interest. Compared to controls, the population of E. coli O157:H7 was reduced by 2.5-5 logs. The corresponding highest reductions in the three major amines were MeIQ, 58%; MeIQx, 72%; and PhIP, 78%. The results show that carvacrol concurrently reduced E. coli O157:H7 and amines in a widely consumed meat product. Possible mechanisms of the beneficial effects and dietary significance of the results are discussed.

  4. Effect of continuous ohmic heating to inactivate Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes in orange juice and tomato juice.

    PubMed

    Lee, S-Y; Sagong, H-G; Ryu, S; Kang, D-H

    2012-04-01

    The purpose of this study was to investigate the efficacy of continuous ohmic heating for reducing Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes in orange juice and tomato juice. Orange juice and tomato juice were treated with electric field strengths in the range of 25-40 V cm(-1) for different treatment times. The temperature of the samples increased with increasing treatment time and electric field strength. The rate of temperature change for tomato juice was higher than for orange juice at all voltage gradients applied. Higher electric field strength or longer treatment time resulted in a greater reduction of pathogens. Escherichia coli O157:H7 was reduced by more than 5 log after 60-, 90- and 180-s treatments in orange juice with 40, 35 and 30 V cm(-1) electric field strength, respectively. In tomato juice, treatment with 25 V cm(-1) for 30 s was sufficient to achieve a 5-log reduction in E. coli O157:H7. Similar results were observed in Salm. Typhimurium and L. monocytogenes. The concentration of vitamin C in continuous ohmic heated juice was significantly higher than in conventionally heated juice (P < 0·05). Continuous ohmic heating can be effective in killing foodborne pathogens on orange juice and tomato juice with lower degradation of quality than conventional heating. These results suggest that continuous ohmic heating might be effectively used to pasteurize fruit and vegetable juices in a short operating time and that the effect of inactivation depends on applied electric field strengths, treatment time and electric conductivity. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

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

    PubMed Central

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

    1985-01-01

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

  6. Effect of anaerobic and stationary phase growth conditions on the heat shock and oxidative stress responses in Escherichia coli K-12.

    PubMed

    Díaz-Acosta, Alondra; Sandoval, María L; Delgado-Olivares, Luis; Membrillo-Hernández, Jorge

    2006-06-01

    The natural living style of Escherichia coli occurs in the gastrointestinal tract, where most of its existence is spent under anaerobic conditions and in stationary phase of growth. Here we report on the heat shock response of E. coli K-12 cells growing in the presence or absence of oxygen. An rpoH mutant (impaired in the synthesis of the sigma(32) transcriptional factor) exhibited an increased sensitivity to heat shock but only in the exponential phase of aerobic growth, suggesting that in anaerobic growth conditions, and in aerobic stationary phase, sigma(32)-independent mechanisms are playing a prime role in protecting cells from heat stress. Our results demonstrated that sigma(S) is not involved in this protection system. Studies on the kinetics of synthesis of Heat shock proteins (Hsp) after an abrupt rise in temperature demonstrated that in the absence of oxygen, the synthesis of Hsp is triggered faster and is sustained for a longer period of time compared to aerobic growth conditions. Finally, the heated cells in the exponential phase of aerobic growth displayed a high concentration of oxidatively damaged proteins in the presence of 4 mM H(2)O(2), in sharp contrast to cultures of stationary phase or anaerobic growth.

  7. Sensitivity of Escherichia coli O157:H7 to commercially available alkaline cleaners and subsequent resistance to heat and sanitizers.

    PubMed

    Sharma, Manan; Beuchat, Larry R

    2004-03-01

    The effects of seven commercially available alkaline cleaners used in the food processing industry, 0.025 M NaOH, and 0.025 M KOH on viability of wild-type (EDL 933) and rpoS-deficient (FRIK 816-3) strains of Escherichia coli O157:H7 in logarithmic and stationary phases of growth were determined. Cells were treated at 4 or 23 degrees C for 2, 10, or 30 min. Cleaners 2, 4, 6, and 7, which contained hypochlorite and <11% NaOH and/or KOH (pH 11.2 to 11.7), killed significantly higher numbers of cells than treatment with cleaner 3, containing sodium metasilicate (pH 11.4) and <10% KOH, and cleaner 5, containing ethylene glycol monobutyl ether (pH 10.4). There were no differences in the sensitivities of logarithmic and stationary-phase cells to the alkaline cleaners. Treatment with KOH or NaOH (pH 12.2) was not as effective as four out of seven commercial cleaners in killing E. coli O157:H7, indicating that chlorine and other cleaner components have bactericidal activity at high pH. Stationary-phase cells of strain EDL 933 that had been exposed to cleaner 7 at 4 or 23 degrees C and strain FRIK 816-3 exposed to cleaner 7 at 23 degrees C had significantly higher D(55 degrees C) (decimal reduction time, minutes at 55 degrees C) values than control cells or cells exposed to cleaner 5, indicating that exposure to cleaner 7 confers cross-protection to heat. Cells of EDL 933 treated with cleaner 7 at 12 degrees C showed significantly higher D(55 degrees C) values than cells of FRIK 816-3, indicating that rpoS may play a role in cross-protection. Stationary-phase cells treated with cleaner 5 or cleaner 7 at 4 or 12 degrees C were not cross-protected against subsequent exposure to sanitizers containing quaternary ammonium compounds or sodium hypochlorite, or to cetylpyridinium chloride and benzalkonium chloride.

  8. Sensitivity of Escherichia coli O157:H7 to Commercially Available Alkaline Cleaners and Subsequent Resistance to Heat and Sanitizers

    PubMed Central

    Sharma, Manan; Beuchat, Larry R.

    2004-01-01

    The effects of seven commercially available alkaline cleaners used in the food processing industry, 0.025 M NaOH, and 0.025 M KOH on viability of wild-type (EDL 933) and rpoS-deficient (FRIK 816-3) strains of Escherichia coli O157:H7 in logarithmic and stationary phases of growth were determined. Cells were treated at 4 or 23°C for 2, 10, or 30 min. Cleaners 2, 4, 6, and 7, which contained hypochlorite and <11% NaOH and/or KOH (pH 11.2 to 11.7), killed significantly higher numbers of cells than treatment with cleaner 3, containing sodium metasilicate (pH 11.4) and <10% KOH, and cleaner 5, containing ethylene glycol monobutyl ether (pH 10.4). There were no differences in the sensitivities of logarithmic and stationary-phase cells to the alkaline cleaners. Treatment with KOH or NaOH (pH 12.2) was not as effective as four out of seven commercial cleaners in killing E. coli O157:H7, indicating that chlorine and other cleaner components have bactericidal activity at high pH. Stationary-phase cells of strain EDL 933 that had been exposed to cleaner 7 at 4 or 23°C and strain FRIK 816-3 exposed to cleaner 7 at 23°C had significantly higher D55°C (decimal reduction time, minutes at 55°C) values than control cells or cells exposed to cleaner 5, indicating that exposure to cleaner 7 confers cross-protection to heat. Cells of EDL 933 treated with cleaner 7 at 12°C showed significantly higher D55°C values than cells of FRIK 816-3, indicating that rpoS may play a role in cross-protection. Stationary-phase cells treated with cleaner 5 or cleaner 7 at 4 or 12°C were not cross-protected against subsequent exposure to sanitizers containing quaternary ammonium compounds or sodium hypochlorite, or to cetylpyridinium chloride and benzalkonium chloride. PMID:15006806

  9. 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

  10. Effect of frequency and waveform on inactivation of Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium in salsa by ohmic heating.

    PubMed

    Lee, Su-Yeon; Ryu, Sangryeol; Kang, Dong-Hyun

    2013-01-01

    The effect of frequency of alternating current during ohmic heating on electrode corrosion, heating rate, inactivation of food-borne pathogens, and quality of salsa was investigated. The impact of waveform on heating rate was also investigated. Salsa was treated with various frequencies (60 Hz to 20 kHz) and waveforms (sine, square, and sawtooth) at a constant electric field strength of 12.5 V/cm. Electrode corrosion did not occur when the frequency exceeded 1 kHz. The heating rate of the sample was dependent on frequency up to 500 Hz, but there was no significant difference (P > 0.05) in the heating rate when the frequency was increased above 1 kHz. The electrical conductivity of the sample increased with a rise in the frequency. At a frequency of 60 Hz, the square wave produced a lower heating rate than that of sine and sawtooth waves. The heating rate between waveforms was not significantly (P > 0.05) different when the frequency was >500 Hz. As the frequency increased, the treatment time required to reduce Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium to below the detection limit (1 log CFU/g) decreased without affecting product quality. These results suggest that ohmic heating can be effectively used to pasteurize salsa and that the effect of inactivation is dependent on frequency and electrical conductivity rather than waveform.

  11. Effect of Frequency and Waveform on Inactivation of Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium in Salsa by Ohmic Heating

    PubMed Central

    Lee, Su-Yeon; Ryu, Sangryeol

    2013-01-01

    The effect of frequency of alternating current during ohmic heating on electrode corrosion, heating rate, inactivation of food-borne pathogens, and quality of salsa was investigated. The impact of waveform on heating rate was also investigated. Salsa was treated with various frequencies (60 Hz to 20 kHz) and waveforms (sine, square, and sawtooth) at a constant electric field strength of 12.5 V/cm. Electrode corrosion did not occur when the frequency exceeded 1 kHz. The heating rate of the sample was dependent on frequency up to 500 Hz, but there was no significant difference (P > 0.05) in the heating rate when the frequency was increased above 1 kHz. The electrical conductivity of the sample increased with a rise in the frequency. At a frequency of 60 Hz, the square wave produced a lower heating rate than that of sine and sawtooth waves. The heating rate between waveforms was not significantly (P > 0.05) different when the frequency was >500 Hz. As the frequency increased, the treatment time required to reduce Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium to below the detection limit (1 log CFU/g) decreased without affecting product quality. These results suggest that ohmic heating can be effectively used to pasteurize salsa and that the effect of inactivation is dependent on frequency and electrical conductivity rather than waveform. PMID:23023752

  12. Combined effect of ultrasound, heat, and pressure on Escherichia coli O157:H7, polyphenol oxidase activity, and anthocyanins in blueberry (Vaccinium corymbosum) juice.

    PubMed

    Zhu, Jinyan; Wang, Yuehua; Li, Xinghe; Li, Bin; Liu, Suwen; Chang, Nan; Jie, Ding; Ning, Chong; Gao, Haiyan; Meng, Xianjun

    2017-07-01

    The objective of this study was to evaluate the effect of different treatments-heat treatment (HT), sonication (SC), thermosonication (TS), manosonication (MS), manothermal (MT), and manothermosonication (MTS) on Escherichia coli O157:H7, polyphenol oxidase (PPO), and anthocyanin content in blueberry juice. First, samples were treated at different temperatures (30, 40, 50, 60, 70, and 80°C) and power intensities (280, 420, 560, and 700W) for 10min. Subsequently, samples were treated using combinations of power intensity and mild temperature for 10min. For further study, samples were treated using HT (80°C), TS (40°C, 560W), MT (350MPa, 40°C), MS (560W, 5min/350MPa), or MTS (560W, 5min, 40°C/350MPa, 40°C) for 5, 10, 15, 20min for each treatment, and the results compared between treatments. HT significantly reduced PPO activation (2.05% residual activity after only 5min), and resulted in a 2.00-log reduction in E. coli O157:H7 and an 85.25% retention of anthocyanin. Escherichia coli O157:H7 was slightly inactivated by TS after 5min (0.17-log reduction), while residual PPO activity was 23.36% and anthocyanin retention was 98.48%. However, Escherichia coli O157:H7 was rapidly inactivated by MTS (5.85-log reduction) after 5min, while anthocyanin retention was 97.49% and residual PPO activity dropped to 10.91%. The destruction of E. coli cells as a result of these treatments were confirmed using SEM and TEM. Therefore, a combination of sonication, high pressure, and mild heat allows the safety of blueberry juice to be maintained without compromising the retention of desirable antioxidant compounds. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Immunostimulant patches containing Escherichia coli LT enhance immune responses to DNA- and recombinant protein-based Alzheimer's disease vaccines.

    PubMed

    Davtyan, Hayk; Ghochikyan, Anahit; Hovakimyan, Armine; Petrushina, Irina; Yu, Jianmei; Flyer, David; Madsen, Peter Juul; Pedersen, Lars Ostergaard; Cribbs, David H; Agadjanyan, Michael G

    2014-03-15

    Immunotherapeutic approaches to treating Alzheimer's disease (AD) using vaccination strategies must overcome the obstacle of achieving adequate responses to vaccination in the elderly. Here we demonstrate for the first time that application of the Escherichia coli heat-labile enterotoxin adjuvant-laden immunostimulatory patches (LT-IS) dramatically enhances the onset and magnitude of immune responses to DNA- and protein-based vaccines for Alzheimer's disease following intradermal immunization via gene gun and conventional needles, respectively. Our studies suggest that the immune activation mediated by LT-IS offers improved potency for generating AD-specific vaccination responses that should be investigated as an adjuvant in the clinical arena.

  14. Potential pathogens, antimicrobial patterns and genotypic diversity of Escherichia coli isolates in constructed wetlands treating swine wastewater.

    PubMed

    Ibekwe, A M; Murinda, Shelton E; DebRoy, Chitrita; Reddy, Gudigopura B

    2016-02-01

    Escherichia coli populations originating from swine houses through constructed wetlands were analyzed for potential pathogens, antimicrobial susceptibility patterns, and genotypic diversity. Escherichia coli isolates (n = 493) were screened for the presence of the following virulence genes: stx1, stx2 and eae (Shiga toxin-producing E. coli [STEC]), heat-labile enterotoxin (LT) genes and heat stable toxin STa and STb (enterotoxigenic E. coli (ETEC), cytotoxin necrotizing factors 1 and 2 (cnf1 and cnf2 [necrotoxigenic E. coli- NTEC]), as well as O and H antigens, and the presence of the antibiotic resistance genes blaTEM, blaSHV, blaCMY-2, tet A, tet B, tet C, mph(A), aadA, StrA/B, sul1, sul2 and sul3. The commensal strains were further screened for 16 antimicrobials and characterized by BOX AIR-1 PCR for unique genotypes. The highest antibiotic resistance prevalence was for tetracycline, followed by erythromycin, ampicillin, streptomycin, sulfisoxazole and kanamycin. Our data showed that most of the isolates had high distribution of single or multidrug-resistant (MDR) genotypes. Therefore, the occurrence of MDR E. coli in the wetland is a matter of great concern due to possible transfer of resistance genes from nonpathogenic to pathogenic strains or vice versa in the environment.

  15. Characterization of Immunological Cross-Reactivity between Enterotoxigenic Escherichia coli Heat-Stable Toxin and Human Guanylin and Uroguanylin

    PubMed Central

    Taxt, Arne M.; Diaz, Yuleima; Bacle, Amélie; Grauffel, Cédric; Reuter, Nathalie; Aasland, Rein; Sommerfelt, Halvor

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC) expressing the heat-stable toxin (ST) (human-type [STh] and porcine-type [STp] variants) is among the five most important enteric pathogens in young children living in low- and middle-income countries. ST mediates diarrheal disease through activation of the guanylate cyclase C (GC-C) receptor and is an attractive vaccine target with the potential to confer protection against a wide range of ETEC strains. However, immunological cross-reactivity to the endogenous GC-C ligands guanylin and uroguanylin is a major concern because of the similarities to ST in amino acid sequence, structure, and function. We have investigated the presence of similar epitopes on STh, STp, guanylin, and uroguanylin by analyzing these peptides in eight distinct competitive enzyme-linked immunosorbent assays (ELISAs). A fraction (27%) of a polyclonal anti-STh antibody and an anti-STh monoclonal antibody (MAb) cross-reacted with uroguanylin, the latter with a 73-fold-lower affinity. In contrast, none of the antibodies raised against STp, one polyclonal antibody and three MAbs, cross-reacted with the endogenous peptides. Antibodies raised against guanylin and uroguanylin showed partial cross-reactivity with the ST peptides. Our results demonstrate, for the first time, that immunological cross-reactions between ST and the endogenous peptides can occur. However, the partial nature and low affinity of the observed cross-reactions suggest that the risk of adverse effects from a future ST vaccine may be low. Furthermore, our results suggest that this risk may be reduced or eliminated by basing an ST immunogen on STp or a selectively mutated variant of STh. PMID:24778111

  16. Regulation of intestinal guanylate cyclase by the heat-stable enterotoxin of Escherichia coli (STa) and protein kinase C.

    PubMed Central

    Crane, J K; Wehner, M S; Bolen, E J; Sando, J J; Linden, J; Guerrant, R L; Sears, C L

    1992-01-01

    The heat-stable enterotoxin of Escherichia coli (STa) stimulates membrane-bound guanylate cyclase in intestinal epithelium and induces fluid and ion secretion. Using the T84 human colon carcinoma cell line as a model, we observed that phorbol esters markedly enhanced STa-stimulated cyclic GMP accumulation in T84 cells (C. S. Weikel, C. L. Spann, C. P. Chambers, J. K. Crane, J. Linden, and E. L. Hewlett, Infect. Immun. 58:1402-1407, 1990). In this study we document that the phorbol ester treatment increases 125I-STa-binding sites as well as membrane-bound guanylate cyclase activity in T84 cells and provide evidence that both effects are mediated by phosphorylation. Guanylate cyclase activity was increased approximately 50% in membranes prepared from intact T84 cells treated with phorbol-12,13-dibutyrate (beta-PDB) and after treatment of homogenates with beta-PDB in a manner dependent on ATP, MgCl2, and cytosol. Similarly, treatment of membranes with purified bovine brain protein kinase C in the presence of appropriate cofactors and beta-PDB resulted in an increase in STa-stimulated guanylate cyclase activity of about 70%. Likewise, the number of 125I-STa-binding sites was increased by about 25 to 40% in membranes prepared from intact cells or homogenates treated with beta-PDB; no effect on binding affinity (Kd = 0.15 nM) was noted. These experiments suggest that protein kinase C may phosphorylate the STa receptor-guanylate cyclase or a closely related protein and increase guanylate cyclase activity. The stimulatory effects of protein kinase C on STa-sensitive guanylate cyclase are opposite in direction to the profound inhibitory effects of the kinase on atrial natriuretic peptide-stimulated guanylate cyclase, demonstrating differential regulation by protein kinases within the guanylate cyclase-receptor family. PMID:1360449

  17. Escherichia coli heat-stable toxin b impairs intestinal epithelial barrier function by altering tight junction proteins.

    PubMed

    Ngendahayo Mukiza, Clément; Dubreuil, J Daniel

    2013-08-01

    Escherichia coli heat-stable toxin b (STb) causes diarrhea in animals. STb binds to sulfatide, its receptor, and is then internalized. In the cytoplasm, through a cascade of events, STb triggers the opening of ion channels, allowing ion secretion and water loss and leading to diarrhea. Tight junctions (TJs) are well known for controlling paracellular traffic of ions and water by forming a physical intercellular barrier in epithelial cells, and some bacterial toxins are known to affect adversely TJs. The present study aimed at determining the effect of STb on TJs. T84 cells were treated for 24 h with purified STb and a nontoxic STb mutant (D30V). Transepithelial resistance (TER), paracellular flux marker, and confocal microscopy were used to analyze the effect of STb on TJs. Purified STb caused a significant reduction of TER parallel to an increase in paracellular permeability compared to the results seen in untreated cells or mutant D30V. The increased paracellular permeability was associated with a marked alteration of F-actin stress fibers. F-actin filament dissolution and condensation were accompanied by redistribution and/or fragmentation of ZO-1, claudin-1, and occludin. These changes were also observed following treatment of T84 cells with an 8-amino-acid peptide found in the STb sequence corresponding to a consensus sequence of Vibrio cholerae Zot toxin. These effects were not observed with a scrambled peptide or mutant D30V. Our findings indicate that STb induces epithelial barrier dysfunction through changes in TJ proteins that could contribute to diarrhea.

  18. Escherichia coli Heat-Stable Toxin b Impairs Intestinal Epithelial Barrier Function by Altering Tight Junction Proteins

    PubMed Central

    Ngendahayo Mukiza, Clément

    2013-01-01

    Escherichia coli heat-stable toxin b (STb) causes diarrhea in animals. STb binds to sulfatide, its receptor, and is then internalized. In the cytoplasm, through a cascade of events, STb triggers the opening of ion channels, allowing ion secretion and water loss and leading to diarrhea. Tight junctions (TJs) are well known for controlling paracellular traffic of ions and water by forming a physical intercellular barrier in epithelial cells, and some bacterial toxins are known to affect adversely TJs. The present study aimed at determining the effect of STb on TJs. T84 cells were treated for 24 h with purified STb and a nontoxic STb mutant (D30V). Transepithelial resistance (TER), paracellular flux marker, and confocal microscopy were used to analyze the effect of STb on TJs. Purified STb caused a significant reduction of TER parallel to an increase in paracellular permeability compared to the results seen in untreated cells or mutant D30V. The increased paracellular permeability was associated with a marked alteration of F-actin stress fibers. F-actin filament dissolution and condensation were accompanied by redistribution and/or fragmentation of ZO-1, claudin-1, and occludin. These changes were also observed following treatment of T84 cells with an 8-amino-acid peptide found in the STb sequence corresponding to a consensus sequence of Vibrio cholerae Zot toxin. These effects were not observed with a scrambled peptide or mutant D30V. Our findings indicate that STb induces epithelial barrier dysfunction through changes in TJ proteins that could contribute to diarrhea. PMID:23716609

  19. Heterologous expression of betaine aldehyde dehydrogenase gene from Ammopiptanthus nanus confers high salt and heat tolerance to Escherichia coli.

    PubMed

    Yu, Hao-Qiang; Wang, Ying-Ge; Yong, Tai-Ming; She, Yue-Hui; Fu, Feng-Ling; Li, Wan-Chen

    2014-10-01

    Betaine aldehyde dehydrogenase (BADH) catalyzes the synthesis of glycine betaine, a regulator of osmosis, and therefore BADH is considered to play a significant role in response of plants to abiotic stresses. Here, based on the conserved residues of the deduced amino acid sequences of the homologous BADH genes, we cloned the AnBADH gene from the xerophytic leguminous plant Ammopiptanthus nanus by using reverse transcription PCR and rapid amplification of cDNA ends. The full-length cDNA is 1,868 bp long without intron, and contains an open reading frame of 1512 bp, and 3'- and 5'-untranslated regions of 294 and 62 bp. It encodes a 54.71 kDa protein of 503 amino acids. The deduced amino acid sequence shares high homology, conserved amino acid residues and sequence motifs crucial for the function with the BADHs in other leguminous species. The sequence of the open reading frame was used to construct a prokaryotic expression vector pET32a-AnBADH, and transform Escherichia coli. The transformants expressed the heterologous AnBADH gene under the induction of isopropyl β-D-thiogalactopyranoside, and demonstrated significant enhancement of salt and heat tolerance under the stress conditions of 700 mmol L(-1) NaCl and 55°C high temperature. This result suggests that the AnBADH gene might play a crucial role in adaption of A. nanus to the abiotic stresses, and have the potential to be applied to transgenic operations of commercially important crops for improvement of abiotic tolerance.

  20. Patterns of loss of enterotoxigenicity by Escherichia coli isolated from adults with diarrhea: suggestive evidence for an interrelationship with serotype.

    PubMed Central

    Evans, D J; Evans, D G; DuPont, H L; Orskov, F; Orskov, I

    1977-01-01

    Enterotoxigenic Escherichia coli isolates obtained in Mexico from adult subjects with diarrhea and from healthy controls were examined for the production of heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT) after serial passage in the laboratory. Isolates were found to be either stable for the production of ST and LT or unstable with respect to ST, LT, or both. Unilateral loss of either ST or LT production allowed classification of E. coli isolates into four groups according to stability/instability of enterotoxin production. Fewer serotypes, with more representative isolates, were in group I (stable) than in group IV (completely unstable). Isolates from Dacca, Bangladesh, could be similarly classified into stability groups. There is an apparent relationship between serotype, stability of enterotoxin production, particularly LT, and isolation from diarrhea cases as opposed to isolation from healthy controls. PMID:328392

  1. 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.

  2. Detection of fimbrial and toxin genes in Escherichia coli and their prevalence in piglets with diarrhoea. The application of colony hybridization assay, polymerase chain reaction and phenotypic assays.

    PubMed

    Ojeniyi, B; Ahrens, P; Meyling, A

    1994-03-01

    Two different genotypic methods, colony hybridization and polymerase chain reaction (PCR), were applied to detect enterotoxin, verotoxin and fimbrial genes in 708 Escherichia coli (E. coli) strains from piglets with diarrhoea. The results were compared with those obtained by phenotypic methods. DNA fragments specific for each of the enterotoxins LT, STa and STb, the verotoxins VT1, VT2 and VT2v, and for each of the fimbrial genes K88 (F4), K99 (F5), 987P (F6), F41 and F107, respectively, were used as probes in a colony hybridization assay of the E. coli strains. A PCR assay was used as genotypic test for the verotoxin gene. An Enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies was performed to test for the presence of K88 and K99 fimbriae, and a Vero cell test was performed to test for verotoxin production. Toxin detection kits were applied to detect the E. coli heat-labile enterotoxin (LT) and the heat-stable (STa) enterotoxin. The correlation between the results obtained by genotypic and phenotypic methods was 97.7-100%. The prevalence of the different fimbrial and toxin genes in E. coli strains from piglets with neonatal and postweaning diarrhoea were recorded. The verotoxin and the fimbrial F107 genes were found to be more frequent in postweaning E. coli strains than in neonatal E. coli strains.

  3. Heterogeneous expression of DnaK gene from Alicyclobacillus acidoterrestris improves the resistance of Escherichia coli against heat and acid stress.

    PubMed

    Xu, Xixi; Jiao, Lingxia; Feng, Xin; Ran, Junjian; Liang, Xinhong; Zhao, Ruixiang

    2017-12-01

    Alicyclobacillus acidoterrestris, an acidophilic and thermophilic bacteria, is an important microbial resource for stress resistance genes screening. In this study, DnaK gene from A. acidoterrestris was subcloned to construct the recombinant plasmid pET28a-DnaK. The successful construction of the plasmid was verified by double-enzyme digestion and sequencing analysis. The recombinant plasmid was transformed into Escherichia coli BL21 and isopropy-β-D-thiogalactoside (IPTG) was used to induce recombinant E. coli to express DnaK gene. A 70 kD fusion protein was identified by SDS-PAGE, which suggested that DnaK gene from A. acidoterrestris was successfully expressed. The recombinant and wild BL21 were treated with high temperatures of 54, 56 and 58 °C at pH values of 5.0-7.0 to compare the effects of heterogeneous expression of the DnaK gene from A. acidoterrestris on the stress resistance. The experimental results showed that survival rate of recombinant BL21-DnaK has been improved considerably under heat and acid stresses in contrast with the wild BL21, and D-values of recombinant BL21 were 14.7-72% higher than that of wild BL21, which demonstrated that heterogeneous expression of DnaK gene from A. acidoterrestris could significantly enhance the resistance of host bacteria E. coli against heat and acid stresses.

  4. Efficacy of UV, acidified sodium hypochlorite, and mild heat for decontamination of surface and infiltrated Escherichia coli O157:H7 on green onions and baby spinach.

    PubMed

    Durak, M Zeki; Churey, John J; Worobo, Randy W

    2012-07-01

    Produce-associated foodborne illnesses outbreaks have highlighted the need for more effective decontamination methods to ensure the safety of fresh produce. The main objective of this study was to evaluate the individual and combined efficacies of germicidal UV light (12.5 to 500 mJ/cm(2)), acidified sodium hypochlorite (ASC 10 to 200 ppm), and mild heat (40 to 50°C) for decontaminating green onions and baby spinach infected with Escherichia coli O157:H7. Samples were inoculated by spot and dip inoculation methods to mimic surface and infiltrated E. coli O157:H7 contamination, respectively. In green onions and baby spinach, the individual efficacies of UV, ASC, and mild-heat treatments varied based on the produce type and contamination method. Following analysis of the efficacies of the single treatments, a combined treatment with 125 mJ/cm(2) UV and 200 ppm of ASC at 50°C was selected for spot-inoculated green onions, and a combined treatment with 125 mJ/cm(2) UV and 200 ppm of ASC at 20°C was selected for spot- and dip-inoculated baby spinach. While a >5-log reduction was achieved with the combination treatment for spot-inoculated green onions with an initial contamination level of 7.2 log CFU per spot, the same treatment reduced E. coli O157:H7 populations below the detection limit (<1 log) on green onions spot inoculated at a lower contamination level (4.3 log CFU per spot). On spot- and dip-inoculated baby spinach, the combined treatment reduced E. coli O157:H7 populations by 2.8 log CFU per spot and 2.6 log CFU/g, respectively. The combined treatment of 500 mJ/cm(2) UV and 200 ppm of ASC at 50°C selected for the decontamination of dip-inoculated green onions resulted in a 2.2-log CFU/g reduction. These findings suggest that when foodborne pathogens contaminate produce and subsequently infiltrate, attach to, or become localized into protected areas, the individual or combined applications of UV, ASC, and mild-heat treatments have limited decontamination

  5. Decontamination method using heat and relative humidity for radish seeds achieves a 7-log reduction of Escherichia coli O157:H7 without affecting product quality.

    PubMed

    Kim, Y B; Kim, H W; Song, M K; Rhee, M S

    2015-05-18

    We developed a novel decontamination method to inactivate Escherichia coli O157:H7 on radish seeds without adversely affecting seed germination or product quality. The use of heat (55, 60, and 65 °C) combined with relative humidity (RH; 25, 45, 65, 85, and 100%) for 24h was evaluated for effective microbial reduction and preservation of seed germination rates. A significant two-way interaction of heat and RH was observed for both microbial reduction and germination rate (P<0.0001). Increases in heat and RH were associated with corresponding reductions in E. coli O157:H7 and in germination rate (P<0.05). The order of lethality for the different treatments was generally as follows: no treatment <55 °C/25-65% RH ≒60 °C/25-45% RH ≒65 °C/25% RH <55 °C/85% RH =60 °C/65% RH <55 °C/100% RH =60 °C/85-100% RH =65 °C/45-100% RH. The most effective condition, 65 °C/45% RH, completely inactivated E. coli O157:H7 on the seeds (7.0 log CFU/g reduction) and had no significant effect on the germination rate (85.4%; P>0.05) or product quality. The method uses only heat and relative humidity without chemicals, and is thus applicable as a general decontamination procedure in spout producing plants where the use of growth chambers is the norm.

  6. Optimization of heat and relative humidity conditions to reduce Escherichia coli O157:H7 contamination and maximize the germination of radish seeds.

    PubMed

    Song, M K; Kim, H W; Rhee, M S

    2016-06-01

    We previously reported that a combination of heat and relative humidity (RH) had a marked bactericidal effect on Escherichia coli O157:H7 on radish seeds. Here, response surface methodology with a Box-Behnken design was used to build a model to predict reductions in E. coli O157:H7 populations based on three independent variables: heating temperature (55 °C, 60 °C, or 65 °C), RH (40%, 60%, and 80%), and holding time (8, 15, or 22 h). Optimum treatment conditions were selected using a desirability function. The predictive model for microbial reduction had a high regression coefficient (R(2) = 0.97), and the accuracy of the model was verified using validation data (R(2) = 0.95). Among the three variables examined, heating temperature (P < 0.0001) and RH (P = 0.004) were the most significant in terms of bacterial reduction and seed germination, respectively. The optimum conditions for microbial reduction (6.6 log reduction) determined by ridge analysis were as follows: 64.5 °C and 63.2% RH for 17.7 h. However, when both microbial reduction and germination rate were taken into consideration, the desirability function yielded optimal conditions of 65 °C and 40% RH for 8 h (6.6 log reduction in the bacterial population; 94.4% of seeds germinated). This study provides comprehensive data that improve our understanding of the effects of heating temperature, RH, and holding time on the E. coli O157:H7 population on radish seeds. Radish seeds can be exposed to these conditions before sprouting, which greatly increases the microbiological safety of the products.

  7. Influence of Low-Shear Modeled Microgravity on Heat Resistance, Membrane Fatty Acid Composition, and Heat Stress-Related Gene Expression in Escherichia coli O157:H7 ATCC 35150, ATCC 43889, ATCC 43890, and ATCC 43895

    PubMed Central

    Kim, H. W.

    2016-01-01

    ABSTRACT We previously showed that modeled microgravity conditions alter the physiological characteristics of Escherichia coli O157:H7. To examine how microgravity conditions affect bacterial heat stress responses, D values, membrane fatty acid composition, and heat stress-related gene expression (clpB, dnaK, grpE, groES, htpG, htpX, ibpB, and rpoH), E. coli O157:H7 ATCC 35150, ATCC 43889, ATCC 43890, and ATCC 43895 were cultured under two different conditions: low-shear modeled microgravity (LSMMG, an analog of spaceflight conditions) and normal gravity (NG, Earth-like conditions). When 24-h cultures were heated to 55°C, cells cultured under LSMMG conditions showed reduced survival compared with cells cultured under NG conditions at all time points (P < 0.05). D values of all tested strains were lower after LSMMG culture than after NG culture. Fourteen of 37 fatty acids examined were present in the bacterial membrane: nine saturated fatty acids (SFA) and five unsaturated fatty acids (USFA). The USFA/SFA ratio, a measure of membrane fluidity, was higher under LSMMG conditions than under NG conditions. Compared with control cells grown under NG conditions, cells cultured under LSMMG conditions showed downregulation of eight heat stress-related genes (average, −1.9- to −3.7-fold). The results of this study indicate that in a simulated space environment, heat resistance of E. coli O157:H7 decreased, and this might be due to the synergistic effects of the increases in membrane fluidity and downregulated relevant heat stress genes. IMPORTANCE Microgravity is a major factor that represents the environmental conditions in space. Since infectious diseases are difficult to deal with in a space environment, comprehensive studies on the behavior of pathogenic bacteria under microgravity conditions are warranted. This study reports the changes in heat stress resistance of E. coli O157:H7, the severe foodborne pathogen, under conditions that mimic microgravity. The results

  8. Bacteriostasis of Escherichia coli by milk. IV. The bacteriostatic antibody of human milk.

    PubMed Central

    Dolby, J. M.; Honour, P.

    1979-01-01

    Bacteriostatic activity for milk-sensitive and milk-resistant strains of Escherichia coli is reduced when IgA is removed from milk by precipitation. Lysozyme is not involved in bacteriostasis and can be removed without loss of activity; heavy bentonite absorption however removes some lactoferrin causing partial loss of activity. The heat-labile antigen eliciting bacteriostatic antibody for E. coli is present in milk-sensitive and milk-resistant strains and in some other Enterobacteriaceae, e.g. salmonella; it cross reacts with the antigen in others, e.g. proteus and enterobacter. The antibody is therefore likely to be present in all human milk as a result of the normal commensal gut flora and with widespread activity. PMID:385767

  9. Inactivation of viruses in labile blood derivatives. II. Physical methods

    SciTech Connect

    Horowitz, B.; Wiebe, M.E.; Lippin, A.; Vandersande, J.; Stryker, M.H.

    1985-11-01

    The thermal inactivation of viruses in labile blood derivatives was evaluated by addition of marker viruses (VSV, Sindbis, Sendai, EMC) to anti-hemophilic factor (AHF) concentrates. The rate of virus inactivation at 60 degrees C was decreased by at least 100- to 700-fold by inclusion of 2.75 M glycine and 50 percent sucrose, or 3.0 M potassium citrate, additives which contribute to retention of protein biologic activity. Nonetheless, at least 10(4) infectious units of each virus was inactivated within 10 hours. Increasing the temperature from 60 to 70 or 80 degrees C caused a 90 percent or greater loss in AHF activity. An even greater decline in the rate of virus inactivation was observed on heating AHF in the lyophilized state, although no loss in AHF activity was observed after 72 hours of heating at 60 degrees C. Several of the proteins present in lyophilized AHF concentrates displayed an altered electrophoretic mobility as a result of exposure to 60 degrees C for 24 hours. Exposure of lyophilized AHF to irradiation from a cobalt 60 source resulted in an acceptable yield of AHF at 1.0, but not at 2.0, megarads. At 1 megarad, greater than or equal to 6.0 logs of VSV and 3.3 logs of Sindbis virus were inactivated.

  10. Heat shock in Escherichia coli alters the protein-binding properties of the chaperonin groEL by inducing its phosphorylation.

    PubMed

    Sherman MYu; Goldberg, A L

    1992-05-14

    When bacterial or eukaryotic cells are exposed to high temperatures or other harsh conditions, they respond by synthesis of a specific set of heat-shock proteins. Certain heat-shock proteins such as groEL, called 'chaperonins', can prevent misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under harmful conditions. We report here a new aspect of the heat-shock response in Escherichia coli: at high temperatures a fraction of groEL becomes modified covalently, altering its interaction with unfolded proteins. The heat-modified form can be eluted with ATP from an unfolded protein more easily than normal groEL. The critical heat-induced modification seems to be phosphorylation, which is reversed on return to low temperature. Treatment of the modified groEL with phosphatases caused its apparent size, charge and binding properties to resemble those of the unmodified form. Thus during heat shock some groEL is reversibly phosphorylated, which allows its ATP-dependent release from protein substrates in the absence of its usual cofactor (groES), and probably promotes the repair of damaged polypeptides.

  11. Concentration-dependent inhibition of Escherichia coli O157:H7 and heterocyclic amines in heated ground beef patties by apple, olive, and onion powders and clove bud oil

    USDA-ARS?s Scientific Manuscript database

    Meats need to be sufficiently heated to inactivate foodborne pathogens such as Escherichia coli O157:H7. High-temperature heat treatment used to prepare well-done meats could, however, increase the formation of potentially carcinogenic heterocyclic amines (HCAs). The objective of this study was to d...

  12. Inactivation of Escherichia coli O157:H7 in fruit juices by combined treatments of citrus fruit essential oils and heat.

    PubMed

    Espina, Laura; Somolinos, María; Ouazzou, Abdenour Ait; Condón, Santiago; García-Gonzalo, Diego; Pagán, Rafael

    2012-09-17

    This work approaches the possibility of combining mild heat treatments with citrus fruit essential oils (EOs) to improve the effectiveness of heat treatments and thus to reduce treatment intensity. Concentrations between 10 and 200 μL/L of lemon, mandarin, or orange EO were tested at 54 °C for 10 min in laboratory media, determining that 200 μL/L of each EO was necessary to achieve a 5 log(10) reduction of the initial Escherichia coli O157:H7 concentration. A relationship could be established between sublethally injured cells after the heat treatment and inactivated cells after the combined process. In apple juice, the synergism in the inactivation of E. coli O157:H7 when adding 200 μL/L of lemon EO might suppose a reduction in the treatment temperature (of 4.5 °C) or in the treatment time (by 5.7 times) within the range of temperature assayed (54-60 °C). Addition of 75 μL/L of lemon EO was determined to achieve the same synergistic effect of the combined treatment when the initial inoculum was reduced from 3×10(7) to 3×10(4) CFU/mL. Since the addition of lemon EO did not decrease the hedonic acceptability of apple juice, the proposed combined treatment could be further studied and optimized for the production of new minimally processed juices. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Effect of Escherichia coli heat-stable enterotoxin, cholera toxin and theophylline on ion transport in porcine colon

    PubMed Central

    Argenzio, R. A.; Whipp, S. C.

    1981-01-01

    1. The effect of heat-stable enterotoxin (ST) of Escherichia coli, cholera toxin (CT), and theophylline (a phosphodiesterase inhibitor) on ion and water transport was studied with an in vivo isolated loop system of the pig colon. 2. All three agents abolished net Na absorption as a result of a decrease in the lumen to blood Na flux alone. With all three agents, net Cl absorption was reduced, but not abolished, and net HCO3 secretion was elicited. Luminal pCO2 was reduced with CT and theophylline from that observed in normal Ringer alone. 3. Theophylline resulted in a prompt and sustained increase in both cyclic adenosine monophosphate (cyclic AMP) and cyclic guanosine monophosphate (cyclic GMP) levels in colonic mucosa studied in vitro. ST selectively elevated cyclic GMP, whereas CT selectively elevated cyclic AMP. These responses paralleled the time course and magnitude of response of the transepithelial electrical potential difference (ψLB) measured in vivo. 4. Ion replacement studies in the presence or absence of theophylline showed that in the absence of Na, Cl absorption was slightly reduced and HCO3 secretion was elicited; no further additive effects of theophylline in the absence of luminal Na were observed. In the absence of luminal Cl, net Na absorption was abolished and HCO3 was absorbed; theophylline resulted in significant net Na and HCO3 secretion. Theophylline also increased ψLB in the absence of either luminal Na or Cl. 5. Results suggest that in the presence of theophylline or enterotoxin, the coupled Na—H and Cl—HCO3 exchange processes that are normally responsible for at least half of the net NaCl absorption by this tissue are interrupted. Active HCO3 secretion is observed and Cl absorption under these conditions can be entirely explained as a consequence of ψLB. Thus, these studies indicate that the colon may participate in the production of diarrhoea of enterotoxigenic origin. They also suggest an important functional role of cyclic

  14. Potentiating the Heat Inactivation of Escherichia coli O157:H7 in Ground Beef Patties by Natural Antimicrobials

    PubMed Central

    Surendran Nair, Meera; Lau, Patrick; Belskie, Kaylin; Fancher, Samantha; Chen, Chi-Hung; Karumathil, Deepti Prasad; Yin, Hsin-Bai; Liu, Yanyan; Ma, Fulin; Upadhyaya, Indu; Upadhyay, Abhinav; Mancini, Richard; Venkitanarayanan, Kumar

    2016-01-01

    Escherichia coli O157: H7 (EHEC) is a major foodborne pathogen largely transmitted to humans through the consumption of undercooked ground beef. This study investigated the efficacy of two food-grade, plant-derived antimicrobials, namely rutin (RT), and resveratrol (RV) with or without chitosan (CH) in enhancing EHEC inactivation in undercooked hamburger patties. Further, the effect of aforementioned treatments on beef color and lipid oxidation was analyzed. Additionally, the deleterious effects of these antimicrobial treatments on EHEC was determined using scanning electron microscopy (SEM). Ground beef was inoculated with a five-strain mixture of EHEC (7.0 log CFU/g), followed by the addition of RT (0.05%, 0.1% w/w) or RV (0.1, 0.2% w/w) with or without CH (0.01% w/w). The meat was formed into patties (25 g) and stored at 4°C for 5 days. On days 1, 3, and 5, the patties were cooked (65°C, medium rare) and surviving EHEC was enumerated. The effect of these treatments on meat color and lipid oxidation during storage was also determined as per American Meat Science Association guidelines. The study was repeated three times with duplicate samples of each treatment. Both RT and RV enhanced the thermal destruction of EHEC, and reduced the pathogen load by at least 3 log CFU/g compared to control (P < 0.05). The combination of RT or RV with CH was found to be more effective, and reduced EHEC by 5 log CFU/g (P < 0.05). EHEC counts in uncooked patties did not decline during storage for 5 days (P > 0.05). Moreover, patties treated with RV plus CH were more color stable with higher a∗ values (P < 0.05). SEM results revealed that heat treatment with antimicrobials (CH + RV 0.2%) resulted in complete destruction of EHEC cells and extrusion of intracellular contents. Results suggest that the aforementioned antimicrobials could be used for enhancing the thermal inactivation of EHEC in undercooked patties; however, detailed sensory studies are warranted. PMID:26870000

  15. Potentiating the Heat Inactivation of Escherichia coli O157:H7 in Ground Beef Patties by Natural Antimicrobials.

    PubMed

    Surendran Nair, Meera; Lau, Patrick; Belskie, Kaylin; Fancher, Samantha; Chen, Chi-Hung; Karumathil, Deepti Prasad; Yin, Hsin-Bai; Liu, Yanyan; Ma, Fulin; Upadhyaya, Indu; Upadhyay, Abhinav; Mancini, Richard; Venkitanarayanan, Kumar

    2016-01-01

    Escherichia coli O157: H7 (EHEC) is a major foodborne pathogen largely transmitted to humans through the consumption of undercooked ground beef. This study investigated the efficacy of two food-grade, plant-derived antimicrobials, namely rutin (RT), and resveratrol (RV) with or without chitosan (CH) in enhancing EHEC inactivation in undercooked hamburger patties. Further, the effect of aforementioned treatments on beef color and lipid oxidation was analyzed. Additionally, the deleterious effects of these antimicrobial treatments on EHEC was determined using scanning electron microscopy (SEM). Ground beef was inoculated with a five-strain mixture of EHEC (7.0 log CFU/g), followed by the addition of RT (0.05%, 0.1% w/w) or RV (0.1, 0.2% w/w) with or without CH (0.01% w/w). The meat was formed into patties (25 g) and stored at 4°C for 5 days. On days 1, 3, and 5, the patties were cooked (65°C, medium rare) and surviving EHEC was enumerated. The effect of these treatments on meat color and lipid oxidation during storage was also determined as per American Meat Science Association guidelines. The study was repeated three times with duplicate samples of each treatment. Both RT and RV enhanced the thermal destruction of EHEC, and reduced the pathogen load by at least 3 log CFU/g compared to control (P < 0.05). The combination of RT or RV with CH was found to be more effective, and reduced EHEC by 5 log CFU/g (P < 0.05). EHEC counts in uncooked patties did not decline during storage for 5 days (P > 0.05). Moreover, patties treated with RV plus CH were more color stable with higher a(∗) values (P < 0.05). SEM results revealed that heat treatment with antimicrobials (CH + RV 0.2%) resulted in complete destruction of EHEC cells and extrusion of intracellular contents. Results suggest that the aforementioned antimicrobials could be used for enhancing the thermal inactivation of EHEC in undercooked patties; however, detailed sensory studies are warranted.

  16. Inactivation of Salmonella enterica serovar Typhimurium and Escherichia coli O157:H7 in peanut butter cracker sandwiches by radio-frequency heating.

    PubMed

    Ha, Jae-Won; Kim, Sung-Youn; Ryu, Sang-Ryeol; Kang, Dong-Hyun

    2013-05-01

    A multistate outbreak of Salmonella enterica serovar Typhimurium recently occurred in the USA, which was traced back to various food products made with contaminated peanut butter. This study was conducted to investigate the efficacy of radio-frequency (RF) heating to inactivate S. Typhimurium and Escherichia coli O157:H7 in peanut butter cracker sandwiches using creamy and chunky commercial peanut butter and to determine the effect on quality by measuring color changes and sensory evaluation. Samples were treated for a maximum time of 90 s in a 27.12 MHz RF heating system. Samples were prepared in the form of peanut butter cracker sandwiches and placed in the middle of two parallel-plate electrodes. After 90 s of RF treatment, the log reductions of S. Typhimurium and E. coli O157:H7 were 4.29 and 4.39 log CFU/g, respectively, in creamy peanut butter. RF treatment of chunky peanut butter for 90 s also significantly (P < 0.05) reduced levels of S. Typhimurium and E. coli O157:H7 by 4.55 log CFU/g and 5.32 log CFU/g. Color values and sensory characteristics of the RF treated peanut butter and crackers were not significantly (P > 0.05) different from the control. These results suggest that RF heating can be applied to control pathogens in peanut butter products without affecting quality.

  17. Lability of Secondary Organic Particulate Matter

    SciTech Connect

    Liu, Pengfei; Li, Yong Jie; Wang, Yan; Giles, Mary K.; Zaveri, Rahul A.; Bertram, Allan K.; Martin, Scot T.

    2016-10-24

    Accurate simulations of the consenctrations of atmospheric organic particulate matter (PM) are needed for predicting energy flow in the Earth’s climate system. In the past, simulations of organic PM widely assume equilibrium partitioning of semivolatile organic compounds (SVOCs) between the PM and surrounding vapor. Herein, we test this assumption by measuring evaporation rates and associated vapor mass concentration of organic films representative of atmospheric PM. For films representing anthropogenic PM, evaporation rates and vapor mass concentrations increased above a threshold relative humidity (RH), indicating equilibrium partitioning above a transition RH but not below. In contrast for films representing biogenic PM, no threshold was observed, indicating equilibrium partitioning at all RHs. The results suggest that the mass lability of atmospheric organic PM can differ in consequential ways among Earth’s natural biomes, polluted regions, and regions of land-use change, and these differences need to be considered when simulating atmospheric organic PM.

  18. Fate and lability of silver in soils: Effect of ageing

    EPA Science Inventory

    The fate and lability of added soluble Ag in soils over time was examined by measurement of labile metal (E-value) by isotopic dilution using the 110mAg radioactive isotope and the solid-phase speciation of Ag by X-ray absorption near edge structure (XANES) spectrosco...

  19. THE ASSAY AND PROPERTIES OF LABILE FACTOR (FACTOR V)

    PubMed Central

    Quick, Armand J.

    1960-01-01

    Human oxalated plasma stored at 4° C. until the prothrombin time is increased beyond 60 sec. is a reliable medium for assaying labile factor (factor V) because its response to added labile factor corresponds quantitatively to that of plasma from patients with congenital deficiency of this factor. Such an agreement is not obtained with plasma stored at 37°C. The stability of labile factor is closely associated with ionized calcium. The addition of thrombin to fresh oxalated plasma causes an apparent hyperactivity of labile factor, but this is completely removed by adsorption with Ca3(PO)2. Oxalated plasma when adsorbed with Ca3(PO4)2 before treatment with thrombin does not develop this adventitious activity, nor does it occur in stored plasma treated with thrombin. The seemingly high labile factor activity in serum can be explained by the activation of this factor which is independent of labile factor but acts synergistically with it. The true labile factor concentration can be determined only after the accelerator is removed by adsorption with Ca3(PO4)2. A close agreement between the consumption of prothrombin and the loss of labile factor during clotting is observed. PMID:13738700

  20. Fate and lability of silver in soils: Effect of ageing

    EPA Science Inventory

    The fate and lability of added soluble Ag in soils over time was examined by measurement of labile metal (E-value) by isotopic dilution using the 110mAg radioactive isotope and the solid-phase speciation of Ag by X-ray absorption near edge structure (XANES) spectrosco...

  1. Effect of Power Levels on Inactivation of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes in Tomato Paste Using 915-Megahertz Microwave and Ohmic Heating.

    PubMed

    Kim, Sang-Soon; Sung, Hye-Jung; Kwak, Hyo-Sun; Joo, In-Sun; Lee, Jeong-Su; Ko, Gwangpyo; Kang, Dong-Hyun

    2016-09-01

    The effect of power levels on inactivation of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes in tomato paste was investigated using 915-MHz microwave heating (MW) and ohmic heating (OH). Heating uniformity, pathogen inactivation, and quality aspects were determined with 1.8-, 2.1-, 2.4-, and 3.0-kW MW and corresponding OH. GInaFit was used to analyze pathogen inactivation. The heating uniformity of MW-treated samples was inferior to that of OH-treated samples at low power levels of 1.8 to 2.4 kW but improved as the power level increased. Pathogen inactivation of MW-treated samples was significantly higher than that of OH-treated samples at low power levels of 1.8 to 2.4 kW (P < 0.05) but was not significantly different at the highest power level of 3.0 kW (P > 0.05). Quality aspects (color, pH, and lycopene content), except for L*, of MW-treated samples were not significantly degraded (P > 0.05) by increased power levels. Our results indicate that increasing power levels of MW ensures heating uniformity and microbiological safety and preserves quality aspects of tomato paste.

  2. Genetic mapping of the labile (lab) gene: a recessive locus causing irregular spikelet fertility in labile-barley (Hordeum vulgare convar. labile).

    PubMed

    Youssef, Helmy M; Koppolu, Ravi; Rutten, Twan; Korzun, Viktor; Schweizer, Patrick; Schnurbusch, Thorsten

    2014-05-01

    The recessive labile locus mapped on chromosome 5HL causes irregular spikelet fertility and controls floret development as well as row-type in barley. The labile-barley displays a variable number of fertile spikelets at each rachis internode (0-3 fertile spikelets/rachis internode) which is intermediate between that observed in two- or six-rowed types. Previous re-sequencing of Vrs1 in 219 labile-barley (Hordeum vulgare L. convar. labile) accessions showed that all carried a six-rowed specific allele. We therefore hypothesized that this seemingly random reduction in spikelet fertility is most likely caused by the labile (lab) locus, which we aimed to phenotypically and genetically define. Here, we report a detailed phenotypic analysis of spikelet fertility in labile-barleys in comparison to two- and six-rowed genotypes using scanning electron microscopy analysis. We found that the first visible morphological deviation occurred during the stamen primordium stage, when we regularly observed the appearance of arrested central floral primordia in labile but not in two- or six-rowed barleys. At late stamen and early awn primordium stages, lateral florets in two-rowed and only some in labile-barley showed retarded development and reduction in size compared with fully fertile lateral florets in six-rowed barley. We used two F2 mapping populations to generate whole genome genetic linkage maps and ultimately locate the lab locus as a recessive Mendelian trait to a 4.5-5.8 cM interval at approximately 80 cM on chromosome 5HL. Our results will help identifying the role of the lab gene in relation to other spikelet fertility factors in barley.

  3. Natural and experimental infection with an attaching and effacing strain of Escherichia coli in calves.

    PubMed Central

    Moxley, R A; Francis, D H

    1986-01-01

    Gnotobiotic calves were inoculated with an O5:K4:H-, urease-positive strain of Escherichia coli isolated from a 2-day-old calf with diarrhea. The calves developed elevated temperatures and passed loose mucoid feces, with or without blood. The E. coli strain was negative for heat-stable and heat-labile enterotoxins but produced high levels of Shiga-like toxin. Bacteria attached diffusely to the epithelium of the large intestine and multifocally to the epithelium of the ileum. The duodenum and jejunum were not affected. At the sites of bacterial attachment, microvilli were effaced, enterocytes were degenerate, and necrosis and exfoliation had occurred. These results confirm a previous report from England that calves may naturally contract infections similar to those caused by enteropathogenic E. coli strains pathogenic to humans or rabbits. This suggests that the calf bacterial strains, like some enteropathogenic E. coli strains, produce high levels of Shiga-like toxin and cause attachment and effacement lesions in the colonic epithelium of the infected host. Images PMID:3525410

  4. Inactivation of Escherichia coli O157:H7 on Radish Seeds by Sequential Treatments with Chlorine Dioxide, Drying, and Dry Heat without Loss of Seed Viability ▿

    PubMed Central

    Bang, Jihyun; Kim, Haeyoung; Kim, Hoikyung; Beuchat, Larry R.; Ryu, Jee-Hoon

    2011-01-01

    We developed and validated a treatment to inactivate Escherichia coli O157:H7 on radish seeds without decreasing seed viability. Treatments with aqueous ClO2 followed by drying and dry-heat treatments were evaluated for efficacy to inactivate the pathogen. Conditions to dry radish seeds after treatment with water (control) or ClO2 were established. When treated seeds with high water activity (aw) (>0.99) were stored at 45°C and 23% relative humidity (RH), the aw decreased to <0.30 within 24 h. Drying high-aw seeds before exposing them to dry-heat treatment (≥60°C) was essential to preserve seed viability. The germination rate of radish seeds which had been immersed in water for 5 min, dried at 45°C and 23% RH for 24 h, and heated at 70°C for 48 h or at 80°C for 24 h was not significantly decreased (P ≤ 0.05) compared to that of untreated radish seeds. Sequential treatments with ClO2 (500 μg/ml, 5 min), drying (45°C, 23% RH, 24 h), and dry heating (70°C, 23% RH, 48 h) eliminated E. coli O157:H7 (5.9 log CFU/g) on radish seeds and, consequently, sprouts produced from them without decreasing the germination rate. These sequential treatments are recommended for application to radish seeds intended for sprout production. PMID:21803896

  5. Survival of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. on catfish fillets exposed to microwave heating in a continuous mode.

    PubMed

    Sheen, Shiowshuh; Huang, Lihan; Sommers, Christopher

    2012-08-01

    Microwave (MW) heating using continuous power output with feedback control and a modified ingredient formulation may provide better and consistent cooking of foods. Currently, household units with build-in inverter power supply units are available. These new generation MW ovens provide continuous, adjustable output and cooking, in contrast to the traditional rectifier-based ovens that rely on the on-off mechanism for control. This study attempted to apply a feedback power control (termed as modified or "smart" MW oven) and phosphate treatment to further improve heating uniformity and enhance food quality and safety. Listeria monocytogenes (Lm, 4-strain cocktail), Escherichia coli O157:H7 (Ec, 5-strain cocktail), and Salmonella spp. (Sal, 6-strain cocktail), surface inoculated onto catfish fillets (75 × 100 × 15 mm; weight 110 g), were heated using the modified MW oven to study the inactivation of the pathogens. The sensitivity of these 3 bacteria to MW heating was in the order of Ec (most), Lm, and Sal (least). Greater than 4 to 5 log CFU reductions of Ec, Lm, or Sal counts on catfish fillet surfaces were inactivated within 2 min of 1250 W MW heating, where the fillet surface temperature increased from 10 to 20 °C to 80 to 90 °C. MW heating caused degradation of catfish fillet texture, which was noticeable as early as 10 to 15 s after the heating started, as evidenced by bumping sounds. Bumping can be significantly reduced by soaking fillets in phosphate solution. However, the results may need verification if applied in different MW ovens and/or with foods positioned away the geometric oven center. This study successfully demonstrated the feasibility of applying MW energy to eliminate foodborne pathogens on fish fillets. The results demonstrated in this report with the "smart" microwave oven design may enhance microwaveable food safety and quality, and therefore promote the microwaveable food business. © 2012 Institute of Food Technologists®

  6. A biomarker for the identification of cattle fecal pollution in water using the LTIIa toxin gene from enterotoxigenic Escherichia coli.

    PubMed

    Khatib, L A; Tsai, Y L; Olson, B H

    2002-06-01

    This research describes a method based on PCR to identify cattle fecal pollution in water using a portion of the heat labile toxin IIA (LTIIa) gene from enterotoxigenic Escherichia coli (ETEC). We describe the development of the primers and target. DNA extracts (221) from different animal fecal and human sewage samples were screened and showed no cross-reactivity. Minimum detection limits using centrifugation and filtration methods to concentrate E. coli seeded into stream, ocean, and secondary effluent waters were found to be at femtogram and attogram levels, respectively. Stability of the biomarker in stream, ocean, and secondary effluent waters was 2-4 weeks for all water types. Finally, 33 farm lagoon and waste samples were collected and 31 tested to validate the method; 93% were positive for the LTIIa trait when >1,000 E. coli were screened and 100% positive when >10(5) E. coli were screened. Prevalence of the toxin gene in the E. coli population affected the outcome of the analyses. The cow biomarker can be used in watershed studies to identify cattle waste with great accuracy if the appropriate numbers of E. coli are screened.

  7. A heat shock operon in Coxiella burnetti produces a major antigen homologous to a protein in both mycobacteria and Escherichia coli.

    PubMed Central

    Vodkin, M H; Williams, J C

    1988-01-01

    A gene library from the DNA of Coxiella burnetii has been constructed in the cosmid vector pHC79. A particular clone, pJB196, reacted strongly with Coxiella-specific antibodies elicited in a number of different species of animals. This clone produced two abundant C. burnetii-specific polypeptides, a 14-kilodalton nonimmunoreactive protein and a 62-kilodalton immunoreactive protein. Sequencing identified two open reading frames, encoding polypeptides of 10.5 and 58.3 kilodaltons. The only transcriptional control element observed on the 5' side of the initiation codon resembled a heat shock promoter. This heat shock promoter was functionally regulated in Escherichia coli, since both proteins were produced by growth conditions at 37 degrees C and neither protein was detected at 23 degrees C. There were four sequences from the literature that were highly homologous (greater than 50%) to the 62-kilodalton protein from C. burnetii. Three were from Mycobacterium species and represent the immunodominant antigen of this genus. The other was from E. coli, detected as a gene that complements or suppresses a temperature-sensitive RNase activity. Since the recombinant protein was immunogenic, it may serve as an efficacious vaccine against C. burnetii and other pathogenic microorganisms that express the conserved antigen. Images PMID:3343219

  8. Native lysozyme and dry-heated lysozyme interactions with membrane lipid monolayers: lateral reorganization of LPS monolayer, model of the Escherichia coli outer membrane.

    PubMed

    Derde, Melanie; Nau, Françoise; Lechevalier, Valérie; Guérin-Dubiard, Catherine; Paboeuf, Gilles; Jan, Sophie; Baron, Florence; Gautier, Michel; Vié, Véronique

    2015-01-01

    Lysozyme is mainly described active against Gram-positive bacteria, but is also efficient against some Gram-negative species. Especially, it was recently demonstrated that lysozyme disrupts Escherichia coli membranes. Moreover, dry-heating changes the physicochemical properties of the protein and increases the membrane activity of lysozyme. In order to elucidate the mode of insertion of lysozyme into the bacterial membrane, the interaction between lysozyme and a LPS monolayer mimicking the E. coli outer membrane has been investigated by tensiometry, ellipsometry, Brewster angle microscopy and atomic force microscopy. It was thus established that lysozyme has a high affinity for the LPS monolayer, and is able to insert into the latter as long as polysaccharide moieties are present, causing reorganization of the LPS monolayer. Dry-heating increases the lysozyme affinity for the LPS monolayer and its insertion capacity; the resulting reorganization of the LPS monolayer is different and more drastic than with the native protein. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. The effect of heating rate on Escherichia coli metabolism, physiological stress, transcriptional response, and production of temperature-induced recombinant protein: a scale-down study.

    PubMed

    Caspeta, Luis; Flores, Noemí; Pérez, Néstor O; Bolívar, Francisco; Ramírez, Octavio T

    2009-02-01

    At the laboratory scale, sudden step increases from 30 to 42 degrees C can be readily accomplished when expressing heterologous proteins in heat-inducible systems. However, for large scale-cultures only slow ramp-type increases in temperature are possible due to heat transfer limitations, where the heating rate decreases as the scale increases. In this work, the transcriptional and metabolic responses of a recombinant Escherichia coli strain to temperature-induced synthesis of pre-proinsulin in high cell density cultures were examined at different heating rates. Heating rates of 6, 1.7, 0.8, and 0.4 degrees C/min were tested in a scale-down approach to mimic fermentors of 0.1, 5, 20, and 100 m(3), respectively. The highest yield and concentration of recombinant protein was obtained for the slowest heating rate. As the heating rate increased, the yield and maximum recombinant protein concentration decreased, whereas a larger fraction of carbon skeletons was lost as acetate, lactate, and formate. Compared to 30 degrees C, the mRNA levels of selected heat-shock genes at 38 and 42 degrees C, as quantified by qRT-PCR, increased between 2- to over 42-fold when cultures were induced at 6, 1.7, and 0.8 degrees C/min, but no increase was observed at 0.4 degrees C/min. Only small increases (between 1.5- and 4-fold) in the expression of the stress genes spoT and relA were observed at 42 degrees C for cultures induced at 1.7 and 6 degrees C/min, suggesting that cells subjected to slow temperature increases can adapt to stress. mRNA levels of genes from the transcription-translation machinery (tufB, rpoA, and tig) decreased between 40% and 80% at 6, 1.7 and 0.8 degrees C/min, whereas a transient increase occurred for 0.4 degrees C/min at 42 degrees C. mRNA levels of the gene coding for pre-proinsulin showed a similar profile to transcripts of heat-shock genes, reflecting a probable analogous induction mechanism. Altogether, the results obtained indicate that slow heating rates

  10. Generating controlled reducing environments in aerobic recombinant Escherichia coli fermentations: effects on cell growth, oxygen uptake, heat shock protein expression, and in vivo CAT activity.

    PubMed

    Gill, R T; Cha, H J; Jain, A; Rao, G; Bentley, W E

    1998-07-20

    The independent control of culture redox potential (CRP) by the regulated addition of a reducing agent, dithiothreitol (DTT) was demonstrated in aerated recombinant Escherichia coli fermentations. Moderate levels of DTT addition resulted in minimal changes to specific oxygen uptake, growth rate, and dissolved oxygen. Excessive levels of DTT addition were toxic to the cells resulting in cessation of growth. Chloramphenicol acetyltransferase (CAT) activity (nmoles/microgram total protein min.) decreased in batch fermentation experiments with respect to increasing levels of DTT addition. To further investigate the mechanisms affecting CAT activity, experiments were performed to assay heat shock protein expression and specific CAT activity (nmoles/microgram CAT min.). Expression of such molecular chaperones as GroEL and DnaK were found to increase after addition of DTT. Additionally, sigma factor 32 (sigma32) and several proteases were seen to increase dramatically during addition of DTT. Specific CAT activity (nmoles/microgram CAT min. ) varied greatly as DTT was added, however, a minimum in activity was found at the highest level of DTT addition in E. coli strains RR1 [pBR329] and JM105 [pROEX-CAT]. In conjunction, cellular stress was found to reach a maximum at the same levels of DTT. Although DTT addition has the potential for directly affecting intracellular protein folding, the effects felt from the increased stress within the cell are likely the dominant effector. That the effects of DTT were measured within the cytoplasm of the cell suggests that the periplasmic redox potential was also altered. The changes in specific CAT activity, molecular chaperones, and other heat shock proteins, in the presence of minimal growth rate and oxygen uptake alterations, suggest that the ex vivo control of redox potential provides a new process for affecting the yield and conformation of heterologous proteins in aerated E. coli fermentations.

  11. Lability of secondary organic particulate matter

    PubMed Central

    Liu, Pengfei; Li, Yong Jie; Wang, Yan; Gilles, Mary K.; Zaveri, Rahul A.; Bertram, Allan K.

    2016-01-01

    The energy flows in Earth’s natural and modified climate systems are strongly influenced by the concentrations of atmospheric particulate matter (PM). For predictions of concentration, equilibrium partitioning of semivolatile organic compounds (SVOCs) between organic PM and the surrounding vapor has widely been assumed, yet recent observations show that organic PM can be semisolid or solid for some atmospheric conditions, possibly suggesting that SVOC uptake and release can be slow enough that equilibrium does not prevail on timescales relevant to atmospheric processes. Herein, in a series of laboratory experiments, the mass labilities of films of secondary organic material representative of similar atmospheric organic PM were directly determined by quartz crystal microbalance measurements of evaporation rates and vapor mass concentrations. There were strong differences between films representative of anthropogenic compared with biogenic sources. For films representing anthropogenic PM, evaporation rates and vapor mass concentrations increased above a threshold relative humidity (RH) between 20% and 30%, indicating rapid partitioning above a transition RH but not below. Below the threshold, the characteristic time for equilibration is estimated as up to 1 wk for a typically sized particle. In contrast, for films representing biogenic PM, no RH threshold was observed, suggesting equilibrium partitioning is rapidly obtained for all RHs. The effective diffusion rate Dorg for the biogenic case is at least 103 times greater than that of the anthropogenic case. These differences should be accounted for in the interpretation of laboratory data as well as in modeling of organic PM in Earth’s atmosphere. PMID:27791063

  12. Attributional lability in depression and paranoia.

    PubMed

    Bentall, Richard P; Kaney, Sue

    2005-11-01

    Attributions play an important role in depression and paranoia. However, contrary to most attributional models of psychopathology, there is evidence that attributional style is not a stable trait but is affected by recent experiences. Paranoid, depressed, and healthy participants were exposed to a mild stressor in the form of the requirement to complete an anagram task, which included insoluble items. Before and afterwards, they completed measures of attributional style for negative events and a contingency judgment task. Replicating previous findings, paranoid patients initially showed low internality scores for negative events and high judgments of control on the contingency judgment task, whereas the depressed participants showed high internality for negative events and low judgments of control. Following the anagram task, both clinical groups showed increased internality for hypothetical negative events. The paranoid participants also gave increased estimates of control on the contingency judgment task. Attributional style is more labile in paranoid and depressed patients than in healthy individuals. Implications for attributional theories of psychopathology are discussed.

  13. Micellar electrokinetic chromatography with acid labile surfactant.

    PubMed

    Stanley, Bob; Lucy, Charles A

    2012-02-24

    We present a study of a degradable surfactant, sodium 4-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propane sulfonate that is also known as an acid-labile surfactant (ALS). The performance of ALS as a pseudostationary phase is assessed and compared with established pseudostationary phases such as sodium dodecyl sulphate (SDS), volatile surfactants and polymeric micelles. ALS achieves separation efficiency of 100,000-145,000 theoretical plates and relative standard deviation (RSD) of electrophoretic mobility (n=5) of less than 3%. Retention factors with ALS are strongly correlated with those with SDS. This is shown by the R2=0.79 for all eleven analytes and an R2=0.992 for specifically the non-hydrogen bonding (NHB) analytes. However, ALS displays different selectivity than SDS for hydrogen bond donor (HBD) and hydrogen bond acceptor (HBA) solutes (R2 of 0.74 and 0.88, respectively). ALS is degraded to less surface active compounds in acidic solution. These less surface-active compounds are more compatible with the electrospray ionization mass spectrometry (ESI-MS). ALS has a half-life of 48 min at pH 4. ALS has the potential to couple micellar electrokinetic chromatography (MEKC) with the ESI-MS. ALS can be used as a pseudostationary phase for a high efficiency separation and later acid hydrolyzed to enable an ESI-MS analysis. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Practical use of labile protein as an index of hair.

    PubMed

    Inoue, Takafumi; Kizawa, Kenji; Ito, Mayumi; Shinkai, Masakazu; Iwamoto, Yoshimichi

    2004-01-01

    Because of small fluctuations, it is difficult to evaluate hair damage caused by bleaching using previously utilized hair damage indexes. Application of commercial bleaching products elevates partially extractable labile hair protein amounts in the range of 0.4-1.2 mg/g of hair. Within this range, the level of labile protein fluctuates greatly, depending on the extent of bleaching. In the current study, it was found that the effects of alkaline constituents and various peptides contained in bleaching lotions on hair damage could be evaluated by measuring labile protein amounts without employing harsher bleaching conditions.

  15. Use of caprylic acid to control pathogens (Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium) in apple juice at mild heat temperature.

    PubMed

    Kim, S A; Rhee, M S

    2015-11-01

    The aim of this study was to examine the effects of caprylic acid (CA) on pathogens in apple juice having intrinsic organic acids, and to determine any synergistic effects. Bactericidal effects of CA were examined against Escherichia coli O157:H7 and Salmonella Typhimurium present in apple juice at mild heating temperatures. Apple juice containing each of the pathogens was treated with CA (0·1, 0·2, 0·4, 0·6 or 0·8 mmol l(-1)) at 50 or 55°C. Treatment with 0·8 mmol l(-1) (0·013%) CA at 50°C for 5 min or with 0·6 mmol l(-1) (0·010%) CA at 55°C for 5 min resulted in the complete eradication of E. coli O157:H7 (initial population: 7·25-7·34 log CFU ml(-1)). Salmonella Typhimurium were more sensitive than E. coli O157:H7: all bacteria (7·81-7·55 log CFU ml(-1)) were eradicated by treatment with 0·2 mmol l(-1) (0·0032%) CA at 55°C for 5 min or with 0·6 mmol l(-1) CA at 50°C for 5 min. By contrast, when pH-adjusted apple juice (pH 7·0) was treated with 0·8 mmol l(-1) CA, there was no significant difference in bactericidal effects between CA-treated samples and controls (heat treatment alone or heat + 0·1% ethanol treatment). This result suggested that acidic pH in the apple juice boost the antibacterial effects of CA. CA treatment did not affect (P > 0·05) the pH, colour or °Brix of the apple juice. This study highlights the utility of CA as a natural antibacterial agent that can eliminate micro-organisms from apple juice at very low concentrations (≤0·013%) and temperatures (≤55°C) within a short time (≤10 min). The results of our study may contribute to the development of an efficient method for improving the microbiological safety of apple juice. © 2015 The Society for Applied Microbiology.

  16. Lability of secondary organic particulate matter

    SciTech Connect

    Liu, Pengfei; Li, Yong Jie; Wang, Yan; Gilles, Mary K.; Zaveri, Rahul A.; Bertram, Allan K.; Martin, Scot T.

    2016-10-24

    We report the energy flows in Earth’s natural and modified climate systems are strongly influenced by the concentrations of atmospheric particulate matter (PM). For predictions of concentration, equilibrium partitioning of semivolatile organic compounds (SVOCs) between organic PM and the surrounding vapor has widely been assumed, yet recent observations show that organic PM can be semisolid or solid for some atmospheric conditions, possibly suggesting that SVOC uptake and release can be slow enough that equilibrium does not prevail on timescales relevant to atmospheric processes. Herein, in a series of laboratory experiments, the mass labilities of films of secondary organic material representative of similar atmospheric organic PM were directly determined by quartz crystal microbalance measurements of evaporation rates and vapor mass concentrations. There were strong differences between films representative of anthropogenic compared with biogenic sources. For films representing anthropogenic PM, evaporation rates and vapor mass concentrations increased above a threshold relative humidity (RH) between 20% and 30%, indicating rapid partitioning above a transition RH but not below. Below the threshold, the characteristic time for equilibration is estimated as up to 1 wk for a typically sized particle. In contrast, for films representing biogenic PM, no RH threshold was observed, suggesting equilibrium partitioning is rapidly obtained for all RHs. The effective diffusion rate Dorg for the biogenic case is at least 103 times greater than that of the anthropogenic case. In conclusion, these differences should be accounted for in the interpretation of laboratory data as well as in modeling of organic PM in Earth’s atmosphere.

  17. Lability of secondary organic particulate matter

    DOE PAGES

    Liu, Pengfei; Li, Yong Jie; Wang, Yan; ...

    2016-10-24

    We report the energy flows in Earth’s natural and modified climate systems are strongly influenced by the concentrations of atmospheric particulate matter (PM). For predictions of concentration, equilibrium partitioning of semivolatile organic compounds (SVOCs) between organic PM and the surrounding vapor has widely been assumed, yet recent observations show that organic PM can be semisolid or solid for some atmospheric conditions, possibly suggesting that SVOC uptake and release can be slow enough that equilibrium does not prevail on timescales relevant to atmospheric processes. Herein, in a series of laboratory experiments, the mass labilities of films of secondary organic material representativemore » of similar atmospheric organic PM were directly determined by quartz crystal microbalance measurements of evaporation rates and vapor mass concentrations. There were strong differences between films representative of anthropogenic compared with biogenic sources. For films representing anthropogenic PM, evaporation rates and vapor mass concentrations increased above a threshold relative humidity (RH) between 20% and 30%, indicating rapid partitioning above a transition RH but not below. Below the threshold, the characteristic time for equilibration is estimated as up to 1 wk for a typically sized particle. In contrast, for films representing biogenic PM, no RH threshold was observed, suggesting equilibrium partitioning is rapidly obtained for all RHs. The effective diffusion rate Dorg for the biogenic case is at least 103 times greater than that of the anthropogenic case. In conclusion, these differences should be accounted for in the interpretation of laboratory data as well as in modeling of organic PM in Earth’s atmosphere.« less

  18. Advanced application of bovine intestinal epithelial cell line for evaluating regulatory effect of lactobacilli against heat-killed enterotoxigenic Escherichia coli-mediated inflammation

    PubMed Central

    2013-01-01

    Background Previously, a bovine intestinal epithelial cell line (BIE cells) was successfully established. This work hypothesized that BIE cells are useful in vitro model system for the study of interactions of microbial- or pathogen-associated molecular patterns (MAMPs or PAMPs) with bovine intestinal epithelial cells and for the selection of immunoregulatory lactic acid bacteria (LAB). Results All toll-like receptor (TLR) genes were expressed in BIE cells, being TLR4 one of the most strongly expressed. We demonstrated that heat-stable PAMPs of enterotoxigenic Escherichia coli (ETEC) significantly enhanced the production of IL-6, IL-8, IL-1α and MCP-1 in BIE cells by activating both NF-κB and MAPK pathways. We evaluated the capacity of several lactobacilli strains to modulate heat-stable ETEC PAMPs-mediated inflammatory response in BIE cells. Among these strains evaluated, Lactobacillus casei OLL2768 attenuated heat-stable ETEC PAMPs-induced pro-inflammatory response by inhibiting NF-κB and p38 signaling pathways in BIE cells. Moreover, L. casei OLL2768 negatively regulated TLR4 signaling in BIE cells by up-regulating Toll interacting protein (Tollip) and B-cell lymphoma 3-encoded protein (Bcl-3). Conclusions BIE cells are suitable for the selection of immunoregulatory LAB and for studying the mechanisms involved in the protective activity of immunobiotics against pathogen-induced inflammatory damage. In addition, we showed that L. casei OLL2768 functionally modulate the bovine intestinal epithelium by attenuating heat-stable ETEC PAMPs-induced inflammation. Therefore L. casei OLL2768 is a good candidate for in vivo studying the protective effect of LAB against intestinal inflammatory damage induced by ETEC infection or heat-stable ETEC PAMPs challenge in the bovine host. PMID:23497067

  19. Investigation of optimum ohmic heating conditions for inactivation of Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes in apple juice.

    PubMed

    Park, Il-Kyu; Ha, Jae-Won; Kang, Dong-Hyun

    2017-05-19

    Control of foodborne pathogens is an important issue for the fruit juice industry and ohmic heating treatment has been considered as one of the promising antimicrobial interventions. However, to date, evaluation of the relationship between inactivation of foodborne pathogens and system performance efficiency based on differing soluble solids content of apple juice during ohmic heating treatment has not been well studied. This study aims to investigate effective voltage gradients of an ohmic heating system and corresponding sugar concentrations (°Brix) of apple juice for inactivating major foodborne pathogens (E. coli O157:H7, S. Typhimurium, and L. monocytogenes) while maintaining higher system performance efficiency. Voltage gradients of 30, 40, 50, and 60 V/cm were applied to 72, 48, 36, 24, and 18 °Brix apple juices. At all voltage levels, the lowest heating rate was observed in 72 °Brix apple juice and a similar pattern of temperature increase was shown in18-48 °Brix juice samples. System performance coefficients (SPC) under two treatment conditions (30 V/cm in 36 °Brix or 60 V/cm in 48 °Brix juice) were relatively greater than for other combinations. Meanwhile, 5-log reductions of the three foodborne pathogens were achieved after treatment for 60 s in 36 °Brix at 30 V/cm, but this same reduction was observed in 48 °Brix juice at 60 V/cm within 20 s without affecting product quality. With respect to both bactericidal efficiency and SPC values, 60 V/cm in 48 °Brix was the most effective ohmic heating treatment combination for decontaminating apple juice concentrates.

  20. Why is firefly oxyluciferin a notoriously labile substance?

    PubMed

    Maltsev, Oleg V; Nath, Naba K; Naumov, Panče; Hintermann, Lukas

    2014-01-13

    The chemistry of firefly bioluminescence is important for numerous applications in biochemistry and analytical chemistry. The emitter of this bioluminescent system, firefly oxyluciferin, is difficult to handle. The cause of its lability was clarified while its synthesis was reinvestigated. A side product was identified and characterized by NMR spectroscopy and X-ray crystallography. The reason for the lability of oxyluciferin is now ascribed to autodimerization of the coexisting enol and keto forms in a Mannich-type reaction.

  1. R plasmid in Escherichia coli O103 coding for colonization of the rabbit intestinal tract.

    PubMed Central

    Reynaud, A; Federighi, M; Licois, D; Guillot, J F; Joly, B

    1991-01-01

    One rabbit pathogenic Escherichia coli strain, belonging to serogroup O103, harbors a self-transferable 117-kb plasmid (pREC-1) encoding resistance to several antibiotics. The role of this R plasmid in the colonization of the digestive tract in specific-pathogen-free (E. coli O103-free) rabbits was studied. Five-week-old rabbits were inoculated with the wild-type strain, with its variant cured of the plasmid, with an E. coli K-12 strain, or with an untypeable E. coli strain from a healthy rabbit. No symptoms and no mortality were observed in animals inoculated with strains without the plasmid pREC-1, but 87.5% of the rabbits infected by the wild strain died, generally with bloody diarrhea, between days 5 and 15 postinfection. The weight gain of animals was strongly reduced. Transfer of the plasmid to the cured strain or to nonvirulent strains led these strains to induce the same pathology but with a lower mortality. Colonization of the gut by the O103 strain and symptoms of bloody diarrhea are thus related to the presence of the pREC-1 plasmid. The GV strain, which does not produce classical heat-labile enterotoxin or heat-stable enterotoxin and is not invasive, could be considered an enteropathogenic E. coli-like strain. The presence of a conjugative plasmid such as pREC-1 encoding both antibiotic resistance and virulence determinants in O103 E. coli from rabbits could represent a prominent epidemiological hazard under selective pressure by antibiotic therapy. Images PMID:2037350

  2. Freshwater processing of terrestrial dissolved organic matter: What governs lability?

    NASA Astrophysics Data System (ADS)

    D'Andrilli, J.; Smith, H. J.; Junker, J. R.; Scholl, E. A.; Foreman, C. M.

    2016-12-01

    Aquatic and terrestrial ecosystems are linked through the transfer of energy and materials. Allochthonous organic matter (OM) is central to freshwater ecosystem function, influencing local food webs, trophic state, and nutrient availability. In order to understand the nature and fate of OM from inland headwaters to the open ocean, it is imperative to understand the links between OM lability and ecosystem function. Thus, biological, chemical, and physical factors need to be evaluated together to inform our understanding of environmental lability. We performed a laboratory processing experiment on naturally occurring OM leachates from riparian leaves, grasses, and pine needles. Measures of water chemistry, OM optical and molecular characterization, bacterial abundances, microbial assemblage composition, respiration, and C:N:P were integrated to discern the nature and fate of labile and recalcitrant OM in a freshwater stream. Peak processing of all OM sources in the stream water occurred after two days, with spikes in bacterial cell abundances, respiration rates, microbial assemblage shifts, and maximum C utilization. Respiration rates and microbial assemblages were dependent on the degree of lability of the OM molecular composition. Within the first few days, no differences in respiration rates were observed between leachate sources, however, beyond day five, the rates diverged with C processing efficiency correlated with OM lability. Originally comprised of amino acid-like, labile fluorescent species, the inoculated stream water OM became more recalcitrant after 16 days, indicating humification processing over time. Our study highlights the importance of interdisciplinary approaches for understanding the processing and fate of OM in aquatic ecosystems.

  3. Effects of age and ambient temperature on the responses of infant mice to heat-stable enterotoxin of Escherichia coli: assay modifications.

    PubMed Central

    Moon, H W; Fung, P Y; Whipp, S C; Isaacson, R E

    1978-01-01

    The response of infant mice to heat-stable enterotoxin from Escherichia coli was affected by the age of the mice (2, 4, 6, and 8 days) and by the ambient temperature (25, 30, and 37 degrees C) after exposure to the enterotoxin. The younger mice and/or mice held at lower temperatures tended to accumulate intestinal fluid (high gut weight/body weight ratios), but older mice and/or mice held at higher temperatures tended to respond with diarrhea and low gut weight/body weight ratios. The standard infant mouse assay forheat-stable E. coli enterotoxin can be simplified, without loss of sensitivity or reliability, by holding the mice at 37 degrees C after exposure and using diarrhea as the index of response. Diarrhea can be detected easily by incorporating dye in the inocula and (at the end of the assay) checking for dye mixed with feces on the rear quarters of the mice or on a sheet of white paper placed under them during incubation. PMID:352935

  4. Design of a Photoreactive Analogue of the Escherichia coli Heat-Stable Enterotoxin STIb: Use in Identifying Its Receptor on Rat Brush Border Membranes

    NASA Astrophysics Data System (ADS)

    Gariepy, Jean; Schoolnik, Gary K.

    1986-01-01

    The Escherichia coli heat-stable enterotoxin, STIb was prepared by solid-phase peptide synthesis and purified to homogeneity by high-pressure liquid chromatography. This analogue was iodinated and shown to bind specifically to rat intestinal membranes. The radiolabeled peptide was derivatized at the amino terminus with the photoreactive heterobifunctional crosslinking agent N-hydroxysuccinimidyl p-benzoylbenzoate. This photoreactive probe also exhibited binding specificity. It was mixed with rat intestinal brush border membranes and photolyzed in the presence or absence of excess unlabeled STIb. Polyacrylamide gel electrophoresis performed in the presence of sodium dodecyl sulfate and 2-mercaptoethanol indicated that the peptide probe was cross-linked specifically to two molecular species of 57 and 75 kDa. One or both of these molecules appear to constitute the enterotoxin receptor or to be in close proximity to it.

  5. Protective role of Mycobacterium leprae small heat-shock protein in heterologous hosts, Escherichia coli and Mycobacterium smegmatis, grown under stress.

    PubMed

    Maheshwari, Jayapal Jeya; Dharmalingam, Kuppamuthu

    2013-07-01

    The aim of this study is to examine the in vivo role of a small heat-shock protein (sHsp18) from Mycobacterium leprae in the survival of heterologous recombinant hosts carrying the gene encoding this protein under different environmental conditions that are normally encountered by M. leprae during its infection of the human host. Using an Escherichia coli system where shsp18 expression is controlled by its native promoter, we show that expression of shsp18 is induced under low oxygen tension, nutrient depletion and oxidative stress, all of which reflect the natural internal environment of the granulomas where the pathogen resides for long periods. We demonstrate the in vivo chaperone activity of sHsp18 through its ability to confer survival advantage to recombinant E. coli at heat-shock temperatures. Additional evidence for the protective role of sHsp18 was obtained when Mycobacterium smegmatis harbouring a copy of shsp18 was found to multiply better in human macrophages. Furthermore, the autokinase activity of sHsp18 protein demonstrated for what is believed to be the first time in this study implies that some of the functions of sHsp18 might be controlled by the phosphorylation state of this protein. Results from this study suggest that shsp18 might be one of the factors that facilitate the survival and persistence of M. leprae under stress and autophosphorylation of sHsp18 protein could be a mechanism used by this protein to sense changes in the external environment.

  6. Screening for toxigenic Escherichia coli in stool samples of diarrhoeal patients by polymerase chain reaction.

    PubMed

    Nawaz, Benish; Ali, Anum; Syed, Muhammad Noman; Khan, Abdul Basit

    2014-09-01

    Escherichia coli (E. coli) are normal flora of the intestines of most animals, including humans. Most strains are harmless and beneficial to host by preventing the establishment of pathogenic bacteria within the intestine. However, some E. coli strains can cause a wide variety of intestinal and extra-intestinal diseases, such as diarrhoea, urinary tract infections, septicaemia, neonatal meningitis and renal complications. Several virulence factors including toxins, adhesins, serine proteases, etc. have been reported in these highly adapted clones. The present study was designed to enumerate toxin genotype through PCR assay in local clinical isolates of E. coli. A total of 37 E. coli strains were collected from different clinical laboratories of Karachi and examined for the presence of shiga toxin 1 (stx1) and shiga toxin 2 (stx2) genes of Eenterohemorrhagic E. Coli (EHEC) and heat stable (st) and healt labile (lt) toxin genes of enterotoxigenic E. Coli (ETEC). It was observed that 16 strains out of 37 carried one or more type of toxin genes. The presence of stx1 gene was significantly higher as it was positive in 10 isolates compared to others toxins. Two in above stx1 positive strains were also carrying for stx2 gene. Six out of 37 isolates were positive for lt gene, and none of the strains are carrying st gene. Although, the study was carried out with fewer isolates, yet it demonstrated the trend of dispersion of toxin genes and findings can be used to correlate the gastro-intestinal infections and their complications in Pakistan.

  7. Some Characteristics of the Outer Membrane Material Released by Growing Enterotoxigenic Escherichia coli

    PubMed Central

    Gankema, Henk; Wensink, Jan; Guinée, Pieter A. M.; Jansen, Wim H.; Witholt, Bernard

    1980-01-01

    The high-molecular-weight material released into the medium by Escherichia coli AP1, an enterotoxigenic strain of porcine origin, has been isolated and resolved into two clearly distinct fractions, based on sucrose density gradient and differential centrifugation, chemical analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and freeze-fracture electron microscopy. These two fractions, referred to as “medium vesicles” and “medium lipopolysaccharides”, were compared with the cellular outer and cytoplasmic membranes, the periplasmic fraction, and the cytoplasmic fraction. The medium vesicles closely resembled outer membrane and accounted for 3 to 5% of the total cellular outer membrane. They contained most of the heat-labile enterotoxin (LT) activity released into the medium by E. coli AP1. The medium lipopolysaccharide consisted mostly of lipopolysaccharide and a small amount of outer membrane and contained relatively little LT activity. Based on experiments with E. coli K-12 strains, in which about 5% of the newly synthesized outer membrane is lost from areas of outer membrane synthesis, it is proposed that enterotoxigenic E. coli strains release LT as part of such newly synthesized outer membrane fragments and that released outer membrane fragments may function as physiologically significant LT carriers. ImagesFig. 4Fig. 1Fig. 2Fig. 2Fig. 3 PMID:7011982

  8. 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

  9. Overcoming Enterotoxigenic Escherichia coli Pathogen Diversity: Translational Molecular Approaches to Inform Vaccine Design

    PubMed Central

    Fleckenstein, James M.; Rasko, David A.

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC) are a genetically diverse E. coli pathovar that share in the ability to produce heat-labile toxin and/or heat-stable toxins. While these pathogens contribute substantially to the burden of diarrheal illness in developing countries, at present, there is no suitable broadly protective vaccine to prevent these common infections. Most vaccine development attempts to date have followed a classical approach involving a relatively small group of antigens. The extraordinary underlying genetic plasticity of E. coli has confounded the antigen valency requirements based on this approach. The recent discovery of additional virulence proteins within this group of pathogens, as well as the availability of whole-genome sequences from hundreds of ETEC strains to facilitate identification of conserved molecules, now permits a reconsideration of the classical approaches, and the exploration of novel antigenic targets to complement existing strategies overcoming antigenic diversity that has impeded progress toward a broadly protective vaccine. Progress to date in antigen discovery and methods currently available to explore novel immunogens are outlined here. PMID:27076141

  10. Overcoming Enterotoxigenic Escherichia coli Pathogen Diversity: Translational Molecular Approaches to Inform Vaccine Design.

    PubMed

    Fleckenstein, James M; Rasko, David A

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC) are a genetically diverse E. coli pathovar that share in the ability to produce heat-labile toxin and/or heat-stable toxins. While these pathogens contribute substantially to the burden of diarrheal illness in developing countries, at present, there is no suitable broadly protective vaccine to prevent these common infections. Most vaccine development attempts to date have followed a classical approach involving a relatively small group of antigens. The extraordinary underlying genetic plasticity of E. coli has confounded the antigen valency requirements based on this approach. The recent discovery of additional virulence proteins within this group of pathogens, as well as the availability of whole-genome sequences from hundreds of ETEC strains to facilitate identification of conserved molecules, now permits a reconsideration of the classical approaches, and the exploration of novel antigenic targets to complement existing strategies overcoming antigenic diversity that has impeded progress toward a broadly protective vaccine. Progress to date in antigen discovery and methods currently available to explore novel immunogens are outlined here.

  11. Influence of water activity on inactivation of Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes in peanut butter by microwave heating.

    PubMed

    Song, Won-Jae; Kang, Dong-Hyun

    2016-12-01

    This study evaluated the efficacy of a 915 MHz microwave with 3 different electric power levels to inactivate three pathogens in peanut butter with different aw. Peanut butter inoculated with Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium and Listeria monocytogenes (0.3, 0.4, and 0.5 aw) were treated with a 915 MHz microwave with 2, 4, and 6 kW for up to 5 min. Six kW 915 MHz microwave treatment for 5 min reduced these three pathogens by 1.97 to >5.17 log CFU/g. Four kW 915 MHz microwave processing for 5 min reduced these pathogens by 0.41-1.98 log CFU/g. Two kW microwave heating did not inactivate pathogens in peanut butter. Weibull and Log-Linear + Shoulder models were used to describe the survival curves of three pathogens because they exhibited shouldering behavior. Td and T5d values were calculated based on the Weibull and Log-Linear + Shoulder models. Td values of the three pathogens were similar to D-values of Salmonella subjected to conventional heating at 90 °C but T5d values were much shorter than those of conventional heating at 90 °C. Generally, increased aw resulted in shorter T5d values of pathogens, but not shorter Td values. The results of this study can be used to optimize microwave heating pasteurization system of peanut butter.

  12. Evaluation of ascorbic acid in protecting labile folic acid derivatives.

    PubMed

    Wilson, S D; Horne, D W

    1983-11-01

    The use of ascorbic acid as a reducing agent to protect labile, reduced derivatives of folic acid has been evaluated by high-performance liquid chromatographic separations and Lactobacillus casei microbiological assay of eluate fractions. Upon heating for 10 min at 100 degrees C, solutions of tetrahydropteroylglutamic acid (H4PteGlu) in 2% sodium ascorbate gave rise to 5,10-methylene-H4PteGlu and 5-methyl-H4PteGlu. H2PteGlu acid gave rise to 5-methyl-H4PteGlu and PteGlu. 10-Formyl-H4PteGlu gave rise to 5-formyl-H4PteGlu and 10-formyl-PteGlu. 5-Formyl-H4-PteGlu gave rise to a small amount of 10-formyl-PteGlu. 5-Methyl-H4PteGlu and PteGlu appeared stable to these conditions. These interconversions were not seen when solutions of these folate derivatives were kept at 0 degrees C in 1% ascorbate. These observations indicate that elevated temperatures are necessary for the interconversions of folates in ascorbate solutions. Assays of ascorbic acid solutions indicated the presence of formaldehyde (approximately equal to 6 mM). This was confirmed by the identification of 3,5-diacetyl-1,4-dihydrolutidine by UV, visible, and fluorescence spectroscopy and by thin-layer chromatography of chloroform extracts of the reaction mixture of ascorbic acid solutions, acetylacetone, and ammonium acetate. These results indicate that solutions of sodium ascorbate used at elevated temperatures are not suitable for extracting tissue for the subsequent assay of the individual folic acid derivatives.

  13. Evaluation of ascorbic acid in protecting labile folic acid derivatives.

    PubMed Central

    Wilson, S D; Horne, D W

    1983-01-01

    The use of ascorbic acid as a reducing agent to protect labile, reduced derivatives of folic acid has been evaluated by high-performance liquid chromatographic separations and Lactobacillus casei microbiological assay of eluate fractions. Upon heating for 10 min at 100 degrees C, solutions of tetrahydropteroylglutamic acid (H4PteGlu) in 2% sodium ascorbate gave rise to 5,10-methylene-H4PteGlu and 5-methyl-H4PteGlu. H2PteGlu acid gave rise to 5-methyl-H4PteGlu and PteGlu. 10-Formyl-H4PteGlu gave rise to 5-formyl-H4PteGlu and 10-formyl-PteGlu. 5-Formyl-H4-PteGlu gave rise to a small amount of 10-formyl-PteGlu. 5-Methyl-H4PteGlu and PteGlu appeared stable to these conditions. These interconversions were not seen when solutions of these folate derivatives were kept at 0 degrees C in 1% ascorbate. These observations indicate that elevated temperatures are necessary for the interconversions of folates in ascorbate solutions. Assays of ascorbic acid solutions indicated the presence of formaldehyde (approximately equal to 6 mM). This was confirmed by the identification of 3,5-diacetyl-1,4-dihydrolutidine by UV, visible, and fluorescence spectroscopy and by thin-layer chromatography of chloroform extracts of the reaction mixture of ascorbic acid solutions, acetylacetone, and ammonium acetate. These results indicate that solutions of sodium ascorbate used at elevated temperatures are not suitable for extracting tissue for the subsequent assay of the individual folic acid derivatives. PMID:6415653

  14. Emotional Lability and Affective Synchrony in Borderline Personality Disorder

    PubMed Central

    Schoenleber, Michelle; Berghoff, Christopher R.; Tull, Matthew T.; DiLillo, David; Messman-Moore, Terri; Gratz, Kim L.

    2015-01-01

    Extant research on emotional lability in borderline personality disorder (BPD) has focused almost exclusively on lability of individual emotions or emotion types, with limited research considering how different types of emotions shift together over time. Thus, this study examined the temporal dynamics of emotion in BPD at the level of both individual emotions (i.e., self-conscious emotions [SCE], anger, and anxiety) and mixed emotions (i.e., synchrony between emotions). One hundred forty-four women from the community completed a diagnostic interview and laboratory study involving five emotion induction tasks (each of which was preceded and followed by a 5-min resting period or neutral task). State ratings of SCE, anger, and anxiety were provided at 14 time points (before and after each laboratory task and resting period). Hierarchical linear modeling results indicate that women with BPD reported greater mean levels of SCE and Anxiety (but not Anger), and greater lability of Anxiety. Women with BPD also exhibited greater variability in lability of all three emotions (suggestive of within-group differences in the relevance of lability to BPD). Results also revealed synchrony (i.e., positive relations) between each possible pair of emotions, regardless of BPD status. Follow-up regression analyses suggest the importance of accounting for lability when examining the role of synchrony in BPD, as the relation of SCE-Anger synchrony to BPD symptom severity was moderated by Anger and SCE lability. Specifically, synchronous changes in SCE and Anger were associated with greater BPD symptom severity when large shifts in SCE were paired with minor shifts in Anger. PMID:27362623

  15. Comparative Genomics and Characterization of Hybrid Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC) Strains

    PubMed Central

    Nyholm, Outi; Halkilahti, Jani; Wiklund, Gudrun; Okeke, Uche; Paulin, Lars; Auvinen, Petri; Haukka, Kaisa; Siitonen, Anja

    2015-01-01

    Background Shigatoxigenic Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) cause serious foodborne infections in humans. These two pathogroups are defined based on the pathogroup-associated virulence genes: stx encoding Shiga toxin (Stx) for STEC and elt encoding heat-labile and/or est encoding heat-stable enterotoxin (ST) for ETEC. The study investigated the genomics of STEC/ETEC hybrid strains to determine their phylogenetic position among E. coli and to define the virulence genes they harbor. Methods The whole genomes of three STEC/ETEC strains possessing both stx and est genes were sequenced using PacBio RS sequencer. Two of the strains were isolated from the patients, one with hemolytic uremic syndrome, and one with diarrhea. The third strain was of bovine origin. Core genome analysis of the shared chromosomal genes and comparison with E. coli and Shigella spp. reference genomes was performed to determine the phylogenetic position of the STEC/ETEC strains. In addition, a set of virulence genes and ETEC colonization factors were extracted from the genomes. The production of Stx and ST were studied. Results The human STEC/ETEC strains clustered with strains representing ETEC, STEC, enteroaggregative E. coli, and commensal and laboratory-adapted E. coli. However, the bovine STEC/ETEC strain formed a remote cluster with two STECs of bovine origin. All three STEC/ETEC strains harbored several other virulence genes, apart from stx and est, and lacked ETEC colonization factors. Two STEC/ETEC strains produced both toxins and one strain Stx only. Conclusions This study shows that pathogroup-associated virulence genes of different E. coli can co-exist in strains originating from different phylogenetic lineages. The possibility of virulence genes to be associated with several E. coli pathogroups should be taken into account in strain typing and in epidemiological surveillance. Development of novel hybrid E. coli strains may cause a new public health risk, which

  16. Effect of pomegranate powder on the heat inactivation of Escherichia coli O157:H4 in ground chicken

    USDA-ARS?s Scientific Manuscript database

    Health concerns have led to a search for natural plant-based antimicrobials. Ellagic acid has been shown to have antimicrobial activity against foodborne pathogens. The objective of this study was to assess the effect of a high-ellagic acid commercial pomegranate on the heat resistance of Escherich...

  17. Differential degradation for small heat shock proteins IbpA and IbpB is synchronized in Escherichia coli: implications for their functional cooperation in substrate refolding.

    PubMed

    Shi, Xiaodong; Yan, Linxuan; Zhang, Hanlin; Sun, Kai; Chang, Zengyi; Fu, Xinmiao

    2014-09-26

    Small heat shock proteins (sHSPs), as a conserved family of ATP-independent molecular chaperones, are known to bind non-native substrate proteins and facilitate the substrate refolding in cooperation with ATP-dependent chaperones (e.g., DnaK and ClpB). However, how different sHSPs function in coordination is poorly understood. Here we report that IbpA and IbpB, the two sHSPs of Escherichia coli, are coordinated by synchronizing their differential in vivo degradation. Whereas the individually expressed IbpA and IbpB are respectively degraded slowly and rapidly in cells cultured under both heat shock and normal conditions, their simultaneous expression leads to a synchronized degradation at a moderate rate. Apparently, such synchronization is linked to their hetero-oligomerization and cooperation in binding substrate proteins. In addition, truncation of the flexible N- and C-terminal tails dramatically suppresses the IbpB degradation, and somehow accelerates the IbpA degradation. In view of these in vivo data, we propose that the synchronized degradation for IbpA and IbpB are crucial for their synergistic promoting effect on DnaK/ClpB-mediated substrate refolding, conceivably via the formation of IbpA-IbpB-substrate complexes. This scenario may be common for different sHSPs that interact with each other in cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Pathogenic Potential, Genetic Diversity, and Population Structure of Escherichia coli Strains Isolated from a Forest-Dominated Watershed (Comox Lake) in British Columbia, Canada

    PubMed Central

    Mazumder, Asit

    2014-01-01

    Escherichia coli isolates (n = 658) obtained from drinking water intakes of Comox Lake (2011 to 2013) were screened for the following virulence genes (VGs): stx1 and stx2 (Shiga toxin-producing E. coli [STEC]), eae and the adherence factor (EAF) gene (enteropathogenic E. coli [EPEC]), heat-stable (ST) enterotoxin (variants STh and STp) and heat-labile enterotoxin (LT) genes (enterotoxigenic E. coli [ETEC]), and ipaH (enteroinvasive E. coli [EIEC]). The only genes detected were eae and stx2, which were carried by 37.69% (n = 248) of the isolates. Only eae was harbored by 26.74% (n = 176) of the isolates, representing potential atypical EPEC strains, while only stx2 was detected in 10.33% (n = 68) of the isolates, indicating potential STEC strains. Moreover, four isolates were positive for both the stx2 and eae genes, representing potential EHEC strains. The prevalence of VGs (eae or stx2) was significantly (P < 0.0001) higher in the fall season, and multiple genes (eae plus stx2) were detected only in fall. Repetitive element palindromic PCR (rep-PCR) fingerprint analysis of 658 E. coli isolates identified 335 unique fingerprints, with an overall Shannon diversity (H′) index of 3.653. Diversity varied among seasons over the years, with relatively higher diversity during fall. Multivariate analysis of variance (MANOVA) revealed that the majority of the fingerprints showed a tendency to cluster according to year, season, and month. Taken together, the results indicated that the diversity and population structure of E. coli fluctuate on a temporal scale, reflecting the presence of diverse host sources and their behavior over time in the watershed. Furthermore, the occurrence of potentially pathogenic E. coli strains in the drinking water intakes highlights the risk to human health associated with direct and indirect consumption of untreated surface water. PMID:25548059

  19. Pathogenic potential, genetic diversity, and population structure of Escherichia coli strains isolated from a forest-dominated watershed (Comox Lake) in British Columbia, Canada.

    PubMed

    Chandran, Abhirosh; Mazumder, Asit

    2015-03-01

    Escherichia coli isolates (n = 658) obtained from drinking water intakes of Comox Lake (2011 to 2013) were screened for the following virulence genes (VGs): stx1 and stx2 (Shiga toxin-producing E. coli [STEC]), eae and the adherence factor (EAF) gene (enteropathogenic E. coli [EPEC]), heat-stable (ST) enterotoxin (variants STh and STp) and heat-labile enterotoxin (LT) genes (enterotoxigenic E. coli [ETEC]), and ipaH (enteroinvasive E. coli [EIEC]). The only genes detected were eae and stx2, which were carried by 37.69% (n = 248) of the isolates. Only eae was harbored by 26.74% (n = 176) of the isolates, representing potential atypical EPEC strains, while only stx2 was detected in 10.33% (n = 68) of the isolates, indicating potential STEC strains. Moreover, four isolates were positive for both the stx2 and eae genes, representing potential EHEC strains. The prevalence of VGs (eae or stx2) was significantly (P < 0.0001) higher in the fall season, and multiple genes (eae plus stx2) were detected only in fall. Repetitive element palindromic PCR (rep-PCR) fingerprint analysis of 658 E. coli isolates identified 335 unique fingerprints, with an overall Shannon diversity (H') index of 3.653. Diversity varied among seasons over the years, with relatively higher diversity during fall. Multivariate analysis of variance (MANOVA) revealed that the majority of the fingerprints showed a tendency to cluster according to year, season, and month. Taken together, the results indicated that the diversity and population structure of E. coli fluctuate on a temporal scale, reflecting the presence of diverse host sources and their behavior over time in the watershed. Furthermore, the occurrence of potentially pathogenic E. coli strains in the drinking water intakes highlights the risk to human health associated with direct and indirect consumption of untreated surface water.

  20. Exploring the frontier between life and death in Escherichia coli: evaluation of different viability markers in live and heat- or UV-killed cells.

    PubMed

    Villarino, A; Bouvet, O M; Regnault, B; Martin-Delautre, S; Grimont PAD

    2000-11-01

    A number of methods have been proposed to assess the viability of cells without culture. Each method is based on criteria that reflect different levels of cellular integrity or functionality. As a consequence, the interpretation of viability is often ambiguous. The purposes of this work were to evaluate the capacity of current viability markers to distinguish between live and dead Escherichia coli K-12 cells. Methods that assess 'viability' by the demonstration of metabolic activities (esterase activity, active electron transport chain, transport of glucose), cellular integrity (membrane integrity, presence of nucleic acids) or the building up of cellular material (cell elongation) have been evaluated in live and UV- or heat-killed cells. With live cells, viability markers detected cells in counts similar to the colony count. However, these so-called viability markers could stain dead cells for some time after the lethal treatment. For the UV-killed cells, residual activities were detected even after 48 h of storage at 20 degrees C. However, for heat-treated cells, these activities disappeared within hours after heat treatment. Only a combination of fluorescence in situ hybridization with rRNA probes and cell elongation in response to nutrients (in the presence of an inhibitor of cell division) had the ability to differentiate live from dead cells. Problems in the definition of a viable but nonculturable state are in part due to the lack of a clear definition of bacterial death. We consider death as an irreversible state where no growth, cell elongation or protein synthesis may occur.

  1. Parietal cortex and information granularity in labile and stable learning.

    PubMed

    Wang, Xiuzhen; Zhong, Ning; Lu, Shengfu; Liu, Chunnian; Gu, Weiquan

    2010-01-27

    We investigated the effects of rule learning based on information granularity. Using two homogeneous Boolean arithmetic tasks, we examined parietal cortex activity during the calculation of labile and stabilized learning. The results revealed stability-related behavioral advantages in a comparison of granularity-based effects with labile learning of Boolean problems. The functional MRI results revealed that different regions within the parietal cortex exhibited increased activity while solving Boolean problems in both the conditions. The calculation of labile rule learning based on low-granularity Boolean rules was significantly correlated with activation in bilateral parietal cortex, whereas stable rule learning based on high-granularity Boolean rules was correlated with activation in the left parietal cortex.

  2. In vivo emergence of enterotoxigenic Escherichia coli variants lacking genes for K99 fimbriae and heat-stable enterotoxin.

    PubMed Central

    Mainil, J G; Sadowski, P L; Tarsio, M; Moon, H W

    1987-01-01

    Neonatal pigs were inoculated with porcine enterotoxigenic Escherichia coli 431, which carries genes for K99 fimbriae and STaP enterotoxin. Colonies of strain 431 were recovered from feces of pigs for up to 17 days after inoculation and tested for hybridization with gene probes for K99 and STaP. Variants of strain 431 that did not hybridize with the probes were considered to have lost the genes. Variants were recovered from 10 of 13 suckling pigs that survived the infection. Only 0.4% of the isolates recovered during the first 2 days after inoculation were variants. Of the isolates recovered 3 to 5 days after inoculation, 20 to 36% were variants. Variant colonies were detected more frequently among pigs in some litters than in others. The litter with the highest number of variant-shedding pigs had the dam with the highest titer of K99 antibody in her colostrum. Variants also occurred in colostrum-deprived, artificially reared pigs. However, the number of variants detected was lower and they occurred later in the course of the infection in colostrum-deprived pigs than in suckling pigs. More variants were detected and they were detected earlier in colostrum-deprived pigs fed anti-K99 monoclonal antibody than in controls fed anti-K88 monoclonal antibody. Loss of STaP appeared to be secondary to loss of K99 in that some variants lacked only K99 (K99- STaP+) and some lacked both genes (K99- STaP-), but none was of the K99+ STaP- type. Our results confirmed reports of gene loss from enterotoxigenic E. coli during infection. They are consistent with the hypothesis that variants emerge under in vivo selection pressure of K99 antibody and with the speculation that gene loss may be an important component of protection in vaccinated populations. However, the emergence of variants did not appear to play a major role in the recovery of individual pigs from clinical disease. PMID:2890584

  3. Characterization of plasma labile heme in hemolytic conditions.

    PubMed

    Gouveia, Zélia; Carlos, Ana R; Yuan, Xiaojing; Aires-da-Silva, Frederico; Stocker, Roland; Maghzal, Ghassan J; Leal, Sónia S; Gomes, Cláudio M; Todorovic, Smilja; Iranzo, Olga; Ramos, Susana; Santos, Ana C; Hamza, Iqbal; Gonçalves, João; Soares, Miguel P

    2017-10-01

    Extracellular hemoglobin, a byproduct of hemolysis, can release its prosthetic heme groups upon oxidation. This produces metabolically active heme that is exchangeable between acceptor proteins, macromolecules and low molecular weight ligands, termed here labile heme. As it accumulates in plasma labile heme acts in a pro-oxidant manner and regulates cellular metabolism while exerting pro-inflammatory and cytotoxic effects that foster the pathogenesis of hemolytic diseases. Here, we developed and characterized a panel of heme-specific single domain antibodies (sdAbs) that together with a cellular-based heme reporter assay, allow for quantification and characterization of labile heme in plasma during hemolytic conditions. Using these approaches, we demonstrate that when generated during hemolytic conditions labile heme is bound to plasma molecules with an affinity higher than 10(-7) m and that 2-8% (~ 2-5 μm) of the total amount of heme detected in plasma can be internalized by bystander cells, termed here bioavailable heme. Acute, but not chronic, hemolysis is associated with transient reduction of plasma heme-binding capacity, that is, the ability of plasma molecules to bind labile heme with an affinity higher than 10(-7) m. The heme-specific sdAbs neutralize the pro-oxidant activity of soluble heme in vitro, suggesting that these maybe used to counter the pathologic effects of labile heme during hemolytic conditions. Finally, we show that heme-specific sdAbs can be used to visualize cellular heme. In conclusion, we describe a panel of heme-specific sdAbs that when used with other approaches provide novel insights to the pathophysiology of heme. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  4. Repeated Labilization-Reconsolidation Processes Strengthen Declarative Memory in Humans

    PubMed Central

    Forcato, Cecilia; Rodríguez, María L. C.; Pedreira, María E.

    2011-01-01

    The idea that memories are immutable after consolidation has been challenged. Several reports have shown that after the presentation of a specific reminder, reactivated old memories become labile and again susceptible to amnesic agents. Such vulnerability diminishes with the progress of time and implies a re-stabilization phase, usually referred to as reconsolidation. To date, the main findings describe the mechanisms associated with the labilization-reconsolidation process, but little is known about its functionality from a biological standpoint. Indeed, two functions have been proposed. One suggests that destabilization of the original memory after the reminder allows the integration of new information into the background of the original memory (memory updating), and the other suggests that the labilization-reconsolidation process strengthens the original memory (memory strengthening). We have previously reported the reconsolidation of human declarative memories, demonstrating memory updating in the framework of reconsolidation. Here we deal with the strengthening function attributed to the reconsolidation process. We triggered labilization-reconsolidation processes successively by repeated presentations of the proper reminder. Participants learned an association between five cue-syllables and their respective response-syllables. Twenty-four hours later, the paired-associate verbal memory was labilized by exposing the subjects to one, two or four reminders. The List-memory was evaluated on Day 3 showing that the memory was improved when at least a second reminder was presented in the time window of the first labilization-reconsolidation process prompted by the earlier reminder. However, the improvement effect was revealed on Day 3, only when at least two reminders were presented on Day2 and not as a consequence of only retrieval. Therefore, we propose central concepts for the reconsolidation process, emphasizing its biological role and the parametrical constrains

  5. The role of quorum sensing in Escherichia coli (ETEC) virulence factors.

    PubMed

    Sturbelle, Régis Tuchtenhagen; de Avila, Luciana Farias da Costa; Roos, Talita Bandeira; Borchardt, Jéssica Lopes; da Conceição, Rita de Cássia dos Santos; Dellagostin, Odir Antonio; Leite, Fábio Pereira Leivas

    2015-11-18

    Quorum sensing (QS) is a signaling system among bacteria mediated by auto-inducer substances (AI). Whenever the concentration of these molecules reaches a threshold corresponding to a high cell density or quorum, the whole population starts a coordinated expression of specific genes. Studies have shown that epinephrine is also responsible for activating specific bacterial genes. This work aimed to investigate the role of conditioned medium (containing AI), epinephrine and their association on growth, motility, F4 fimbriae and heat-labile toxin (LT) expression on enterotoxigenic Escherichia coli (ETEC, E68). A significant increase in motility, F4 and LT expression, was observed in the ETEC culture supplemented with conditioned medium and epinephrine. These findings suggest that ETEC uses some components of conditioned medium (e.g., AI molecules), host molecules (epinephrine), and their association to modulate the expression of important virulence genes.

  6. Contribution of the highly conserved EaeH surface protein to enterotoxigenic Escherichia coli pathogenesis.

    PubMed

    Sheikh, Alaullah; Luo, Qingwei; Roy, Koushik; Shabaan, Salwa; Kumar, Pardeep; Qadri, Firdausi; Fleckenstein, James M

    2014-09-01

    Enterotoxigenic Escherichia coli (ETEC) strains are among the most common causes of diarrheal illness worldwide. These pathogens disproportionately afflict children in developing countries, where they cause substantial morbidity and are responsible for hundreds of thousands of deaths each year. Although these organisms are important targets for enteric vaccines, most development efforts to date have centered on a subset of plasmid-encoded fimbrial adhesins known as colonization factors and heat-labile toxin (LT). Emerging data suggest that ETEC undergoes considerable changes in its surface architecture, sequentially deploying a number of putative adhesins during its interactions with the host. We demonstrate here that one putative highly conserved, chromosomally encoded adhesin, EaeH, engages the surfaces of intestinal epithelial cells and contributes to bacterial adhesion, LT delivery, and colonization of the small intestine. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  7. Formation in vitro of complexes between an abnormal fusion protein and the heat shock proteins from Escherichia coli and yeast mitochondria.

    PubMed Central

    Sherman, M Y; Goldberg, A L

    1991-01-01

    Heat shock proteins (HSPs) of the Hsp70 and GroEL families associate with a variety of cell proteins in vivo. However, the formation of such complexes has not been systematically studied. A 31-kDa fusion protein (CRAG), which contains 12 residues of cro repressor, truncated protein A, and 14 residues of beta-galactosidase, when expressed in Escherichia coli, was found in complexes with DnaK, GrpE, protease La, and GroEL. When an E. coli extract not containing CRAG was applied to an affinity column containing CRAG, DnaK, GroEL, and GrpE were selectively bound. These HSPs did not bind to a normal protein A column. DnaK, GrpE, and the fraction of GroEL could be eluted from the CRAG column with ATP but not with a nonhydrolyzable ATP analog. The ATP-dependent release of DnaK and GroEL also required Mg2+, but GrpE dissociated with ATP alone. The binding and release of DnaK and GroEL were independent events, but the binding of GrpE required DnaK. Inactivation of DnaJ, GrpE, and GroES did not affect the association or dissociation of DnaK or GroEL from CRAG. The DnaK and GrpE proteins could be eluted with 10(-6) M ATP, but 10(-4) M was required for GroEL release. This approach allows a one-step purification of these proteins from E. coli and also the isolation of the DnaK and GroEL homologs from yeast mitochondria. Competition experiments with oligopeptide fragments of CRAG showed that DnaK and GroEL interact with different sites on CRAG and that the cro-derived domain of CRAG contains the DnaK-binding site. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5 FIG. 7 FIG. 8 PMID:1938919

  8. Cloning of a heat-stable chitin deacetylase gene from Aspergillus nidulans and its functional expression in Escherichia coli.

    PubMed

    Wang, Yun; Song, Jin-Zhu; Yang, Qian; Liu, Zhi-Hua; Huang, Xiao-Mei; Chen, Yan

    2010-10-01

    A gene encoding chitin deacetylase was cloned by polymerase chain reaction from Aspergillus nidulans. Sequencing result showed 40% homology to the corresponding gene from Colletotrichum lindemuthianum. The complete gene contains an open reading frame of 747 nucleotides encoding a sequence of 249 amino acid residues. The chitin deacetylase gene was subcloned into a pET28a expression vector and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a His-bind column. The purified chitin deacetylase demonstrated an activity of 0.77 U ml(-1) for the glycol chitin substrates, and its specific activity was 4.17 U mg(-1) for it. The optimal temperature and pH of the purified enzyme were 50 degrees C and 8.0, respectively. When glycol chitin was used as the substrate, K (m) was 4.92 mg ml(-1), and K (cat) showed 6.25 s(-1), thus the ratio of K (cat) and K (m) was 1.27 ml s(-1) mg(-1). The activity of chitin deacetylase was affected by a range of metal ions and ethylenediaminetetraacetic acid.

  9. Labile trace elements in carbonaceous chondrites - A survey

    NASA Technical Reports Server (NTRS)

    Xiao, Xiaoyue; Lipschutz, Michael E.

    1992-01-01

    Data are presented on 14 trace elements, including Co, Au, Ga, Rb, Sb, Ag, Se, Cs, Te, Zn, Cd, Bi, Tl, and In (nearly all of which are moderately or highly labile in meteorites), obtained by radiochemical neutron activation analyses of 42 C2-C6 chondrites, all but three from Antarctica. The data indicate that carbonaceous chondrites of petrographic types 2-6 define compositional continua. It is suggested that carbonaceous C2-C6 chondrites may reflect a mixture of material that formed at low temperatures and that contained cosmic levels of highly labile elements, with material that was devoid of them.

  10. Labile trace elements in carbonaceous chondrites - A survey

    NASA Technical Reports Server (NTRS)

    Xiao, Xiaoyue; Lipschutz, Michael E.

    1992-01-01

    Data are presented on 14 trace elements, including Co, Au, Ga, Rb, Sb, Ag, Se, Cs, Te, Zn, Cd, Bi, Tl, and In (nearly all of which are moderately or highly labile in meteorites), obtained by radiochemical neutron activation analyses of 42 C2-C6 chondrites, all but three from Antarctica. The data indicate that carbonaceous chondrites of petrographic types 2-6 define compositional continua. It is suggested that carbonaceous C2-C6 chondrites may reflect a mixture of material that formed at low temperatures and that contained cosmic levels of highly labile elements, with material that was devoid of them.

  11. Clonal relatedness of Shiga-like toxin-producing Escherichia coli O101 strains of human and porcine origin.

    PubMed Central

    Franke, S; Harmsen, D; Caprioli, A; Pierard, D; Wieler, L H; Karch, H

    1995-01-01

    Shiga-like toxin (SLT)-producing Escherichia coli (SLTEC) O101 has recently been associated with hemorrhagic colitis and hemolytic-uremic syndrome in humans. In this study, SLTEC O101 strains from humans and pigs were characterized for clonal relatedness by nucleotide sequence analysis of their slt genes, DNA finger-printing of genomic DNA, and determination of virulence factors. The slt genes of five E. coli O101 strains were cloned and sequenced. For all strains, the deduced amino acid sequences of the B subunits were identical to those of the SLT-IIe present in the classical SLTEC O139 strains that cause edema disease in pigs. The A subunit revealed more than 99% homology to that of SLT-IIe. DNA fingerprinting revealed a high degree of genetic relatedness between the human and porcine O101 isolates. None of the O101 strains investigated had virulence factors frequently found in porcine (F107 fimbriae or heat-stable or heat-labile enterotoxins) or human SLTEC strains (eaeA or enterohemorrhagic E. coli hemolysin). The absence of virulence factors typical of SLT-I- and SLT-II-producing E. Coli together with the presence of SLT-IIe, a toxin previously seen only in porcine E. coli, suggests a new pathogenic mechanism for E. coli O101 infection of humans. For diagnostic purposes, we recommend the use of PCR primers and DNA probes complementary to slt-IIe to correctly identify such strains and to further evaluate their role in human diseases. PMID:8586696

  12. Clonal relatedness of Shiga-like toxin-producing Escherichia coli O101 strains of human and porcine origin.

    PubMed

    Franke, S; Harmsen, D; Caprioli, A; Pierard, D; Wieler, L H; Karch, H

    1995-12-01

    Shiga-like toxin (SLT)-producing Escherichia coli (SLTEC) O101 has recently been associated with hemorrhagic colitis and hemolytic-uremic syndrome in humans. In this study, SLTEC O101 strains from humans and pigs were characterized for clonal relatedness by nucleotide sequence analysis of their slt genes, DNA finger-printing of genomic DNA, and determination of virulence factors. The slt genes of five E. coli O101 strains were cloned and sequenced. For all strains, the deduced amino acid sequences of the B subunits were identical to those of the SLT-IIe present in the classical SLTEC O139 strains that cause edema disease in pigs. The A subunit revealed more than 99% homology to that of SLT-IIe. DNA fingerprinting revealed a high degree of genetic relatedness between the human and porcine O101 isolates. None of the O101 strains investigated had virulence factors frequently found in porcine (F107 fimbriae or heat-stable or heat-labile enterotoxins) or human SLTEC strains (eaeA or enterohemorrhagic E. coli hemolysin). The absence of virulence factors typical of SLT-I- and SLT-II-producing E. Coli together with the presence of SLT-IIe, a toxin previously seen only in porcine E. coli, suggests a new pathogenic mechanism for E. coli O101 infection of humans. For diagnostic purposes, we recommend the use of PCR primers and DNA probes complementary to slt-IIe to correctly identify such strains and to further evaluate their role in human diseases.

  13. Neuropsychological Correlates of Emotional Lability in Children with ADHD

    ERIC Educational Resources Information Center

    Banaschewski, Tobias; Jennen-Steinmetz, Christine; Brandeis, Daniel; Buitelaar, Jan K.; Kuntsi, Jonna; Poustka, Luise; Sergeant, Joseph A.; Sonuga-Barke, Edmund J.; Frazier-Wood, Alexis C.; Albrecht, Bjorn; Chen, Wai; Uebel, Henrik; Schlotz, Wolff; van der Meere, Jaap J.; Gill, Michael; Manor, Iris; Miranda, Ana; Mulas, Fernando; Oades, Robert D.; Roeyers, Herbert; Rothenberger, Aribert; Steinhausen, Hans-Christoph; Faraone, Stephen V.; Asherson, Philip

    2012-01-01

    Background: Emotional lability (EL) is commonly seen in patients with attention-deficit/hyperactivity disorder (ADHD). The reasons for this association remain currently unknown. To address this question, we examined the relationship between ADHD and EL symptoms, and performance on a range of neuropsychological tasks to clarify whether EL symptoms…

  14. Neuropsychological Correlates of Emotional Lability in Children with ADHD

    ERIC Educational Resources Information Center

    Banaschewski, Tobias; Jennen-Steinmetz, Christine; Brandeis, Daniel; Buitelaar, Jan K.; Kuntsi, Jonna; Poustka, Luise; Sergeant, Joseph A.; Sonuga-Barke, Edmund J.; Frazier-Wood, Alexis C.; Albrecht, Bjorn; Chen, Wai; Uebel, Henrik; Schlotz, Wolff; van der Meere, Jaap J.; Gill, Michael; Manor, Iris; Miranda, Ana; Mulas, Fernando; Oades, Robert D.; Roeyers, Herbert; Rothenberger, Aribert; Steinhausen, Hans-Christoph; Faraone, Stephen V.; Asherson, Philip

    2012-01-01

    Background: Emotional lability (EL) is commonly seen in patients with attention-deficit/hyperactivity disorder (ADHD). The reasons for this association remain currently unknown. To address this question, we examined the relationship between ADHD and EL symptoms, and performance on a range of neuropsychological tasks to clarify whether EL symptoms…

  15. Labile dissolved organic carbon supply limits hyporheic denitrification

    Treesearch

    Jay P. Zarnetske; Roy Haggerty; Steven M. Wondzell; Michelle A. Baker

    2012-01-01

    We used an in situ steady state 15N-labeled nitrate and acetate well-to-wells injection experiment to determine how the availability of labile dissolved organic carbon as acetate influences microbial denitrification in the hyporheic zone of an upland (third-order) agricultural stream.

  16. Mechanism of Stabilization of Labile Compounds by Silk Fibroin Proteins

    DTIC Science & Technology

    2017-04-05

    to stability of labile molecules. In addition , we plan to complete efforts initiated to refine our understanding of electrogelation mechanisms of...paper approaches. Additional processing can remediate interactions with conformational structures of the silk protein to further enhance blood... additional benefits such as preventing exposure to enzymatic and UV stresses. Long-term temperature stability of dried silk formats such as films or

  17. E. Coli

    MedlinePlus

    ... Emergency Room? What Happens in the Operating Room? E. Coli KidsHealth > For Kids > E. Coli A A A What's in this article? What ... Doctor Do? What Can Kids Do? en español E. coli What Is It? E. coli is a common ...

  18. Effect of Lactobacillus rhamnosus NCDC 298 with FOS in Combination on Viability and Toxin Production of Enterotoxigenic Escherichia coli.

    PubMed

    Anand, Santosh; Mandal, Surajit; Tomar, Sudhir Kumar

    2017-09-25

    The present study was to investigate the utilization of prebiotics by Lactobacillus rhamnosus NCDC 298 and its synergistic adversary effect on both population and production of heat-labile (LT) toxin in enterotoxigenic Escherichia coli (ETEC). To select suitable prebiotic in order to enhance functionality, its utilization and the prebiotic activity score was examined. Antivirulence effect on ETEC was inspected by its inactivation rate and heat-labile toxin production in presence of different synbiotic combination. L. rhamnosus NCDC 298 strain grown well on media supplemented with fructooligosaccharides (FOS) and galactooligosaccharides (GOS), whereas significant inactivation of ETEC was observed when FOS was added to the co-culture medium. Significant decrease in LT enterotoxin was seen through GM1 ganglioside enzyme linked immunoassay (GM1 ELISA), when ETEC has grown with L. rhamnosus NCDC 298 and FOS. Short-chain FOS proved to be the most effective substrate, improving antagonistic activity for L. rhamnosus NCDC 298. Both L. rhamnosus NCDC 298 with FOS can be used as an effective synbiotic combination for secretory antidiarrheal fermented dairy formulations.

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

    PubMed

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

    2015-07-01

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

  20. The Escherichia coli heat shock proteins GroEL and GroES modulate the folding of the beta-lactamase precursor.

    PubMed Central

    Laminet, A A; Ziegelhoffer, T; Georgopoulos, C; Plückthun, A

    1990-01-01

    One of the fundamental problems in biochemistry is the role of accessory proteins in the process of protein folding. The Escherichia coli heat shock protein complex GroEL/ES has been suggested to be a 'chaperonin' and be involved in both oligomer assembly as well as protein transport through the membrane. We show here that the folding of the purified precursor of beta-lactamase is inhibited by purified GroEL or the GroEL/ES complex with a stoichiometry of one particle per molecule of pre-beta-lactamase. Purified GroES alone has no effect on folding. After Mg2+ ATP addition folding resumes and the yield of active enzyme is higher than in the absence of GroEL or GroEL/ES. Unexpectedly, GroEL or GroEL/ES, when added to folded pre-beta-lactamase, lead to an apparent net 'unfolding', probably to a collapsed state of the protein, which can be reversed by the addition of Mg2+ ATP. The reversible and Mg2+ ATP-dependent association of GroEL/ES with non-native proteins might explain its postulated role in both protein transport and oligomer assembly. Images Fig. 4. PMID:2192863

  1. Binding of Escherichia coli heat-stable toxin and rise of guanylyl cyclase activity in the brush-border membranes of rabbit intestinal epithelial cells.

    PubMed

    Bhattacharya, J; Chakrabarti, M K

    1999-03-01

    The study examines the age-related differences in the density of Escherichia coli heat-stable enterotoxin (STa) receptors in the small intestine of rabbits. The number of STa receptors was found to be 1.7 x 10(12) in 14-day old rabbits compared to 2.4 x 10(9) in 14-week old rabbits per milligram brush-border membrane protein. The STa-induced guanylyl cyclase activity in the intestinal brush-border membranes was found to be stimulated by 6.2 folds over the basal enzyme activity in 14-day old rabbits, whereas in the 14-week old rabbits, it was 4 folds over the basal activity. Moreover, the enzyme activity remained lower in the adult rabbits compared to the younger ones. Autoradiographic analysis of sodium dodecyl sulphate polyacrylamide gel electrophoresis showed two STa-binding proteins of apparent molecular weights of 140 and 38 kDa in the intestinal brush-border membranes of rabbits.

  2. Non-labile silver species in biosolids remain stable throughout ...

    EPA Pesticide Factsheets

    Increasing commercial use of nanosilver has focussed attention on the fate of silver (Ag) in the wastewater release pathway. This paper reports the speciation and lability of Ag in archived, stockpiled, and contemporary biosolids from the UK, USA and Australia, and indicates that biosolids Ag concentrations have decreased significantly over recent decades. XANES revealed the importance of reduced-sulfur binding environments for Ag speciation in materials ranging from freshly produced sludge to biosolids weathered under ambient environmental conditions for more than 50 years. Isotopic dilution with 110mAg showed that Ag was predominantly non-labile in both fresh and aged biosolids (13.7% mean lability), with E-values ranging from 0.3 to 60 mg/kg and 5 mM CaNO3 extractable Ag from 1.2 to 609 µg/kg (0.002 - 3.4% of the total Ag). This study indicates that at the time of soil application, biosolids Ag will be predominantly Ag-sulfides and characterised by low isotopic lability. This paper presents an overview of biosolids Ag chemistry in historic and contemporary biosolids sourced from the UK, USA and Australia from the 1950s until today by drawing on a unique collection of archived, stockpiled and contemporary biosolids samples. Characteristics of biosolids Ag chemistry determined in this study included total Ag measurement by neutron activation analysis (NAA); the assessment of Ag lability by 110mAg isotopic dilution (E-values); and Ag speciation by X-ray Absorp

  3. Non-labile silver species in biosolids remain stable throughout ...

    EPA Pesticide Factsheets

    Increasing commercial use of nanosilver has focussed attention on the fate of silver (Ag) in the wastewater release pathway. This paper reports the speciation and lability of Ag in archived, stockpiled, and contemporary biosolids from the UK, USA and Australia, and indicates that biosolids Ag concentrations have decreased significantly over recent decades. XANES revealed the importance of reduced-sulfur binding environments for Ag speciation in materials ranging from freshly produced sludge to biosolids weathered under ambient environmental conditions for more than 50 years. Isotopic dilution with 110mAg showed that Ag was predominantly non-labile in both fresh and aged biosolids (13.7% mean lability), with E-values ranging from 0.3 to 60 mg/kg and 5 mM CaNO3 extractable Ag from 1.2 to 609 µg/kg (0.002 - 3.4% of the total Ag). This study indicates that at the time of soil application, biosolids Ag will be predominantly Ag-sulfides and characterised by low isotopic lability. This paper presents an overview of biosolids Ag chemistry in historic and contemporary biosolids sourced from the UK, USA and Australia from the 1950s until today by drawing on a unique collection of archived, stockpiled and contemporary biosolids samples. Characteristics of biosolids Ag chemistry determined in this study included total Ag measurement by neutron activation analysis (NAA); the assessment of Ag lability by 110mAg isotopic dilution (E-values); and Ag speciation by X-ray Absorp

  4. [Detection of verotoxin-producing E. coli in field isolates from domestic and agricultural animals in Sachsen-Anhalt].

    PubMed

    Gallien, P; Klie, H; Lehmann, S; Protz, D; Helmuth, R; Schäfer, R; Ehrler, M

    1994-10-01

    A report is given on the detection of verotoxin-producing E. coli (VTEC) strains from field isolates of healthy or ill cattle (n = 141), pigs (n = 306), sheep (n = 15), cats (n = 29) and dogs (n = 25) in the region of the new federal land Sachsen-Anhalt. 5% of the strains isolated from cattle, 32% from pigs, 20% from sheep, 4% from dogs and 0% from cats have shown VTEC. The E. coli-strains were checked for the presence of other factors of virulence, too. A good correlation (82%) was found between the colonization factor F107 and SLT 2/2v-containing strains from pigs in the region of Sachsen-Anhalt, too. Enterohemolysin was not found in SLT 2/2v-positive strains. 91% of the VTEC, isolated from pigs, produced alpha-Hemolysin. The correlation of SLT-containing strains and the production of enterohemolysin was confirmed for ruminants, only. Plasmidprofilings of VTEC from pigs showed mainly a 60 MDa or a 68 MDa plasmid or both, too. The occurrence of heat labile (LT) and in some cases of heat stable (ST) toxin was also checked, to differentiate the VTEC-strains from the enterotoxigenic E. coli strains (ETEC). These investigations showed, that VTEC produce SLT almost without exception. Correlations and conclusions on the pathogenicity for humans are discussed.

  5. Enterotoxigenic Escherichia coli diarrhea among young children in Jakarta, Indonesia.

    PubMed

    Richie, E; Punjabi, N H; Corwin, A; Lesmana, M; Rogayah, I; Lebron, C; Echeverria, P; Simanjuntak, C H

    1997-07-01

    The incidence of diarrhea and enterotoxigenic Escherichia coli (ETEC) infection was evaluated in children six months to five years of age from an urban community in Jakarta, Indonesia. From January through May 1994, 408 children were monitored in their homes for diarrheal disease. Thirty-six percent (148 of 408) of the study children had at least one episode of diarrhea during the study period. Twenty-nine (19.6%) of the 148 children with diarrhea had ETEC isolated from a rectal swab sample at least once during the surveillance period; five children had ETEC isolated from two distinct episodes of diarrhea, giving a total of 34 episodes of ETEC positive diarrhea in the study group. Ten of 34 episodes were associated with heat-labile toxin, 15 of 34 with heat-stable toxin, and seven of 34 with both toxins. The mean age of children with diarrhea (1.7 years), whether ETEC positive or negative, was significantly lower than those who did not have diarrhea (2.4 years) during the study period; 82% of the children with ETEC were less than two years of age. This study demonstrates a high incidence of ETEC diarrhea among young children in Jakarta, and suggests this site would be suitable for ETEC vaccine efficacy trials.

  6. Quantification of Labile Soil Mercury by Stable Isotope Dilution Techniques

    NASA Astrophysics Data System (ADS)

    Shetaya, Waleed; Huang, Jen-How; Osterwalder, Stefan; Alewell, Christine

    2016-04-01

    Mercury (Hg) is a toxic element that can cause severe health problems to humans. Mercury is emitted to the atmosphere from both natural and anthropogenic sources and can be transported over long distances before it is deposited to aquatic and terrestrial environments. Aside from accumulation in soil solid phases, Hg deposited in soils may migrate to surface- and ground-water or enter the food chain, depending on its lability. There are many operationally-defined extraction methods proposed to quantify soil labile metals. However, these methods are by definition prone to inaccuracies such as non-selectivity, underestimation or overestimation of the labile metal pool. The isotopic dilution technique (ID) is currently the most promising method for discrimination between labile and non-labile metal fractions in soil with a minimum disturbance to soil-solid phases. ID assesses the reactive metal pool in soil by defining the fraction of metal both in solid and solution phases that is isotopically-exchangeable known as the 'E-value'. The 'E-value' represents the metal fraction in a dynamic equilibrium with the solution phase and is potentially accessible to plants. This is carried out by addition of an enriched metal isotope to soil suspensions and quantifying the fraction of metal that is able to freely exchange with the added isotope by measuring the equilibrium isotopic ratio by ICP-MS. E-value (mg kg-1) is then calculated as follows: E-Value = (Msoil/ W) (CspikeVspike/ Mspike) (Iso1IAspike -Iso2IAspikeRss / Iso2IAsoil Rss - Iso1IAsoil) where M is the average atomic mass of the metal in the soil or the spike, W is the mass of soil (kg), Cspike is the concentration of the metal in the spike (mg L-1), Vspike is the volume of spike (L), IA is isotopic abundance, and Rss is the equilibrium ratio of isotopic abundances (Iso1:Iso2). Isotopic dilution has been successfully applied to determine E-values for several elements. However, to our knowledge, this method has not yet

  7. Influence of moisture content on inactivation of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in powdered red and black pepper spices by radio-frequency heating.

    PubMed

    Jeong, Seul-Gi; Kang, Dong-Hyun

    2014-04-17

    The influence of moisture content during radio-frequency (RF) heating on heating rate, dielectric properties, and inactivation of foodborne pathogens was investigated. The effect of RF heating on the quality of powdered red and black pepper spices with different moisture ranges was also investigated. Red pepper (12.6%, 15.2%, 19.1%, and 23.3% dry basis, db) and black pepper (10.1%, 17.2%, 23.7%, and 30.5% db) inoculated with Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium were treated in a RF heating system with 27.12 MHz. The heating rate of the sample was dependent on moisture content up to 19.1% (db) of red pepper and 17.2% (db) of black pepper, but there was a significant decrease in the heating rate when the moisture content was increased beyond these levels. The dielectric properties of both samples increased with a rise in moisture content. As the moisture content increased, treatment time required to reduce E. coli O157:H7 and S. Typhimurium by more than 7 log CFU/g (below the detection limit, 1 log CFU/g) decreased and then increased again without affecting product quality when the moisture content exceeded a level corresponding to the peak heating rate. RF treatment significantly (P<0.05) reduced moisture content of both spices. These results suggest that RF heating can be effectively used to not only control pathogens but also reduce moisture levels in spices and that the effect of inactivation is dependent on moisture content. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Virulence factors of lactose-negative Escherichia coli strains isolated from children with diarrhea in Somalia.

    PubMed Central

    Nicoletti, M; Superti, F; Conti, C; Calconi, A; Zagaglia, C

    1988-01-01

    Lactose-negative Escherichia coli strains were isolated at high frequency from children with diarrhea in Somalia during a 2-year study on diarrheal diseases. Sixty-four of these strains, considered to be a representative sample, were characterized for virulence factors, plasmid profiles, and antibiotic resistance. Of these strains, 5 were recognized as enteroinvasive E. coli (they were serotyped as O135:K-:H-), 6 belonged to classical enteropathogenic E. coli serotypes, 9 were able to adhere to tissue culture cells (of these, 4 showed a pattern of localized adherence and 1 was an enteropathogenic strain), 18 were both adherent and hemolytic, and 8 were simply hemolytic. None hybridized with 32P-labeled heat-labile or heat-stable (a and b) enterotoxin gene probes or produced moderate or high-level cytotoxic effects on HeLa cells. Of the 64 strains examined, 24 produced mannose-resistant hemagglutination with human, chicken, and monkey erythrocytes. One of these was serotyped as O4:K-:H8, and a rabbit O antiserum raised against this strain allowed us to establish that 23 strains had the same O antigen. The 23 O4 strains were hemolytic and were not enterotoxic for rabbit ileal loops, and intact bacteria were able to destroy tissue culture cell monolayers very rapidly. The uniformity of the antibiotic resistance pattern and of the plasmid DNA content, together with the fact that they were isolated in different years and in different children, suggests that the O4 strains must be epidemiologically relevant in Somalia. A possible diarrheagenic role for the adherent-hemolytic E. coli strains is also discussed. Images PMID:3281977

  9. Escherichia coli-induced temporal and differential secretion of heat-shock protein 70 and interleukin-1β by human fetal membranes in a two-compartment culture system.

    PubMed

    Osorio-Caballero, M; Perdigón-Palacio, C; García-López, G; Flores-Herrera, O; Olvera-Sánchez, S; Morales-Méndez, I; Sosa-González, I; Acevedo, J F; Guzmán-Grenfell, A M; Molina-Hernández, A; Díaz, N F; Flores-Herrera, H

    2015-03-01

    Escherichia coli is recognized as an etiological bacteria associated with chorioamnionitis and the preterm premature rupture of fetal membranes. This pathological condition induces pro-inflammatory cytokines and degradative metalloproteinases, which are considered biological markers secreted in an acute stage of infection. Heat-shock proteins (HSPs) are an important component of the innate immunity response and are found in different pathological conditions. They have not been previously measured in human fetal membranes in response to infectious conditions. We hypothesized that the choriodecidual tissue and amniotic epithelium secreted temporal and differential Hsp-60, Hsp-70, and interleukin (IL)-1β mediated by E. coli infection. Fetal membranes were mounted in a two-compartment culture system and infected with two passes of live E. coli at different doses (10², 10⁴, 10⁵, and 10⁶ colony-forming units (CFU)/mL) and intervals of incubation (3, 6, and 24 h). The culture medium was collected, and Hsp-60, Hsp-70, and IL-1β were assessed using the enzyme-linked immunosorbent assay (ELISA) method. After 3 and 6 h of infection, E. coli induced an increase in Hsp-70 secretion in the choriodecidual tissue. However, after 24 h of incubation, Hsp-70 was downregulated and we observed an increase in IL-1β secretion. By contrast, E. coli induced a lower Hsp-60 secretion in the amnion compared to Hsp-70. Human fetal membranes responded actively to E. coli infection, with an increase in Hsp-70 during the first hours of infection. After 24 h, there was an increase in the liberation of IL-1β. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein

    PubMed Central

    1989-01-01

    When nuclear localization sequences (termed NLS) are placed at the N terminus of cytochrome c1, a mitochondrial inner membrane protein, the resulting hybrid proteins do not assemble into mitochondria when synthesized in the yeast Saccharomyces cerevisiae. Cells lacking mitochondrial cytochrome c1, but expressing the hybrid NLS-cytochrome c1 proteins, are unable to grow on glycerol since the hybrid proteins are associated primarily with the nucleus. A similar hybrid protein with a mutant NLS is transported to and assembled into the mitochondria. To identify proteins that might be involved in recognition of nuclear localization signals, we isolated conditional- lethal mutants (npl, for nuclear protein localization) that missorted NLS-cytochrome c1 to the mitochondria, allowing growth on glycerol. The gene corresponding to one complementation group (NPL1) encodes a protein with homology to DnaJ, an Escherichia coli heat shock protein. npl1-1 is allelic to sec63, a gene that affects transit of nascent secretory proteins across the endoplasmic reticulum. Rothblatt, J. A., R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman. 1989. J. Cell Biol. 109:2641-2652. The npl1 mutants reported here also weakly affect translocation of preprocarboxypeptidaseY across the ER membrane. A normally nuclear hybrid protein containing a NLS fused to invertase and a nucleolar protein are not localized to the nucleus in npl1/sec63 cells at the nonpermissive temperature. Thus, NPL1/SEC63 may act at a very early common step in localization of proteins to the nucleus and the ER. Alternatively, by affecting ER and nuclear envelope assembly, npl1 may indirectly alter assembly of proteins into the nucleus. PMID:2556404

  11. Photo-lability of deep ocean dissolved black carbon

    NASA Astrophysics Data System (ADS)

    Stubbins, A.; Niggemann, J.; Dittmar, T.

    2012-01-01

    Dissolved black carbon (DBC), defined here as condensed aromatics isolated from seawater via PPL solid phase extraction and quantified as benzene polycarboxylic acid oxidation products, is a significant component of the oceanic dissolved organic carbon (DOC) pool. These condensed aromatics are widely distributed in the open ocean and appear to be tens of thousands of years old. As such DBC is regarded as highly refractory. In the current study, the photo-lability of DBC, DOC and coloured dissolved organic matter (CDOM; ultraviolet-visible absorbance) were determined over the course of a 28 d irradiation of North Atlantic Deep Water under a solar simulator. During the irradiation DBC fell from 1044 ± 164 nM C to 55 ± 15 nM C, a 20-fold decrease in concentration. Dissolved black carbon photo-degradation was more rapid and more extensive than for bulk CDOM and DOC. Further, the photo-lability of components of the DBC pool increased with their degree of aromatic condensation. These trends indicate that a continuum of compounds of varying photo-lability exists within the marine DOC pool. In this continuum, photo-lability scales with aromatic character, specifically the degree of condensation. Scaling the rapid photo-degradation of DBC to rates of DOC photo-mineralisation for the global ocean leads to an estimated photo-chemical half-life for oceanic DBC of less than 800 yr. This is more than an order of magnitude shorter than the apparent age of DBC in the ocean. Photo-degradation is therefore posited as the primary sink for oceanic DBC and the survival of DBC molecules in the oceans for millennia appears to be facilitated not by their inherent inertness but by the rate at which they are cycled through the surface ocean's photic zone.

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

    DTIC Science & Technology

    1981-01-01

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

  13. E. Coli

    MedlinePlus

    ... We Are Organization Director, Anthony Fauci, M.D. History What We ... Escherichia coli ( E. coli ) bacteria live in the intestines of people and animals, and are key to a healthy intestinal tract. ...

  14. Labile disulfide bonds are common at the leucocyte cell surface

    PubMed Central

    Metcalfe, Clive; Cresswell, Peter; Ciaccia, Laura; Thomas, Benjamin; Barclay, A. Neil

    2011-01-01

    Redox conditions change in events such as immune and platelet activation, and during viral infection, but the biochemical consequences are not well characterized. There is evidence that some disulfide bonds in membrane proteins are labile while others that are probably structurally important are not exposed at the protein surface. We have developed a proteomic/mass spectrometry method to screen for and identify non-structural, redox-labile disulfide bonds in leucocyte cell-surface proteins. These labile disulfide bonds are common, with several classes of proteins being identified and around 30 membrane proteins regularly identified under different reducing conditions including using enzymes such as thioredoxin. The proteins identified include integrins, receptors, transporters and cell–cell recognition proteins. In many cases, at least one cysteine residue was identified by mass spectrometry as being modified by the reduction process. In some cases, functional changes are predicted (e.g. in integrins and cytokine receptors) but the scale of molecular changes in membrane proteins observed suggests that widespread effects are likely on many different types of proteins including enzymes, adhesion proteins and transporters. The results imply that membrane protein activity is being modulated by a ‘redox regulator’ mechanism. PMID:22645650

  15. Concentration-dependent inhibition of Escherichia coli O157:H7 and heterocyclic amines in heated ground beef patties by apple and olive extracts, onion powder and clove bud oil.

    PubMed

    Rounds, Liliana; Havens, Cody M; Feinstein, Yelena; Friedman, Mendel; Ravishankar, Sadhana

    2013-08-01

    The effects of plant compounds on Escherichia coli O157:H7 and two major heat-induced heterocyclic amines (HCAs) MeIQx and PhIP in grilled ground beef patties were determined. Ground beef with added apple and olive extracts, onion powder, and clove bud oil was inoculated with E. coli O157:H7 (10⁷ CFU/g) and cooked to reach 45 °C at the geometric center, flipped and then cooked for another 5 min. Cooled samples were taken for microbiological and HCA analyses. Olive extract at 3% reduced E. coli O157:H7 to below detection. Reductions of up to 1 log were achieved with apple extract. Olive and apple extracts reduced MeIQx by up to 49.1 and 50.9% and PhIP by up to 50.6 and 65.2%, respectively. Onion powder reduced MeIQx and PhIP by 47 and 80.7%, respectively. Inactivation of E. coli O157:H7 and suppression of HCAs in grilled meat were achieved by optimized amounts of selected plant compounds.

  16. Efficiency of sodium hypochlorite, fumaric acid, and mild heat in killing native microflora and Escherichia coli O157:H7, Salmonella typhimurium DT104, and Staphylococcus aureus attached to fresh-cut lettuce.

    PubMed

    Kondo, Nozomi; Murata, Masatsune; Isshiki, Kenji

    2006-02-01

    The effect of the disinfectant sodium hypochlorite (NaClO), with or without mild heat (50 degrees C) and fumaric acid, on native bacteria and the foodborne pathogens Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella Typhimurium DT104 attached to iceberg lettuce leaves was examined. The retail lettuce examined consistently harbored 6 to 7 log CFU/g of native bacteria throughout the study period. Inner leaves supported 1 to 2 log CFU/g fewer bacteria than outer leaves. About 70% of the native bacterial flora was removed by washing five times with 0.85% NaCl. S. aureus, E. coli, and Salmonella allowed to attach to lettuce leaves for 5 min were more easily removed by washing than when allowed to attach for 1 h or 2 days, with more S. aureus being removed than E. coli or Salmonella Typhimurium. An increase of time for attachment of pathogens from 5 min to 2 days leads to decreased efficiency of the washing and sanitizing treatment. Treatment with fumaric acid (50 mM for 10 min at room temperature) was the most effective, although it caused browning of the lettuce, with up to a 2-log reduction observed. The combination of 200 ppm of sodium hypochlorite and mild heat treatment at 50 degrees C for 1 min reduced the pathogen populations by 94 to 98% (1.2- to 1.7-log reduction) without increasing browning.

  17. Binding of Escherichia coli Does Not Protect Tulane Virus from Heat-Inactivation Regardless the Expression of HBGA-Like Molecules

    PubMed Central

    Li, Qianqian; Wang, Dapeng; Yang, David; Shan, Lei; Tian, Peng

    2017-01-01

    Histo-blood group antigens (HBGAs) are considered as receptors/co-receptors for human norovirus (HuNoV). It has been reported that binding of HuNoV-derived virus-like particles (VLPs) to HBGA-like molecules-expressing bacteria increased the stability of VLPs to heat-denaturation (HD). In this study, we tested for HBGA-like-binding-conveyed protection against HD on viral replication using Tulane virus (TV) and Escherichia coli O86:H2 (O86:H2), with E. coli K-12 (K-12) used as a control. Expression of HBGA type B was confirmed by ELISA in O86:H2 but not in K-12. Binding of TV was confirmed by ELISA in O86:H2 (P/N = 2.23) but not in K-12 (P/N = 1.90). Pre-incubation of TV with free HBGA could completely inhibit its ability to bind to O86:H2 (p = 0.004), while producing no significant change in its ability to bind K-12 (p = 0.635). We utilized a bacterial-capture-RT-qPCR procedure to confirm that both bacterial strains were capable of binding TV, and that O86:H2 exhibited fivefold greater binding capacity than K-12. Pre-incubation of TV with free HBGA would partially inhibit the binding of TV to O86:H2 (p = 0.047). In contrast, not only did pre-incubation of TV with free HBGA not inhibit the binding of TV to K-12, binding was slightly enhanced (p = 0.13). The viral infectivity assay allowed us to conduct a direct evaluation of the ability of HBGA-like-bound bacteria to confer HD protection to TV. Prior to inoculate to LLC-MK2 cells, TV was incubated with each bacterial strain at ratios of 1:0, 1:1 and 100:1, then both partially and fully HD. The viral amplification was quantitated by RT-qPCR 48 h later. The binding of bacteria to TV reduced viral replication in a dose-dependent matter. We found that neither bound O86:H2 nor K-12 conferred protection of TV against partial or full HD conditions. Partial HD reduction of viral replication was not significantly impacted by the binding of either bacterial strain, with infectivity losses of 99.03, 99.42, 96.32, 96.10, and 98

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

    PubMed Central

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

    1973-01-01

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

  19. The Epidemiology of Travelers’ Diarrhea in Incirlik, Turkey: A Region with a Predominance of Heat-Stabile Toxin Producing Enterotoxigenic Escherichia coli

    DTIC Science & Technology

    2010-01-01

    1999). For Campylobacter spp., hippurate hydrolysis was used to differentiate isolates into Campylobacter jejwzi and non-C. jejuni following...53% (n = I 08) of the cases, with ETEC (82/202, 40.6%) and Campylobacter spp. (25/ 202, 12.4%) (C. jejuni , n = 15; Campylobacter coli, n = 9; other...Escltericltia coli ( ETEC) was the predominant pathogen ( 41 %). followed by Campylobacter spp. ( 12%). The most common ETEC phenotype recovered was stable toxin

  20. Effect of electropermeabilization by ohmic heating for inactivation of Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes in buffered peptone water and apple juice.

    PubMed

    Park, Il-Kyu; Kang, Dong-Hyun

    2013-12-01

    The effect of electric field-induced ohmic heating for inactivation of Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes in buffered peptone water (BPW) (pH 7.2) and apple juice (pH 3.5; 11.8 °Brix) was investigated in this study. BPW and apple juice were treated at different temperatures (55°C, 58°C, and 60°C) and for different times (0, 10, 20, 25, and 30 s) by ohmic heating compared with conventional heating. The electric field strength was fixed at 30 V/cm and 60 V/cm for BPW and apple juice, respectively. Bacterial reduction resulting from ohmic heating was significantly different (P<0.05) from that resulting from conventional heating at 58°C and 60°C in BPW and at 55°C, 58°C, and 60°C in apple juice for intervals of 0, 10, 20, 25, and 30 s. These results show that electric field-induced ohmic heating led to additional bacterial inactivation at sublethal temperatures. Transmission electron microscopy (TEM) observations and the propidium iodide (PI) uptake test were conducted after treatment at 60°C for 0, 10, 20, 25 and 30 s in BPW to observe the effects on cell permeability due to electroporation-caused cell damage. PI values when ohmic and conventional heating were compared were significantly different (P<0.05), and these differences increased with increasing levels of inactivation of three food-borne pathogens. These results demonstrate that ohmic heating can more effectively reduce bacterial populations at reduced temperatures and shorter time intervals, especially in acidic fruit juices such as apple juice. Therefore, loss of quality can be minimized in a pasteurization process incorporating ohmic heating.

  1. Effect of Electropermeabilization by Ohmic Heating for Inactivation of Escherichia coli O157:H7, Salmonella enterica Serovar Typhimurium, and Listeria monocytogenes in Buffered Peptone Water and Apple Juice

    PubMed Central

    Park, Il-Kyu

    2013-01-01

    The effect of electric field-induced ohmic heating for inactivation of Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes in buffered peptone water (BPW) (pH 7.2) and apple juice (pH 3.5; 11.8 °Brix) was investigated in this study. BPW and apple juice were treated at different temperatures (55°C, 58°C, and 60°C) and for different times (0, 10, 20, 25, and 30 s) by ohmic heating compared with conventional heating. The electric field strength was fixed at 30 V/cm and 60 V/cm for BPW and apple juice, respectively. Bacterial reduction resulting from ohmic heating was significantly different (P < 0.05) from that resulting from conventional heating at 58°C and 60°C in BPW and at 55°C, 58°C, and 60°C in apple juice for intervals of 0, 10, 20, 25, and 30 s. These results show that electric field-induced ohmic heating led to additional bacterial inactivation at sublethal temperatures. Transmission electron microscopy (TEM) observations and the propidium iodide (PI) uptake test were conducted after treatment at 60°C for 0, 10, 20, 25 and 30 s in BPW to observe the effects on cell permeability due to electroporation-caused cell damage. PI values when ohmic and conventional heating were compared were significantly different (P < 0.05), and these differences increased with increasing levels of inactivation of three food-borne pathogens. These results demonstrate that ohmic heating can more effectively reduce bacterial populations at reduced temperatures and shorter time intervals, especially in acidic fruit juices such as apple juice. Therefore, loss of quality can be minimized in a pasteurization process incorporating ohmic heating. PMID:23995939

  2. Substitution of a highly conserved histidine in the Escherichia coli heat shock transcription factor, sigma32, affects promoter utilization in vitro and leads to overexpression of the biofilm-associated flu protein in vivo.

    PubMed

    Kourennaia, Olga V; Dehaseth, Pieter L

    2007-12-01

    The heat shock sigma factor (sigma(32) in Escherichia coli) directs the bacterial RNA polymerase to promoters of a specific sequence to form a stable complex, competent to initiate transcription of genes whose products mitigate the effects of exposure of the cell to high temperatures. The histidine at position 107 of sigma(32) is at the homologous position of a tryptophan residue at position 433 of the main sigma factor of E. coli, sigma(70). This tryptophan is essential for the strand separation step leading to the formation of the initiation-competent RNA polymerase-promoter complex. The heat shock sigma factors of all gammaproteobacteria sequenced have a histidine at this position, while in the alpha- and deltaproteobacteria, it is a tryptophan. In vitro the alanine-for-histidine substitution at position 107 (H107A) destabilizes complexes between the GroE promoter and RNA polymerase containing sigma(32), implying that H107 plays a role in formation or maintenance of the strand-separated complex. In vivo, the H107A substitution in sigma(32) impedes recovery from heat shock (exposure to 42 degrees C), and it also leads to overexpression at lower temperatures (30 degrees C) of the Flu protein, which is associated with biofilm formation.

  3. Labile complexes facilitate cadmium uptake by Caco-2 cells.

    PubMed

    Verheyen, L; Degryse, F; Niewold, T; Smolders, E

    2012-06-01

    The Free Ion Activity Model (FIAM) predicts that metal uptake in biota is related to the free ion activity in the external solution and that metal complexes do not contribute. However, studies with plants have shown that labile metal complexes enhance metal bioavailability when the uptake is rate-limited by transport of the free ion in solution to the uptake site. Here, the role of labile complexes of Cd on metal bioavailability was assessed using Caco-2 cells, the cell model for intestinal absorption. At low Cd(2+) concentration (1 nM), the CdCl(n)(2-n) complexes contributed to the uptake almost to the same extent as the free ion. At large Cd(2+) concentration (10 μM), the contribution of the complexes was much smaller. At constant Cd(2+) concentration, Cd intake in the cells from solutions containing synthetic ligands such as EDTA increased as the dissociation rate of the cadmium complexes increased, and correlated well with the Cd diffusion flux in solution measured with the Diffusive Gradient in Thin Films technique (DGT). The Cd intake fluxes in the cells were well predicted assuming that the specific uptake is limited by diffusion of the free Cd(2+) ion to the cell surface. Our results underline that speciation of Cd has a major effect on its uptake by intestinal cells, but the availability is not simply related to the free ion concentration. Labile complexes of Cd enhance metal bioavailability in these cells, likely by alleviating diffusive limitations. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Do Vermont's Floodplains Constitute an Important Source of Labile Carbon?

    NASA Astrophysics Data System (ADS)

    Perdrial, J. N.; Dolan, A.; Kemsley, M.

    2014-12-01

    Floodplains are extremely heterogeneous landscapes with respect to soil and sediment composition and can present an important source of carbon (C) during floods. For example, stream bank soils and sediments are zones of active erosion and deposition of sediment associated C. Due to the presence of plants, riparian soils contain high amounts of C that is exchanged between stream waters and banks. Abandoned channels and meander wetlands that remain hydrologically connected to the main channel contain high amounts of organic matter that can be flushed into the stream during high discharge. This heterogeneity, result of floodplain geomorphology, land cover and use, can profoundly impact the amount and type of dissolved organic matter (DOM) introduced into streams. In order to assess DOM characteristics leached from heterogeneous floodplain soils, aqueous soil extracts were performed on soil samples representative of different land covers (n=20) at four depths. Extracts were analyzed for dissolved organic C and total dissolved nitrogen with a Shimadzu C analyzer. Colored dissolved organic matter characteristics was measured with the Aqualog Fluorescence Spectrometer and quantified with parallel factor analysis (PARAFAC). Preliminary data from three floodplains in Vermont (Connecticut, Missisquoi and Mad River) show a 3D variability of longitudinal, lateral, and vertical extents on water-extractable, mobile C. Dissolved organic carbon concentrations in meander swamp samples were found up to 9 times higher than in those of soils from agricultural field indicative of an important C source. Although C concentrations in adjacent fields were low, high abundance of labile C (indicated by tryptophan-like fluorescence) in water extracts from fields indicates recent biological production of C. This labile C is easily processed by microbes and transformed to the greenhouse gas CO2. These results provide important information on the contribution and lability of different floodplain

  5. 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.

  6. Inflammatory mediators in culture filtrates of Escherichia coli.

    PubMed Central

    Yoshida, T.; Cohen, S.; Bigazzi, P. E.; Kurasuji, T.; Amsden, A.

    1975-01-01

    Escherichia coli, when cultured on a simple medium containing only glucose and inorganic compounds, release soluble factors which have a variety of biologic effects on cells in vitro. These low molecular weight (less than 12,000) substances are capable of: a) reversibly inhibiting the migration of macrophages, b) causing chemotactic attraction of neutrophils, c) inducing blast transformation of lymphocytes, and d) producing cytotoxic effects on mouse fibroblasts in culture. Although these activities are functionally similar to those which have been described for various lymphokines obtained from antigen-activated lymphocyte cultures, lymphocyte and bacterial factors which share a given property do not appear to be identical. For example, the bacterial factor which inhibits macrophage migration is partially heat labile and is dialyzable, characteristics which distinguish it from conventional migration inhibition factor. Nevertheless, similarity of function may imply the existence of some degree of chemical homology which would have importance implications concerning the evolution of host-defense reactions. In any case, as is the situation for the lymphokines, the in vitro behavior of the bacterial factors suggest a role for them in in vivo inflammatory responses. PMID:1103632

  7. Prevalence of adhesin and toxin genes in E. coli strains isolated from diarrheic and non-diarrheic pigs from smallholder herds in northern and eastern Uganda.

    PubMed

    Ikwap, Kokas; Larsson, Jenny; Jacobson, Magdalena; Owiny, David Okello; Nasinyama, George William; Nabukenya, Immaculate; Mattsson, Sigbrit; Aspan, Anna; Erume, Joseph

    2016-08-05

    Enterotoxigenic E. coli (ETEC) significantly contribute to diarrhea in piglets and weaners. The smallholder pig producers in Uganda identified diarrhea as one of the major problems especially in piglets. The aim of this study was to; i) characterize the virulence factors of E. coli strains isolated from diarrheic and non-diarrheic suckling piglets and weaners from smallholder herds in northern and eastern Uganda and ii) identify and describe the post-mortem picture of ETEC infection in severely diarrheic piglets. Rectal swab samples were collected from 83 piglets and weaners in 20 herds and isolated E. coli were characterized by PCR, serotyping and hemolysis. The E. coli strains carried genes for the heat stable toxins STa, STb and EAST1 and adhesins F4 and AIDA-I. The genes for the heat labile toxin LT and adhesins F5, F6, F18 and F41 were not detected in any of the E. coli isolates. Where the serogroup could be identified, E. coli isolates from the same diarrheic pig belonged to the same serogroup. The prevalence of EAST1, STb, Stx2e, STa, AIDA-I, and F4 in the E. coli isolates from suckling piglets and weaners (diarrheic and non-diarrheic combined) was 29, 26.5, 2.4, 1.2, 16, and 8.4 %, respectively. However the prevalence of F4 and AIDA-I in E. coli from diarrheic suckling piglets alone was 22.2 and 20 %, respectively. There was no significant difference in the prevalence of the individual virulence factors in E. coli from the diarrheic and non-diarrheic pigs (p > 0.05). The main ETEC strains isolated from diarrheic and non-diarrheic pigs included F4/STb/EAST1 (7.2 %), F4/STb (1.2 %), AIDA/STb/EAST1 (8 %) and AIDA/STb (8 %). At post-mortem, two diarrheic suckling piglets carrying ETEC showed intact intestinal villi, enterocytes and brush border but with a layer of cells attached to the brush border, suggestive of ETEC infections. This study has shown that the F4 fimbriae is the most predominant in E. coli from diarrheic piglets in the study area and

  8. Photo-lability of deep ocean dissolved black carbon

    NASA Astrophysics Data System (ADS)

    Stubbins, A.; Niggemann, J.; Dittmar, T.

    2012-05-01

    Dissolved black carbon (DBC), defined here as condensed aromatics isolated from seawater via PPL solid phase extraction and quantified as benzenepolycarboxylic acid (BPCA) oxidation products, is a significant component of the oceanic dissolved organic carbon (DOC) pool. These condensed aromatics are widely distributed in the open ocean and appear to be tens of thousands of years old. As such DBC is regarded as highly refractory. In the current study, the photo-lability of DBC, DOC and coloured dissolved organic matter (CDOM; ultraviolet-visible absorbance) were determined over the course of a 28 day irradiation of North Atlantic Deep Water under a solar simulator. During the irradiation DBC fell from 1044 ± 164 nM-C to 55 ± 15 nM-C, a 20-fold decrease in concentration. Dissolved black carbon photo-degradation was more rapid and more extensive than for bulk CDOM and DOC. The concentration of DBC correlated with CDOM absorbance and the quality of DBC indicated by the ratios of different BPCAs correlated with CDOM absorbance spectral slope, suggesting the optical properties of CDOM may provide a proxy for both DBC concentrations and quality in natural waters. Further, the photo-lability of components of the DBC pool increased with their degree of aromatic condensation. These trends indicate that a continuum of compounds of varying photo-lability exists within the marine DOC pool. In this continuum, photo-lability scales with aromatic character, specifically the degree of condensation. Scaling the rapid photo-degradation of DBC to rates of DOC photo-mineralisation for the global ocean leads to an estimated photo-chemical half-life for oceanic DBC of less than 800 years. This is more than an order of magnitude shorter than the apparent age of DBC in the ocean. Consequently, photo-degradation is posited as the primary sink for oceanic DBC and the apparent survival of DBC molecules in the oceans for millennia appears to be facilitated not by their inherent inertness but

  9. 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.

  10. Diarrhea Burden Due to Natural Infection with Enterotoxigenic Escherichia coli in a Birth Cohort in a Rural Egyptian Community

    PubMed Central

    Shaheen, H. I.; Amine, M.; Hassan, K.; Sanders, J. W.; Riddle, M. S.; Armstrong, A. W.; Svennerholm, A. M.; Sebeny, P. J.; Klena, J. D.; Young, S. Y. N.; Frenck, R. W.

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC) is commonly associated with diarrhea in Egyptian children. Children less than 3 years old in Abu Homos, Egypt, had approximately five diarrheal episodes per child every year, and at least one of these episodes was due to ETEC. The epidemiology of ETEC diarrhea among children living in a rural Egyptian community was further evaluated in this study. Between January 2004 and April 2007, 348 neonates were enrolled and followed for 2 years. Children were visited twice weekly, and a stool sample was obtained every 2 weeks regardless of symptomatology. A stool sample was obtained whenever a child had diarrhea. From the routine stool culture, five E. coli-like colonies were selected and screened for heat-labile and heat-stable toxins by GM1 enzyme-linked immunosorbent assay (ELISA) and further typed for colonization factor antigens by dot blot assay. Incidence of ETEC infection was estimated among children with diarrhea (symptomatic) and without diarrhea (asymptomatic). Incidence of diarrhea and ETEC-associated diarrhea was 7.8 and 1.48 per child-year, respectively. High risk of repeated ETEC diarrhea was associated with being over 6 months of age, warm season, male gender, and crowded sleeping conditions. Exclusive breast-feeding was protective for repeated ETEC infection. ETEC-associated diarrhea remains common among children living in the Nile Delta. The protective role of breast-feeding demonstrates the importance of promoting exclusive breast-feeding during, at least, the first 6 months of life. PMID:24829232

  11. Bronchial lability and skin reactivity in siblings of asthmatic children.

    PubMed

    Verity, C M; Vanheule, B; Carswell, F; Hughes, A O

    1984-09-01

    Seventy five children with asthma (42 boys and 33 girls; age range 4 years 2 months to 15 years) and 75 of their siblings (37 boys and 38 girls; age range 4 years 3 months to 17 years 8 months) were studied to elucidate the mechanisms involved in the increased prevalence of asthma in boys, a prevalence that tends to disappear after puberty. Immediate cutaneous hypersensitivity to five allergens and maximum fall in peak expiratory flow rate after six minutes of treadmill running (bronchial lability) were determined in patients and siblings. There was no significant difference between boys and girls in skin test reactivity to single or multiple allergens in the sibling group. The percentage fall in peak expiratory flow rate after exercise was significantly greater in male than in female siblings and when a positive test was defined as a fall after exercise of either 10% or 15% of the rate before exercise the number of positive tests was significantly greater in boys. The results suggest that more boys than girls in this age group have asthma because their bronchial lability is greater, and not because more boys are atopic.

  12. The Labile Side of Iron Supplementation in CKD

    PubMed Central

    Cabantchik, Zvi Ioav

    2015-01-01

    The practice of intravenous iron supplementation has grown as nephrologists have gradually moved away from the liberal use of erythropoiesis-stimulating agents as the main treatment for the anemia of CKD. This approach, together with the introduction of large-dose iron preparations, raises the future specter of inadvertent iatrogenic iron toxicity. Concerns have been raised in original studies and reviews about cardiac complications and severe infections that result from long-term intravenous iron supplementation. Regarding the iron preparations specifically, even though all the currently available preparations appear to be relatively safe in the short term, little is known regarding their long-term safety. In this review we summarize current knowledge of iron metabolism with an emphasis on the sources and potentially harmful effects of labile iron, highlight the approaches to identifying labile iron in pharmaceutical preparations and body fluids and its potential toxic role as a pathogenic factor in the complications of CKD, and propose methods for its early detection in at-risk patients. PMID:25999405

  13. Labile neurotoxin in serum of calves with "nervous" coccidiosis.

    PubMed Central

    Isler, C M; Bellamy, J E; Wobeser, G A

    1987-01-01

    Mouse inoculation was used to test for the presence of a toxin in the serum, cerebrospinal fluid, and intestinal contents collected from cases of bovine enteric coccidiosis, with and without neurological signs, and from control calves. Intravenous inoculation of mice with 10 mL/kg of serum from calves showing nervous signs caused effects significantly different from those caused by the inoculation of serum from calves not showing nervous signs and from control calves. The effect was particularly evident in female mice. At this dosage severe neurological signs such as loss of righting reflex, seizures and death occurred only with serum from calves with "nervous coccidiosis". The results suggest that serum from the calves with neurological signs contains a neurotoxin. This toxin appears to be highly labile. It was not present in the cerebrospinal fluid at levels comparable to those in the serum. The significance of this labile neurotoxin with respect to the pathogenesis of the neurological signs associated with bovine enteric coccidiosis is unknown. PMID:2955865

  14. Non-labile Soil 15Nitrogen Retention beneath Three Tree Species in a Tropical Plantation

    Treesearch

    Jason P. Kaye; Dan Binkley; Xiaoming Zou; John A. Parrotta

    2002-01-01

    Soil organic matter is the largest sink for N additions to forests.Species composition may affect soilNretention by altering the amount or proportion of added N stored in non-labile organic pools. We measured 15N tracer retention in labile and non-labile pools of surface (0–20 cm) mineral soils, 7 yr after the tracer was applied to a 9 yr-old Puerto Rican tree...

  15. An ultrahigh-resolution mass spectrometry index to estimate natural organic matter lability

    PubMed Central

    D'Andrilli, Juliana; Cooper, William T; Foreman, Christine M; Marshall, Alan G

    2015-01-01

    Rationale Determining the chemical constituents of natural organic matter (NOM) by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICRMS) remains the ultimate measure for probing its source material, evolution, and transport; however, lability and the fate of organic matter (OM) in the environment remain controversial. FTICRMS-derived elemental compositions are presented in this study to validate a new interpretative method to determine the extent of NOM lability from various environments. Methods FTICRMS data collected over the last decade from the same 9.4 tesla instrument using negative electrospray ionization at the National High Magnetic Field Laboratory in Tallahassee, Florida, was used to validate the application of a NOM lability index. Solid-phase extraction cartridges were used to isolate the NOM prior to FTICRMS; mass spectral peaks were calibrated internally by commonly identified NOM homologous series, and molecular formulae were determined for NOM composition and lability analysis. Results A molecular lability boundary (MLB) was developed from the FTICRMS molecular data, visualized from van Krevelen diagrams, dividing the data into more and less labile constituents. NOM constituents above the MLB at H/C ≥1.5 correspond to more labile material, whereas NOM constituents below the MLB, H/C <1.5, exhibit less labile, more recalcitrant character. Of all marine, freshwater, and glacial environments considered for this study, glacial ecosystems were calculated to contain the most labile OM. Conclusions The MLB extends our interpretation of FTICRMS NOM molecular data to include a metric of lability, and generally ranked the OM environments from most to least labile as glacial > marine > freshwater. Applying the MLB is useful not only for individual NOM FTICRMS studies, but also provides a lability threshold to compare and contrast molecular data with other FTICRMS instruments that survey NOM from around the world. Copyright © 2015

  16. [Universal implementation of pathogen inactivation in labile blood products is a major step towards transfusion safety].

    PubMed

    Cazenave, Jean-Pierre

    2010-12-01

    Transfusion of labile blood products (red cell concentrates, platelet concentrates and plasma) is vital in the absence of alternatives. Patients and doctors have always feared infections transmitted by blood, blood components and blood-derived drugs. It is potentially dangerous to delay implementation of pathogen inactivation in labile blood products pending a perfect process. Universal implementation of pathogen inactivation in labile blood products is a major step towards transfusion safety.

  17. Non-labile Soil Nitrogen Retention beneath Three Tree Species in a Tropical Plantation

    Treesearch

    Jason P. Kaye; Dan Binkley; Xiaoming Zou; John A. Parrotta

    2002-01-01

    Soil organic matter is the largest sink for N additions to forests. Species composition may affect soilNretention by altering the amount or proportion of added N stored in non-labile organic pools. We measured 15N tracer retention in labile and non-labile pools of surface (0–20 cm) mineral soils, 7 yr after the tracer was applied to a 9 yr-old Puerto Rican tree...

  18. Cellulitis lesions in broiler chickens are induced by Escherichia coli Vacuolating Factor (ECVF).

    PubMed

    Quel, N G; Aragão, A Z B; Salvadori, M R; Farias, A S; Joazeiro, P P; Santos, L M B; Sá, L R M; Ferreira, A J P; Yano, T

    2013-03-23

    Escherichia coli Vacuolating Factor (ECVF) is a heat-labile, vacuolating cytotoxin produced by avian pathogenic E. coli (APEC) isolated from avian cellulitis lesions. In this report, we intend to demonstrate that purified ECVF induces the inflammatory process of cellulitis. Our group is the first to demonstrate the effect of ECVF in a histological analysis by in situ inoculation of broiler chickens with purified ECVF. The animals were inoculated with the APEC AC53 and with purified ECVF subcutaneously on their ventral surface (in the sternum region). The histological analysis showed different grades of an acute inflammatory response in the epidermis, dermis and panniculus. An increase in mRNA expression of the proinflammatory cytokine TNF-α was also demonstrated in the inflamed tissue. When ECVF was systemically administered, increased levels of TNF-α and IL-10 were observed in the serum. These results suggest that ECVF plays a key role in the inflammatory process associated with cellulitis that is mainly mediated by TNF-α. In addition, this inflammation can be downregulated by the anti-inflammatory cytokine IL-10.

  19. The anaerobic ribonucleoside triphosphate reductase from Escherichia coli requires S-adenosylmethionine as a cofactor.

    PubMed Central

    Eliasson, R; Fontecave, M; Jörnvall, H; Krook, M; Pontis, E; Reichard, P

    1990-01-01

    Extracts from anaerobically grown Escherichia coli contain an oxygen-sensitive activity that reduces CTP to dCTP in the presence of NADPH, dithiothreitol, Mg2+ ions, and ATP, different from the aerobic ribonucleoside diphosphate reductase (2'-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) present in aerobically grown E. coli. After fractionation, the activity required at least five components, two heat-labile protein fractions and several low molecular weight fractions. One protein fraction, suggested to represent the actual ribonucleoside triphosphate reductase was purified extensively and on denaturing gel electrophoresis gave rise to several defined protein bands, all of which were stained by a polyclonal antibody against one of the two subunits (protein B1) of the aerobic reductase but not by monoclonal anti-B1 antibodies. Peptide mapping and sequence analyses revealed partly common structures between two types of protein bands but also suggested the presence of an additional component. Obviously, the preparations are heterogeneous and the structure of the reductase is not yet established. The second, crude protein fraction is believed to contain several ancillary enzymes required for the reaction. One of the low molecular weight components is S-adenosylmethionine; a second component is a loosely bound metal. We propose that S-adenosylmethionine together with a metal participates in the generation of the radical required for the reduction of carbon 2' of the ribosyl moiety of CTP. Images PMID:2185465

  20. Cloning and expression of the leukotoxin gene of Pasteurella haemolytica A1 in Escherichia coli K-12.

    PubMed Central

    Lo, R Y; Shewen, P E; Strathdee, C A; Greer, C N

    1985-01-01

    A clone bank of Pasteurella haemolytica A1 was constructed by partial digestion of the genomic DNA with Sau3A and ligation of 5- to 10-kilobase-pair fragments into the BamHI site of the plasmid vector pBR322. After transformation into Escherichia coli K-12, a total of 4 X 10(3) recombinant clones was obtained. These were screened for the production of P. haemolytica soluble antigens by a colony enzyme-linked immunosorbent assay blot method with a rabbit antiserum raised against the soluble antigens. The clones producing P. haemolytica soluble antigens were then analyzed for the production of the leukotoxin by a cytotoxicity assay with cells from a bovine leukemia-derived B-lymphocyte cell line as the target cells. Positive clones were identified, and subsequent restriction analysis of the recombinant plasmids showed that the same 6.3 kilobase pairs of insert DNA was cloned in either of the two orientations into the plasmid vector pBR322. One of the clones was selected for further characterization of the leukotoxin as produced in E. coli. Tests for heat lability and target cell species specificity with canine, porcine, and human peripheral blood lymphocytes indicated that the activity of the cloned leukotoxin was identical to that of the P. haemolytica leukotoxin. Furthermore, the E. coli-produced leukotoxin was also neutralized by bovine or rabbit antiserum known to have antitoxic activity. When cellular proteins from the E. coli clones were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis, a 100,000-dalton protein was identified which corresponded to one of the soluble antigens found in the leukotoxic culture supernatant of P. haemolytica. These results demonstrated that the gene(s) for the P. haemolytica leukotoxin have been cloned and that the leukotoxin was expressed in E. coli. Images PMID:3905610

  1. Mucosa-associated but not luminal Escherichia coli is augmented in Crohn’s disease and ulcerative colitis

    PubMed Central

    2012-01-01

    Background Escherichia coli is believed to participate in the etiology of Crohn’s disease (CD) and possibly of ulcerative colitis (UC), due at least in part to the observed rise in the number of these bacteria in the gut microbiota of CD and UC patients. Nevertheless, it is not fully understood whether this quantitative variation occurs equally throughout the mucosal and luminal spaces of the gut. To assess this question, stools and mucosa biopsies from distinct intestinal sites were cultured aiming at determining their E. coli concentration. The cultures were additionally screened for the presence of some virulence genes of pathogenic E. coli. Results Analyses of clinical materials from 14 controls (38 biopsies and 14 stools samples), 11 CD (25 biopsies and 11 stools samples) and 7 UC patients (18 biopsies and 7 stools samples) indicated no significant variation in the number of E. coli present in stools, but a rise of at least one log10 CFU/mg in biopsies from the ileum of CD patients and the sigmoid and rectum of CD and UC patients. The cultures were screened for the presence of E. coli attaching and effacing (eae), invasion plasmid antigen H (ipaH), aggregative adherence transcriptional activator (aggR), Shiga cytotoxins (stx), and heat labile enterotoxin (elt) and the following serine proteases autotransporters of Enterobacteriaceae (SPATE) genes: plasmid encoded toxin (pet), secreted autotransporter toxin (sat), Shigella extracellular protein (sepA), protein involved in intestinal colonization (pic) and Shigella IgA-like protease homolog (sigA). Six of the 10 genes screened were detected in the total of samples investigated: aggR, eae, pet, sat, sepA and sigA. No difference in the prevalence of any of these markers was observed in cultures from different clinical materials or groups of patients. Methods Bacterial quantitation was carried out following cultures of diluted samples suspensions in MacConkey agar, Wilkins Chalgren agar for anaerobes, E. coli

  2. Pharmacological ascorbate and ionizing radiation (IR) increase labile iron in pancreatic cancer.

    PubMed

    Moser, Justin C; Rawal, Malvika; Wagner, Brett A; Du, Juan; Cullen, Joseph J; Buettner, Garry R

    2013-01-01

    Labile iron, i.e. iron that is weakly bound and is relatively unrestricted in its redox activity, has been implicated in both the pathogenesis as well as treatment of cancer. Two cancer treatments where labile iron may contribute to their mechanism of action are pharmacological ascorbate and ionizing radiation (IR). Pharmacological ascorbate has been shown to have tumor-specific toxic effects due to the formation of hydrogen peroxide. By catalyzing the oxidation of ascorbate, labile iron can enhance the rate of formation of hydrogen peroxide; labile iron can also react with hydrogen peroxide. Here we have investigated the magnitude of the labile iron pool in tumor and normal tissue. We also examined the ability of pharmacological ascorbate and IR to change the size of the labile iron pool. Although a significant amount of labile iron was seen in tumors (MIA PaCa-2 cells in athymic nude mice), higher levels were seen in murine tissues that were not susceptible to pharmacological ascorbate. Pharmacological ascorbate and irradiation were shown to increase the labile iron in tumor homogenates from this murine model of pancreatic cancer. As both IR and pharmacological ascorbate may rely on labile iron for their effects on tumor tissues, our data suggest that pharmacological ascorbate could be used as a radio-sensitizing agent for some radio-resistant tumors.

  3. Affective lability in patients with bipolar disorders is associated with high levels of childhood trauma.

    PubMed

    Aas, Monica; Aminoff, Sofie R; Vik Lagerberg, Trine; Etain, Bruno; Agartz, Ingrid; Andreassen, Ole A; Melle, Ingrid

    2014-08-15

    This study aimed to investigate associations between a history of childhood trauma and levels of affective lability in bipolar patients compared to controls. Forty-two patients and 14 controls were assessed using the Affective Lability Scale (ALS) and the Childhood Trauma Questionnaire (CTQ). Affective Lability Score was significantly associated with scores on the Childhood Trauma Questionnaire. A multivariate regression model indicated a relationship between childhood trauma scores and differences in affective lability between patients and controls. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  4. E. Coli

    MedlinePlus

    ... of Your Teeth El cuidado de los dientes Video: Getting an X-ray E. Coli KidsHealth > For Kids > E. Coli Print A A A What's in ... recalls affecting contaminated vegetables or other products. But kids can ... inside. Don't swallow lake, ocean, or pool water. If the water contains ...

  5. The essential Escherichia coli msgB gene, a multicopy suppressor of a temperature-sensitive allele of the heat shock gene grpE, is identical to dapE.

    PubMed Central

    Wu, B; Georgopoulos, C; Ang, D

    1992-01-01

    The grpE gene product is one of three Escherichia coli heat shock proteins (DnaK, DnaJ, and GrpE) that are essential for both bacteriophage lambda DNA replication and bacterial growth at all temperatures. In an effort to determine the role of GrpE and to identify other factors that it may interact with, we isolated multicopy suppressors of the grpE280 point mutation, as judged by their ability to reverse the temperature-sensitive phenotype of grpE280. Here we report the characterization of one of them, designated msgB. The msgB gene maps at approximately 53 min on the E. coli chromosome. The minimal gene possesses an open reading frame that encodes a protein with a predicted size of 41,269 M(r). This open reading frame was confirmed the correct one by direct amino-terminal sequence analysis of the overproduced msgB gene product. Genetic experiments demonstrated that msgB is essential for E. coli growth in the temperature range of 22 to 37 degrees C. Through a sequence homology search, MsgB was shown to be identical to N-succinyl-L-diaminopimelic acid desuccinylase (the dapE gene product), which participates in the diaminopimelic acid-lysine pathway involved in cell wall biosynthesis. Consistent with this finding, the msgB null allele mutant is viable only when the growth medium is supplemented with diaminopimelic acid. These results suggest that GrpE may have a previously unsuspected function(s) in cell wall biosynthesis in E. coli. Images PMID:1644751

  6. In-silico design, expression, and purification of novel chimeric Escherichia coli O157:H7 OmpA fused to LTB protein in Escherichia coli

    PubMed Central

    Novinrooz, Aytak; Zahraei Salehi, Taghi; Firouzi, Roya; Arabshahi, Sina; Derakhshandeh, Abdollah

    2017-01-01

    E. coli O157:H7, one of the major EHEC serotypes, is capable of developing bloody diarrhea, hemorrhagic colitis (HC), and fatal hemolytic uremic syndrome (HUS) and is accompanied by high annual economic loss worldwide. Due to the increased risk of HC and HUS development following antibiotic therapy, the prevention of infections caused by this pathogen is considered to be one of the most effective ways of avoiding the consequences of this infection. The main aim of the present study was to design, express, and purify a novel chimeric protein to develope human vaccine candidate against E. coli O157:H7 containing loop 2–4 of E. coli O157:H7, outer membrane protein A (OmpA), and B subunit of E. coli heat labile enterotoxin (LTB) which are connected by a flexible peptide linker. Several online databases and bioinformatics software were utilized to choose the peptide linker among 537 analyzed linkers, design the chimeric protein, and optimize the codon of the relative gene encoding this protein. Subsequently, the recombinant gene encoding OmpA-LTB was synthesized and cloned into pET-24a (+) expression vector and transferred to E. coli BL21(DE3) cells. The expression of OmpA-LTB chimeric protein was then carried out by induction of cultured E. coli Bl21 (DE3) cells with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG). The purification of OmpA-LTB was then performed by nickel affinity chromatography. Expression and purification were analyzed by sodium dodecyl sulphate poly acrylamide gel electrophoresis. Moreover, the identity of the expressed protein was analyzed by western blotting. SDS-PAGE and western immunoblotting confirmed the successful expression of a 27 KDa recombinant protein after 24 hours at 37°C post-IPTG induction. OmpA-LTB was then successfully purified, using nickel affinity chromatography under denaturing conditions. The yield of purification was 12 mg per liter of culture media. Ultimately, we constructed the successful design and efficient expression

  7. ESBL-plasmids carrying toxin-antitoxin systems can be “cured” of wild-type Escherichia coli using a heat technique

    PubMed Central

    2013-01-01

    Background Plasmid-encoded extended-spectrum beta-lactamase (ESBL)-enzymes are frequently produced by Escherichia coli. Several ESBL-plasmids contain genes for toxin-antitoxin (TA) systems, which assure the maintenance of plasmids in bacteria and prevent the cells from “post-segregational killing”. These systems limit options to “cure” plasmids of ESBL-wild-type strains due to the death of the bacterial cells. A helpful tool to understand the role of ESBL-plasmids in the dissemination of pandemic multi-resistant E. coli are ESBL-plasmid-“cured”-variants (PCVs) and their comparison to ESBL-wild-type strains. The purpose of this study was to construct PCVs of ESBL-wild-type E. coli strains despite the presence of genes for TA systems. Findings Using enhanced temperatures and brain-heart-infusion broth it was possible to construct viable PCVs of wild-type ESBL-E. coli strains. The occurrence of TA system-genes including hok/sok, srnB/C, vagC/D, pemI/K on ESBL-plasmids of replicon types FIA or FIB was demonstrated by bioinformatic analyses. The loss of the plasmid and the genetic identity of PCV and corresponding wild-type strain was confirmed via different methods including plasmid-profile-analysis, pulsed-field gel electrophoresis and bioinformatics using generated whole genome data of the strains. Conclusions This short report describes the successful construction of viable PCVs of ESBL-wild-type E. coli strains. The results are hence surprising due to the fact that all “cured” ESBL-plasmids contained at least one complete toxin-antitoxin system, whose loss would normally mean the death of bacterial cells. PMID:24245987

  8. Effects of high pressure and mild heat on endogenous microflora and on the inactivation and sublethal injury of Escherichia coli inoculated into fruit juices and vegetable soup.

    PubMed

    Muñoz, Marina; de Ancos, Begoña; Sánchez-Moreno, Concepción; Cano, M Pilar

    2007-07-01

    The objective of this study was to determine the effects of high-pressure treatments and mild temperatures on endogenous microflora and Escherichia coli CECT 515 artificially inoculated into orange and apple juices and vegetable soup. In general, the viability of aerobic bacteria was significantly reduced as pressure and temperature increased. Although the greatest reduction in the concentration of aerobic mesophilic vegetative cells was reached at 350 MPa and 60 degrees C, the same reduction occurred in fruit juices at 350 MPa and 20 degrees C. Yeasts and molds were below the level of detection (1 log CFU/ml) for the fruit juices and did not exceed 2 log CFU/ml for vegetable soup. Foods inoculated with E. coli were subjected to several treatments as indicated by the mathematical model applied in response surface methodology to obtain the maximum information with the minimum number of experiments. The number of tests for a range of pressures (150 to 350 MPa) and temperatures (20 to 60 degrees C) was limited to 11. The models were considered adequate because of satisfactory R2 values. The optimum process parameters (pressure and temperature) for a 6-log reduction of E. coli were obtained at 248.25 MPa and 59.91 degrees C in orange juice, 203.50 MPa and 57.18 degrees C in apple juice, and 269.8 MPa and 59.9 degrees C in vegetable soup. Sublethal injury of E. coli occurred as pressure and temperature increased. Nearly all of the E. coli cells were injured at 350 MPa and 20 degrees C in fruit juices and after all treatments in vegetable soup.

  9. The role of labile sulfur compounds in thermochemical sulfate reduction

    NASA Astrophysics Data System (ADS)

    Amrani, Alon; Zhang, Tongwei; Ma, Qisheng; Ellis, Geoffrey S.; Tang, Yongchun

    2008-06-01

    The reduction of sulfate to sulfide coupled with the oxidation of hydrocarbons to carbon dioxide, commonly referred to as thermochemical sulfate reduction (TSR), is an important abiotic alteration process that most commonly occurs in hot carbonate petroleum reservoirs. In the present study we focus on the role that organic labile sulfur compounds play in increasing the rate of TSR. A series of gold-tube hydrous pyrolysis experiments were conducted with n-octane and CaSO4 in the presence of reduced sulfur (e.g. H2S, S°, organic S) at temperatures of 330 and 356 °C under a constant confining pressure. The in-situ pH was buffered to 3.5 (∼6.3 at room temperature) with talc and silica. For comparison, three types of oil with different total S and labile S contents were reacted under similar conditions. The results show that the initial presence of organic or inorganic sulfur compounds increases the rate of TSR. However, organic sulfur compounds, such as 1-pentanethiol or diethyldisulfide, were significantly more effective in increasing the rate of TSR than H2S or elemental sulfur (on a mole S basis). The increase in rate is achieved at relatively low concentrations of 1-pentanethiol, less than 1 wt% of the total n-octane, which is comparable to the concentration of organic S that is common in many oils (∼0.3 wt%). We examined several potential reaction mechanisms to explain the observed reactivity of organic LSC. First, the release of H2S from the thermal degradation of thiols was discounted as an important mechanism due to the significantly greater reactivity of thiol compared to an equivalent amount of H2S. Second, we considered the generation of olefines in association with the elimination of H2S during thermal degradation of thiols because olefines are much more reactive than n-alkanes during TSR. In our experiments, olefines increased the rate of TSR, but were less effective than 1-pentanethiol and other organic LSC. Third, the thermal decomposition of

  10. Chemical leaching methods and measurements of marine labile particulate Fe

    NASA Astrophysics Data System (ADS)

    Revels, B. N.; John, S.

    2012-12-01

    Iron (Fe) is an essential nutrient for life. Yet its low solubility and concentration in the ocean limits marine phytoplankton productivity in many regions of the world. Dissolved phase Fe (<0.4μm) has traditionally been considered the most biologically accessible form, however, the particulate phase (>0.4μm) may contain an important, labile reservoir of Fe that may also be available to phytoplankton. However, concentration data alone cannot elucidate the sources of particulate Fe to the ocean and to what extent particulate iron may support phytoplankton growth. Isotopic analysis of natural particles may help to elucidate the biogeochemical cycling of Fe, though it is important to find a leaching method which accesses bioavailable Fe. Thirty-three different chemical leaches were performed on a marine sediment reference material, MESS-3. The combinations included four different acids (25% acetic acid, 0.01M HCl, 0.5M HCl, 0.1M H2SO4 at pH2), various redox conditions (0.02M hydroxylamine hydrochloride or 0.02M H2O2), three temperatures (25°C, 60°C, 90°C), and three time points (10 minutes, 2 hours, 24 hours). Leached Fe concentrations varied from 1mg/g to 35mg/g, with longer treatment times, stronger acids, and hotter temperatures generally associated with an increase in leached Fe. δ56Fe in these leaches varied from -1.0‰ to +0.2‰. Interestingly, regardless of leaching method used, there was a very similar relationship between the amount of Fe leached from the particles and the δ56Fe of this iron. Isotopically lighter δ56Fe values were associated with smaller amounts of leached Fe whereas isotopically heavier δ56Fe values were associated with larger amounts of leached Fe. Two alternate hypotheses could explain these data. Either, the particles may contain pools of isotopically light Fe that are easily accessed early in dissolution, or isotopically light Fe may be preferentially leached from the particle due to a kinetic isotope effect during dissolution

  11. [Labile iron pool formation in rat's blood under rhabdomyolysis].

    PubMed

    Shandrenko, S H

    2012-01-01

    The labile nonheme iron pool formation in blood under glycerol induced rhabdomyolysis in rats has been investigated. This iron is not included in transferrin, thereby it is redox-active. Rhabdomyolysis was caused by intramuscular injection of 50% glycerol in a dose of 10 ml/kg. In the first day it has been registered that the blood plasma free heme content increased 10 times and the liver heme-oxigenase activity increased 6 times. Plasma redox-active iron pool formation has been registered by EPR method. Such iron was absent in the control group. This iron pool content in the interval from the 1st to the 6st day was more than 2 mg/l and significantly higher than the transferrin iron level. The plasma iron pool unshielded by transferrin may be one of oxidative stress causes.

  12. Lability of renal papillary tissue composition in the rat.

    PubMed Central

    Atherton, J C

    1978-01-01

    1. The acute effects of (a) a minor operative procedure using ether as the anaesthetic, and (b) the administration of 0.9% saline as a single I.V. injection in the conscious rat, on renal tissue composition were studied in hydropenic and normally hydrated rats. 2. The operative procedure and anaesthesia induced a rapid and transient decrease in papillary osmolality in both hydropenic and normally hydrated animals, the important contributing factor being a significant decrease in urea content. 3. Administration of a small volume of saline caused a rapid decrease in urea content, and an increase in water content. 4. It is concluded that papillary composition is extremely labile, large changes being produced by relatively minor experimental procedures. PMID:624997

  13. The role of labile sulfur compounds in thermochemical sulfate reduction

    USGS Publications Warehouse

    Amrani, A.; Zhang, T.; Ma, Q.; Ellis, G.S.; Tang, Y.

    2008-01-01

    The reduction of sulfate to sulfide coupled with the oxidation of hydrocarbons to carbon dioxide, commonly referred to as thermochemical sulfate reduction (TSR), is an important abiotic alteration process that most commonly occurs in hot carbonate petroleum reservoirs. In the present study we focus on the role that organic labile sulfur compounds play in increasing the rate of TSR. A series of gold-tube hydrous pyrolysis experiments were conducted with n-octane and CaSO4 in the presence of reduced sulfur (e.g. H2S, S??, organic S) at temperatures of 330 and 356 ??C under a constant confining pressure. The in-situ pH was buffered to 3.5 (???6.3 at room temperature) with talc and silica. For comparison, three types of oil with different total S and labile S contents were reacted under similar conditions. The results show that the initial presence of organic or inorganic sulfur compounds increases the rate of TSR. However, organic sulfur compounds, such as 1-pentanethiol or diethyldisulfide, were significantly more effective in increasing the rate of TSR than H2S or elemental sulfur (on a mole S basis). The increase in rate is achieved at relatively low concentrations of 1-pentanethiol, less than 1 wt% of the total n-octane, which is comparable to the concentration of organic S that is common in many oils (???0.3 wt%). We examined several potential reaction mechanisms to explain the observed reactivity of organic LSC. First, the release of H2S from the thermal degradation of thiols was discounted as an important mechanism due to the significantly greater reactivity of thiol compared to an equivalent amount of H2S. Second, we considered the generation of olefines in association with the elimination of H2S during thermal degradation of thiols because olefines are much more reactive than n-alkanes during TSR. In our experiments, olefines increased the rate of TSR, but were less effective than 1-pentanethiol and other organic LSC. Third, the thermal decomposition of

  14. Effects of labile carbon addition on a headwater stream food web

    Treesearch

    Heidi S. Wilcox; J. Bruce Wallace; Judy L. Meyer; Jonathan P. Benstead

    2005-01-01

    We added dextrose for two 8-week periods (summer and autumn) to a highly heterotrophic headwater stream in North Carolina, U.S.A., to examine the responses of its benthic food web to increased labile carbon. We hypothesized that addition of labile carbon would elevate microbial abundance and activity, resulting in greater resource availability and higher...

  15. Phaeochromocytoma presenting with labile blood pressures following coronary artery bypass grafting.

    PubMed

    Roy, James; Akhunji, Zakir; Kushwaha, Virag; Mackie, James; Jepson, Nigel

    2016-12-01

    Pheochromocytomas have been reported prior to and during coronary artery bypass surgery. We present a patient with an undiagnosed pheochromocytoma who presented with labile hypertension following coronary artery bypass surgery. This case calls attention to the inclusion of an undiagnosed pheochromocytoma in the differential diagnosis for all patients who develop labile hypertension in the postoperative period following cardiac surgery. © 2016 Wiley Periodicals, Inc.

  16. Elemental composition and functional groups in soil labile organic matter fractions

    USDA-ARS?s Scientific Manuscript database

    Labile organic matter fractions are major components involved in nutrient cycle in soil. In this chapter, we examine three labile organic matter fraction: light fraction (LF), humic acid (HA) and fulvic acid (HA) in Alabama cotton soils (ultisol) amended with chemical fertilizer (NH4NO3) and poult...

  17. Lability criteria for successive metal complexes in steady-state planar diffusion.

    PubMed

    Salvador, José; Puy, Jaume; Galceran, Josep; Cecília, Joan; Town, Raewyn M; van Leeuwen, Herman P

    2006-01-19

    The lability of sequential metal complexes, ML, ML2, ML3, ... , up to a general 1:n metal/ligand stoichiometric ratio is considered for the case of metal ions (M) being accumulated at a surface (analytical sensor or organism). The analytical solution for the steady-state diffusion of M within a sequential complexation scheme allows quantification of the contribution from the dissociation of all of the complex species to the metal flux through the so-called lability degree, xi. A lability degree for each sequential complexation step is also defined which, due to the sequential character of the complexation scheme, depends not only on the proper kinetic constants of the given complexation step but also on the kinetics of the previous ones. When all contributions from the complexes are diffusion limited, the system is fully labile and xi=1. To provide simple lability criteria, the reaction layer approximation is extended to specifically deal with this sequential complexation scheme, so that a reaction layer thickness is defined when the existence of one particular rate-limiting step is assumed. Expressions for the classical lability parameter, L, are formulated using the reaction layer approximation. The change of the lability of the system as the diffusion layer thickness is modified is analyzed in detail. The contribution of the complex flux reflects the evolution of the system from labile to inert as the thickness of the sensor is appropriately decreased.

  18. Estimating soil labile organic carbon and potential turnover rates using a sequential fumigation–incubation procedure.

    Treesearch

    X.M. Zoua; H.H. Ruanc; Y. Fua; X.D. Yanga; L.Q. Sha

    2005-01-01

    Labile carbon is the fraction of soil organic carbon with most rapid turnover times and its oxidation drives the flux of CO2 between soils and atmosphere. Available chemical and physical fractionation methods for estimating soil labile organic carbon are indirect and lack a clear biological definition. We have modified the well-established Jenkinson and Powlson’s...

  19. Knowledge lability: Within-person changes in parental knowledge and their associations with adolescent problem behavior

    PubMed Central

    Lippold, Melissa A.; Fosco, Gregory; Ram, Nilam; Feinberg, Mark E.

    2015-01-01

    Higher levels of parental knowledge about youth activities has been associated with lower levels of youth risky behavior. Yet little is known about how parental knowledge fluctuates during early adolescence and how those fluctuations are associated with the development of problem behavior. We use the term lability to describe within-person fluctuations in knowledge over time with higher lability indicating greater fluctuations in knowledge from year-to-year. This longitudinal study of rural adolescents (N = 840) investigated if change in parental knowledge across four waves of data from Grades 6 to 8 is characterized by lability, and if greater lability is associated with higher youth substance use, delinquency, and internalizing problems in Grade 9. Our models indicated that only some of the variance in parental knowledge was accounted for by developmental trends. The remaining residual variance reflects within-person fluctuations around these trends, lability, plus measurement and occasion-specific error. Even controlling for level and developmental trends in knowledge, higher knowledge lability (i.e., more fluctuation) was associated with increased risk for later alcohol and tobacco use, and for girls, higher delinquency and internalizing problems. Our findings suggest that lability in parental knowledge has unique implications for adolescent outcomes. The discussion focuses on mechanisms that may link knowledge lability to substance use. Interventions may be most effective if they teach parents to consistently and predictably decrease knowledge across early adolescence. PMID:26381431

  20. Labile proteins accumulated in damaged hair upon permanent waving and bleaching treatments.

    PubMed

    Inoue, Takafumi; Ito, Mayumi; Kizawa, Kenji

    2002-01-01

    We previously found that certain hair proteins were soluble by means of a partial extraction method. In this study, we demonstrate that the amount of soluble proteins internally formed in permed and bleached hair, labile proteins, is a useful index for hair damage assessment. Compared to tensile property changes, this index rose in widely dynamic ranges as the time of either permanent waving or bleaching treatments increased. The amount of labile proteins was much larger than that of proteins eluted into perming and bleaching lotions. However, the labile proteins showed electrophoretic profiles similar to those of the eluted proteins. These results suggest that a portion of the stable proteins in normal hair was transformed into labile proteins upon permanent waving and bleaching treatments. Consequently, permed and bleached hair tends to release the resultant labile proteins.

  1. Enterotoxigenic Escherichia coli and diffusely adherent E. coli as likely causes of a proportion of pathogen-negative travelers' diarrhea--a PCR-based study.

    PubMed

    Meraz, Ismail M; Jiang, Zhi-Dong; Ericsson, Charles D; Bourgeois, A Louis; Steffen, Robert; Taylor, David N; Hernandez, Norma; DuPont, Herbert L

    2008-01-01

    Enteropathogens cannot be identified in 40% to 50% of subjects with travelers' diarrhea (TD). We used polymerase chain reaction (PCR) methods to look for the presence of two bacterial causes of diarrhea in a large group of international travelers after failing to detect a pathogen by conventional tests. DNA was isolated from the diarrheal stool and subjected to PCR from 162 subjects from whom we earlier failed to identify a pathogen in a previous study and included 54 from Antigua, Guatemala, 39 from Guadalajara, Mexico, 29 from Kolkata, India, and 40 from Goa, India. Gene products for enterotoxigenic Escherichia coli (ETEC)--LT (heat-labile enterotoxin) and ST (heat-stable enterotoxin)--and diffusely adherent E. coli (DAEC), afa/dr (Afa fimbrial and Dr nonfimbrial family of adhesins), were used. At least one gene product was identified in diarrhea stool samples of 47 of 162 (29%) subjects. ETEC virulence genes (LT, ST) were found in 34 (21%) samples studied, with rates of occurrence ranging from 8% in Goa to 39% for the samples from Guatemala (p = 0.0006). A large number of ST-only strains explained the high ETEC rate in Guatemala. DAEC afa/dr family of adhesions was identified in between 8 and 14% of the samples. ETEC and DAEC were implicated in nearly one-third of the subjects initially diagnosed as pathogen negative. Direct PCR results from stools are consistent with the previous assumption that most undiagnosed TD is bacterial in nature and also highlights the potential value that PCR can add to studies designed to evaluate treatment and preventive interventions for TD, including vaccines.

  2. Antibacterial and Antidiarrheal Activities of Plant Products against Enterotoxinogenic Escherichia coli

    PubMed Central

    Dubreuil, J. Daniel

    2013-01-01

    Enterotoxigenic Escherichia coli (ETEC) produces two types of enterotoxins: heat-labile (LT) and heat-stable (STa and STb). These molecules are involved in the induction of secretory diarrhea in animals including humans. This condition is currently treated using a fluid replacement therapy and antibiotics. This treatment is often not available to people in developing countries, and several die from the condition provoke by ETEC. Over the years, plants and plant extracts have been use as traditional medicine to treat various gastrointestinal ailments including diarrhea. Many of these plant products have been claimed to be active against diarrhea, however few have been extensively studied. The main objective of this review was to gather the scattered information on the antidiarrheal activities reported for various plant products on ETEC. This includes two major effects: (1) The inhibitory effect on bacterial growth or viability and (2) The interference with ETEC enterotoxins activity upon the intestinal epithelium. We will focus on plant products and extracts for which we have major indications of their biological activity against ETEC and their enterotoxins. Because Vibrio cholerae toxin (CT) is structurally, antigenically and mechanistically related to LT, it will also be discussed in this review. PMID:24212181

  3. Identification of enterotoxigenic Escherichia coli with synthetic alkaline phosphatase-conjugated oligonucleotide DNA probes.

    PubMed Central

    Seriwatana, J; Echeverria, P; Taylor, D N; Sakuldaipeara, T; Changchawalit, S; Chivoratanond, O

    1987-01-01

    Alkaline phosphatase-conjugated (AP) 26-base oligonucleotide DNA probes were compared with the same probes labeled with gamma-32P for the identification of heat-labile (LT) and heat-stable (ST) enterotoxigenic Escherichia coli (ETEC). The AP oligonucleotide probes were as sensitive as the radiolabeled (RL) probes in detecting LT and STA-2 target cell DNA, but the AP ST probe, which differed from STA-1 by two bases, was less sensitive than the RL probe in detecting STA-1 DNA (6.25 versus 0.78 ng). Of 94 ETEC that were identified with the RL probe, the AP probes detected 93% (28 of 30) of ST, 73% (25 of 34) of LT, and 67% (20 of 30) of LTST ETEC. When colony lysates of these ETEC were examined, the AP probes identified all 94 ETEC. In examinations of stool blots, the RL and AP probes were shown to have sensitivities of 71 and 59%, specificities of 91 and 86%, positive predictive values of 87 and 73%, and negative predictive values of 86 and 74%, respectively. AP oligonucleotide probes to detect ETEC were less sensitive in detecting ETEC by colony or stool blot hybridization than the RL probes but could be used by laboratories without access to radioisotopes to examine colony lysates. Images PMID:3305559

  4. Comparative Effect of Heat Shock on Survival of O157:H7 and Non-O157 Shiga Toxigenic Escherichia coli and Salmonella in Lean Beef with or without Moisture-Enhancing Ingredients.

    PubMed

    Vasan, Akhila; Ingham, Steven C; Ingham, Barbara H

    2017-06-01

    Thermal tolerance of pathogenic bacteria has been shown to increase after exposure to sublethal elevated temperatures, or heat shock. We evaluated the effect of heat shock at 48°C on thermal tolerance (D55°C) of cocktails of O157 and non-O157 Shiga toxigenic Escherichia coli (STEC) and Salmonella in lean ground beef with or without moisture-enhancing ingredients. Beef was moisture enhanced to 110% (w) with a 5% NaCl-2.5% sodium tripolyphosphate (w/w) brine. Meat, with or without added brine, was inoculated (∼10(8) CFU/g) and heat shocked at 48°C for 0, 5, or 30 min, followed by isothermal heating at 55°C. Inoculated control samples were unenhanced and were not subject to heat shock. From the linear portion of the log CFU per gram surviving cells over time plots, D55°C-values (minutes) were calculated. D55°C was 20.43, 28.78, and 21.15 min for O157, non-O157, and Salmonella controls, respectively. Overall, heat shock significantly increased D55°C, regardless of pathogen (P < 0.05). After 30 min of heat shock, D55°C increased 89 and 160% for O157 STEC, 32 and 49% for non-O157 STEC, and 29 and 57% for Salmonella, in unenhanced and enhanced samples, respectively, relative to the pathogen control. D55°C for Salmonella was the same or significantly less than for O157 and non-O157 STEC, regardless of heat shock, and was significantly less than for O157 and non-O157 STEC in all trials with moisture-enhanced meat (P < 0.05). Moisture-enhancing ingredients significantly increased D55°C, regardless of pathogen (P < 0.05). We suggest that thermal processes validated against Salmonella may not prove effective against STEC in all cases and that regulators of the beef industry should focus attention on STEC in nonintact moisture-enhanced beef products.

  5. A bacteriophage-encoded J-domain protein interacts with the DnaK/Hsp70 chaperone and stabilizes the heat-shock factor σ32 of Escherichia coli.

    PubMed

    Perrody, Elsa; Cirinesi, Anne-Marie; Desplats, Carine; Keppel, France; Schwager, Françoise; Tranier, Samuel; Georgopoulos, Costa; Genevaux, Pierre

    2012-01-01

    The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ(32), which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ(32) facilitates RB43 bacteriophage proliferation is discussed.

  6. A Bacteriophage-Encoded J-Domain Protein Interacts with the DnaK/Hsp70 Chaperone and Stabilizes the Heat-Shock Factor σ32 of Escherichia coli

    PubMed Central

    Perrody, Elsa; Cirinesi, Anne-Marie; Desplats, Carine; Keppel, France; Schwager, Françoise; Tranier, Samuel; Georgopoulos, Costa; Genevaux, Pierre

    2012-01-01

    The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ32, which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ32 facilitates RB43 bacteriophage proliferation is discussed. PMID:23133404

  7. Lability of trace metals in submerged soils: a column study

    NASA Astrophysics Data System (ADS)

    Nimirciag, Ramona; Ajmone-Marsan, Franco

    2013-04-01

    The reduction of Fe (III) and Mn (IV) and the decomposition of organic matter exert a great influence on the biogeochemical cycles of many trace metals and nutrients in the environment. In the particular case of intermittently submerged soils, metals associated with Fe and Mn oxides become readily available due to the reductive dissolution of Fe and Mn oxides. The effects of oxido-reductive conditions on the release of Cu and Zn from heavy metal contaminated soils and the changes in their chemical speciation were studied. Column experiments were performed, using Rhizon soil moisture samplers inserted at different heights to monitor the mobility and transport of metals in the submerged soil samples. Cu was released in solution immediately, in the first red-ox cycle, either due to the solubilization of Fe and Mn oxides, or to the oxidation of organic matter with which Cu is commonly complexed, or both. During the following reductive half-cycles, the amount of Cu extracted from the soil solution decreased. However, the concentration of Cu in the solution leached from the column, which was percolated in aerobic conditions, increased. Since in the successive red-ox cycles the Eh decreases faster and to lower values, it is possible that Cu might have been removed from pore water by sulfide precipitation during the anaerobic half-cycle and released during the aerobic half-cycle, due to the oxidation of sulfides to sulfates. The release of Zn was similar to the dissolution of Fe and Mn oxyhydroxydes, and the amount extracted by Rhizon and by leaching increased during the four red-ox cycles. The chemical fractionation of the soils was also studied and the results showed that the alternate oxidative-reductive conditions cause, in general, an increase in the lability of trace metals. While Zn speciation suffers little change, Cu showed a much higher exchangeable fraction in the submerged soils, as compared to the initial, not submerged ones. The results of this study indicate

  8. High Sensitivity Combined with Extended Structural Coverage of Labile Compounds via Nanoelectrospray Ionization at Subambient Pressures

    DOE PAGES

    Cox, Jonathan T.; Kronewitter, Scott R.; Shukla, Anil K.; ...

    2014-09-15

    Subambient pressure ionization with nanoelectrospray (SPIN) has proven to be effective in producing ions with high efficiency and transmitting them to low pressures for high sensitivity mass spectrometry (MS) analysis. Here we present evidence that not only does the SPIN source improve MS sensitivity but also allows for gentler ionization conditions. The gentleness of a conventional heated capillary electrospray ionization (ESI) source and the SPIN source was compared by the liquid chromatography mass spectrometry (LC-MS) analysis of colominic acid. Colominic acid is a mixture of sialic acid polymers of different lengths containing labile glycosidic linkages between monomer units necessitating amore » gentle ion source. By coupling the SPIN source with high resolution mass spectrometry and using advanced data processing tools, we demonstrate much extended coverage of sialic acid polymer chains as compared to using the conventional ESI source. Additionally we show that SPIN-LC-MS is effective in elucidating polymer features with high efficiency and high sensitivity previously unattainable by the conventional ESI-LC-MS methods.« less

  9. Simultaneous near-infrared radiant heating and UV radiation for inactivating Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in powdered red pepper (Capsicum annuum L.).

    PubMed

    Ha, Jae-Won; Kang, Dong-Hyun

    2013-11-01

    This study was conducted to investigate the efficacy of the simultaneous application of near-infrared (NIR) heating and UV irradiation for reducing populations of food-borne pathogens, including Salmonella enterica serovar Typhimurium and Escherichia coli O157:H7 in red pepper powder and to clarify the mechanisms of the lethal effect of the NIR-UV combined treatment. Also, the effect of the combination treatment on quality was determined by measuring changes in color and pungency constituents. Simultaneous NIR-UV combined treatment for 5 min achieved 3.34- and 2.78-log CFU reductions in S. Typhimurium and E. coli O157:H7, respectively, which involved 1.86- and 1.31-log CFU reductions, respectively, which were attributed to the synergistic effect. Through qualitative and quantitative analyses, damage to the cell envelope was identified as the main factor contributing to the synergistic lethal effect of NIR-UV combined treatment. Color values and capsaicin and dihydrocapsaicin content of NIR-UV simultaneously treated red pepper powder were not significantly (P > 0.05) different from those of untreated samples. These results suggest that simultaneous application of NIR and UV treatment can be effectively used to control food-borne pathogens in powdered red pepper without affecting quality.

  10. Simultaneous Near-Infrared Radiant Heating and UV Radiation for Inactivating Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium in Powdered Red Pepper (Capsicum annuum L.)

    PubMed Central

    Ha, Jae-Won

    2013-01-01

    This study was conducted to investigate the efficacy of the simultaneous application of near-infrared (NIR) heating and UV irradiation for reducing populations of food-borne pathogens, including Salmonella enterica serovar Typhimurium and Escherichia coli O157:H7 in red pepper powder and to clarify the mechanisms of the lethal effect of the NIR-UV combined treatment. Also, the effect of the combination treatment on quality was determined by measuring changes in color and pungency constituents. Simultaneous NIR-UV combined treatment for 5 min achieved 3.34- and 2.78-log CFU reductions in S. Typhimurium and E. coli O157:H7, respectively, which involved 1.86- and 1.31-log CFU reductions, respectively, which were attributed to the synergistic effect. Through qualitative and quantitative analyses, damage to the cell envelope was identified as the main factor contributing to the synergistic lethal effect of NIR-UV combined treatment. Color values and capsaicin and dihydrocapsaicin content of NIR-UV simultaneously treated red pepper powder were not significantly (P > 0.05) different from those of untreated samples. These results suggest that simultaneous application of NIR and UV treatment can be effectively used to control food-borne pathogens in powdered red pepper without affecting quality. PMID:23956394

  11. Affect lability predicts occurrence of suicidal ideation in bipolar patients: a two-year prospective study.

    PubMed

    Ducasse, D; Jaussent, I; Guillaume, S; Azorin, J M; Bellivier, F; Belzeaux, R; Bougerol, T; Etain, B; Gard, S; Henry, C; Kahn, J P; Leboyer, M; Loftus, J; Passerieux, C; Olié, E; Courtet, P

    2017-05-01

    The aim of our study was to investigate, in bipolar patients, whether affect lability was associated with suicidal ideation incidence during 2-year follow-up, and which subtype of affect lability was associated with suicidal ideation. A total of 319 euthymic or mildly depressed bipolar outpatients recruited in the French FondaMental Advanced Centres of Expertise for Bipolar Disorder were divided into two subgroups according to the occurrence of suicidal ideation during a 2-year follow-up. Affect lability was assessed by the French version of the Affect Lability Scale. Bipolar patients with high affect lability were more likely to report suicidal ideation during follow-up, even after adjustment for age, study level, rapid cycling, current depression level, anxiety disorder, and lifetime history SA (OR = 2.47; 95% CI [1.15-5.30], P = 0.01). The risk of suicidal ideation increased with the level of affect lability. More specifically, the propensity to switch from neutral to elation affect, from anxious to depressive affect (or inversely), and from neutral to anger affect predicted suicidal ideation. Reducing affective lability could become a new therapeutic target of suicidal prevention in bipolar disorder. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Cell-to-cell transformation in Escherichia coli: a novel type of natural transformation involving cell-derived DNA and a putative promoting pheromone.

    PubMed

    Etchuuya, Rika; Ito, Miki; Kitano, Seiko; Shigi, Fukiko; Sobue, Rina; Maeda, Sumio

    2011-01-20

    Escherichia coli is not assumed to be naturally transformable. However, several recent reports have shown that E. coli can express modest genetic competence in certain conditions that may arise in its environment. We have shown previously that spontaneous lateral transfer of non-conjugative plasmids occurs in a colony biofilm of mixed E. coli strains (a set of a donor strain harbouring a plasmid and a plasmid-free recipient strain). In this study, with high-frequency combinations of strains and a plasmid, we constructed the same lateral plasmid transfer system in liquid culture. Using this system, we demonstrated that this lateral plasmid transfer was DNase-sensitive, indicating that it is a kind of transformation in which DNase-accessible extracellular naked DNA is essential. However, this transformation did not occur with purified plasmid DNA and required a direct supply of plasmid from co-existing donor cells. Based on this feature, we have termed this transformation type as 'cell-to-cell transformation'. Analyses using medium conditioned with the high-frequency strain revealed that this strain released a certain factor(s) that promoted cell-to-cell transformation and arrested growth of the other strains. This factor is heat-labile and protease-sensitive, and its roughly estimated molecular mass was between ∼9 kDa and ∼30 kDa, indicating that it is a polypeptide factor. Interestingly, this factor was effective even when the conditioned medium was diluted 10(-5)-10(-6), suggesting that it acts like a pheromone with high bioactivity. Based on these results, we propose that cell-to-cell transformation is a novel natural transformation mechanism in E. coli that requires cell-derived DNA and is promoted by a peptide pheromone. This is the first evidence that suggests the existence of a peptide pheromone-regulated transformation mechanism in E. coli and in Gram-negative bacteria.

  13. Labile aggregation stimulating substance, free fatty acids, and platelet aggregation.

    PubMed

    Gerrard, J M; White, J G; Krivit, W

    1976-01-01

    Labile aggregation stimulating substance (LASS), an intermediate produced during platelet biosynthesis of PGE2 and PGF2alpha, acts as a physiologic intercellular messenger to promote platelet aggregation and the release reaction. The activity is formed by intact cells after physiologic stimulation or can be generated from platelet membrane fractions after combination with arachidonate. In the present investigation, small amounts of polyunsaturated fatty acids added to an incubation mixture of platelet microsomes and arachidonate were found to significantly inhibit subsequent platelet aggregation. Saturated and mono-unsaturated fatty acids in the same concentrations were without effect. However, in higher concentrations mono-unsaturated fatty acids were found to be inhibitory and stearic acid was found to enhance subsequent platelet aggregation. The inhibition caused by the polyunsaturated fatty acid, linoleate, was shown to be the result of an effect on the production of LASS through an interaction with the platelet enzyme responsible for conversion of arachidonate to LASS. In contrast, stearic acid was found to enhance platelet aggregation by acting on the platelets and not directly on LASS production. The results suggest that small changes in the fatty acid composition of platelet phospholipids could significantly influence platelet reactivity.

  14. Anticariogenic and phytochemical evaluation of Eucalyptus globules Labill.

    PubMed Central

    Ishnava, Kalpesh B.; Chauhan, Jenabhai B.; Barad, Mahesh B.

    2012-01-01

    In the present study, in vitro anticariogenic potential of ethyl acetate, hexane and methanol and aqueous extracts of plant leaves of Eucalyptus globules Labill. were evaluated by using four cariogenic bacteria, Lactobacillus acidophilus, Lactobacillus casei, Staphylococcus aureus and Streptococcus mutans. Agar well diffusion method and minimum inhibitory concentration (MIC) were used for this purpose. The ethyl acetate extracted fraction of plant leaves showed good inhibitory effects against all selected bacteria. In Eucalyptus globules, hexane and ethyl acetate extracts found highly effective against, Lactobacillus acidophilus with MIC value of 0.031 and 0.062 mg/mL, respectively. Qualitative phytochemical investigation of above extracts showed the presence of alkaloids, phenolic compounds, steroids, cardiac glycosides and terpenes. Based on the MIC value and bioautography, ethyl acetate of plant leaf was selected for further study. Further investigation on the structure elucidation of the bioactive compound using IR, GC-MS and NMR techniques revealed the presence of alpha-farnesene, a sesquiterpene. Eucalyptus globules plant leaf extracts have great potential as anticariogenic agents that may be useful in the treatment of oral disease. PMID:23961222

  15. Microbial formation of labile organic carbon in Antarctic glacial environments

    NASA Astrophysics Data System (ADS)

    Smith, H. J.; Foster, R. A.; McKnight, D. M.; Lisle, J. T.; Littmann, S.; Kuypers, M. M. M.; Foreman, C. M.

    2017-04-01

    Roughly six petagrams of organic carbon are stored within ice worldwide. This organic carbon is thought to be of old age and highly bioavailable. Along with storage of ancient and new atmospherically deposited organic carbon, microorganisms may contribute substantially to the glacial organic carbon pool. Models of glacial microbial carbon cycling vary from net respiration to net carbon fixation. Supraglacial streams have not been considered in models although they are amongst the largest ecosystems on most glaciers and are inhabited by diverse microbial communities. Here we investigate the biogeochemical sequence of organic carbon production and uptake in an Antarctic supraglacial stream in the McMurdo Dry Valleys using nanometre-scale secondary ion mass spectrometry, fluorescence spectroscopy, stable isotope analysis and incubation experiments. We find that heterotrophic production relies on highly labile organic carbon freshly derived from photosynthetic bacteria rather than legacy organic carbon. Exudates from primary production were utilized by heterotrophs within 24 h, and supported bacterial growth demands. The tight coupling of microbially released organic carbon and rapid uptake by heterotrophs suggests a dynamic local carbon cycle. Moreover, as temperatures increase there is the potential for positive feedback between glacial melt and microbial transformations of organic carbon.

  16. Continuous flow analysis of labile iron in ice-cores.

    PubMed

    Hiscock, William T; Fischer, Hubertus; Bigler, Matthias; Gfeller, Gideon; Leuenberger, Daiana; Mini, Olivia

    2013-05-07

    The important active and passive role of mineral dust aerosol in the climate and the global carbon cycle over the last glacial/interglacial cycles has been recognized. However, little data on the most important aeolian dust-derived biological micronutrient, iron (Fe), has so far been available from ice-cores from Greenland or Antarctica. Furthermore, Fe deposition reconstructions derived from the palaeoproxies particulate dust and calcium differ significantly from the Fe flux data available. The ability to measure high temporal resolution Fe data in polar ice-cores is crucial for the study of the timing and magnitude of relationships between geochemical events and biological responses in the open ocean. This work adapts an existing flow injection analysis (FIA) methodology for low-level trace Fe determinations with an existing glaciochemical analysis system, continuous flow analysis (CFA) of ice-cores. Fe-induced oxidation of N,N'-dimethyl-p-pheylenediamine (DPD) is used to quantify the biologically more important and easily leachable Fe fraction released in a controlled digestion step at pH ~1.0. The developed method was successfully applied to the determination of labile Fe in ice-core samples collected from the Antarctic Byrd ice-core and the Greenland Ice-Core Project (GRIP) ice-core.

  17. Structural Lability of Barley Stripe Mosaic Virus Virions

    PubMed Central

    Semenyuk, Pavel I.; Abashkin, Dmitry A.; Kalinina, Natalya O.; Arutyunyan, Alexsandr M.; Solovyev, Andrey G.; Dobrov, Eugeny N.

    2013-01-01

    Virions of Barley stripe mosaic virus (BSMV) were neglected for more than thirty years after their basic properties were determined. In this paper, the physicochemical characteristics of BSMV virions and virion-derived viral capsid protein (CP) were analyzed, namely, the absorption and intrinsic fluorescence spectra, circular dichroism spectra, differential scanning calorimetry curves, and size distributions by dynamic laser light scattering. The structural properties of BSMV virions proved to be intermediate between those of Tobacco mosaic virus (TMV), a well-characterized virus with rigid rod-shaped virions, and flexuous filamentous plant viruses. The BSMV virions were found to be considerably more labile than expected from their rod-like morphology and a distant sequence relation of the BSMV and TMV CPs. The circular dichroism spectra of BSMV CP subunits incorporated into the virions, but not subunits of free CP, demonstrated a significant proportion of beta-structure elements, which were proposed to be localized mostly in the protein regions exposed on the virion outer surface. These beta-structure elements likely formed during virion assembly can comprise the N- and C-terminal protein regions unstructured in the non-virion CP and can mediate inter-subunit interactions. Based on computer-assisted structure modeling, a model for BSMV CP subunit structural fold compliant with the available experimental data was proposed. PMID:23613760

  18. Metabolically Labile Fumarate Esters Impart Kinetic Selectivity to Irreversible Inhibitors.

    PubMed

    Zaro, Balyn W; Whitby, Landon R; Lum, Kenneth M; Cravatt, Benjamin F

    2016-12-14

    Electrophilic small molecules are an important class of chemical probes and drugs that produce biological effects by irreversibly modifying proteins. Examples of electrophilic drugs include covalent kinase inhibitors that are used to treat cancer and the multiple sclerosis drug dimethyl fumarate. Optimized covalent drugs typically inactivate their protein targets rapidly in cells, but ensuing time-dependent, off-target protein modification can erode selectivity and diminish the utility of reactive small molecules as chemical probes and therapeutics. Here, we describe an approach to confer kinetic selectivity to electrophilic drugs. We show that an analogue of the covalent Bruton's tyrosine kinase (BTK) inhibitor Ibrutinib bearing a fumarate ester electrophile is vulnerable to enzymatic metabolism on a time-scale that preserves rapid and sustained BTK inhibition, while thwarting more slowly accumulating off-target reactivity in cell and animal models. These findings demonstrate that metabolically labile electrophilic groups can endow covalent drugs with kinetic selectivity to enable perturbation of proteins and biochemical pathways with greater precision.

  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. 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

  1. Phenotypic Profiles of Enterotoxigenic Escherichia coli Associated with Early Childhood Diarrhea in Rural Egypt

    PubMed Central

    Shaheen, Hind I.; Khalil, Sami B.; Rao, Malla R.; Elyazeed, Remon Abu; Wierzba, Thomas F.; Peruski, Leonard F.; Putnam, Shannon; Navarro, Armando; Morsy, Badria Z.; Cravioto, Alejandro; Clemens, John D.; Svennerholm, Ann-Mari; Savarino, Stephen J.

    2004-01-01

    Enterotoxigenic Escherichia coli (ETEC) causes substantial diarrheal morbidity and mortality in young children in countries with limited resources. We determined the phenotypic profiles of 915 ETEC diarrheal isolates derived from Egyptian children under 3 years of age who participated in a 3-year population-based study. For each strain, we ascertained enterotoxin and colonization factor (CF) expression, the O:H serotype, and antimicrobial susceptibility. Sixty-one percent of the strains expressed heat-stable enterotoxin (ST) only, 26% expressed heat-labile enterotoxin (LT) alone, and 12% expressed both toxins. The most common CF phenotypes were colonization factor antigen I (CFA/I) (10%), coli surface antigen 6 (CS6) (9%), CS14 (6%), and CS1 plus CS3 (4%). Fifty-nine percent of the strains did not express any of the 12 CFs included in our test panel. Resistance of ETEC strains to ampicillin (63%), trimethoprim-sulfamethoxazole (52%), and tetracycline (43%) was common, while resistance to quinolone antibiotics was rarely detected. As for the distribution of observed serotypes, there was an unusually wide diversity of O antigens and H types represented among the 915 ETEC strains. The most commonly recognized composite ETEC phenotypes were ST CS14 O78:H18 (4%), ST (or LTST) CFA/I O128:H12 (3%), ST CS1+CS3 O6:H16 (2%), and ST CFA/I O153:H45 (1.5%). Temporal plots of diarrheal episodes associated with ETEC strains bearing common composite phenotypes were consistent with discrete community outbreaks either within a single or over successive warm seasons. These data suggest that a proportion of the disease that is endemic to young children in rural Egypt represents the confluence of small epidemics by clonally related ETEC strains that are transiently introduced or that persist in a community reservoir. PMID:15583286

  2. Phenotypic profiles of enterotoxigenic Escherichia coli associated with early childhood diarrhea in rural Egypt.

    PubMed

    Shaheen, Hind I; Khalil, Sami B; Rao, Malla R; Abu Elyazeed, Remon; Wierzba, Thomas F; Peruski, Leonard F; Putnam, Shannon; Navarro, Armando; Morsy, Badria Z; Cravioto, Alejandro; Clemens, John D; Svennerholm, Ann-Mari; Savarino, Stephen J

    2004-12-01

    Enterotoxigenic Escherichia coli (ETEC) causes substantial diarrheal morbidity and mortality in young children in countries with limited resources. We determined the phenotypic profiles of 915 ETEC diarrheal isolates derived from Egyptian children under 3 years of age who participated in a 3-year population-based study. For each strain, we ascertained enterotoxin and colonization factor (CF) expression, the O:H serotype, and antimicrobial susceptibility. Sixty-one percent of the strains expressed heat-stable enterotoxin (ST) only, 26% expressed heat-labile enterotoxin (LT) alone, and 12% expressed both toxins. The most common CF phenotypes were colonization factor antigen I (CFA/I) (10%), coli surface antigen 6 (CS6) (9%), CS14 (6%), and CS1 plus CS3 (4%). Fifty-nine percent of the strains did not express any of the 12 CFs included in our test panel. Resistance of ETEC strains to ampicillin (63%), trimethoprim-sulfamethoxazole (52%), and tetracycline (43%) was common, while resistance to quinolone antibiotics was rarely detected. As for the distribution of observed serotypes, there was an unusually wide diversity of O antigens and H types represented among the 915 ETEC strains. The most commonly recognized composite ETEC phenotypes were ST CS14 O78:H18 (4%), ST (or LTST) CFA/I O128:H12 (3%), ST CS1+CS3 O6:H16 (2%), and ST CFA/I O153:H45 (1.5%). Temporal plots of diarrheal episodes associated with ETEC strains bearing common composite phenotypes were consistent with discrete community outbreaks either within a single or over successive warm seasons. These data suggest that a proportion of the disease that is endemic to young children in rural Egypt represents the confluence of small epidemics by clonally related ETEC strains that are transiently introduced or that persist in a community reservoir.

  3. The epidemiology of travelers' diarrhea in Incirlik, Turkey: a region with a predominance of heat-stabile toxin producing enterotoxigenic Escherichia coli.

    PubMed

    Porter, Chad K; Riddle, Mark S; Tribble, David R; Putnam, Shannon D; Rockabrand, David M; Frenck, Robert W; Rozmajzl, Patrick; Kilbane, Edward; Fox, Ann; Ruck, Richard; Lim, Matthew; Johnston, James; Murphy, Emmett; Sanders, John W

    2010-03-01

    This study evaluated travelers' diarrhea among US military personnel on short-term deployment to Incirlik Air Base, Turkey, from June through September 2002. Upon reporting for care for travelers' diarrhea, subjects were enrolled into the study and completed a series of questionnaires and provided stool specimens for pathogen identification and antimicrobial susceptibility testing. Fifty-three percent of the 202 participating subjects had a pathogen isolated from their stool. Enterotoxigenic Escherichia coli (ETEC) was the predominant pathogen (41%), followed by Campylobacter spp. (12%). The most common ETEC phenotype recovered was stable toxin (ST) CS6 (47% of all ETEC). Most (91.1%) of the cases presented with water diarrhea regardless of isolated pathogen. However, there were some differences in nongastrointestinal symptoms among subjects with Campylobacter spp. All illnesses were well managed with antibiotics with or without loperamide with a median time to the last unformed stool of 9 h (interquartile range, 1-32 h). We found no food or environmental factors associated with a differential risk of infection with a specific pathogen. Travelers' diarrhea among a US military population in and around Incirlik, Turkey, can commonly be attributed to ETEC and Campylobacter spp. The high proportion of ST-only-producing CS6 ETEC in this region highlights the pathogen's worldwide diversity. Future studies of travelers' diarrhea in this population should adapt more novel microbiologic techniques such as polymerase chain reaction and enhanced culture methods to increase the likelihood of identifying pathogenic E. coli. Published by Elsevier Inc.

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

    PubMed

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

    2014-08-01

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

  5. Clonal relatedness of enterotoxigenic Escherichia coli (ETEC) strains expressing LT and CS17 isolated from children with diarrhoea in La Paz, Bolivia.

    PubMed

    Rodas, Claudia; Klena, John D; Nicklasson, Matilda; Iniguez, Volga; Sjöling, Asa

    2011-01-01

    Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveller's and infantile diarrhoea in the developing world. ETEC produces two toxins, a heat-stable toxin (known as ST) and a heat-labile toxin (LT) and colonization factors that help the bacteria to attach to epithelial cells. In this study, we characterized a subset of ETEC clinical isolates recovered from Bolivian children under 5 years of age using a combination of multilocus sequence typing (MLST) analysis, virulence typing, serotyping and antimicrobial resistance test patterns in order to determine the genetic background of ETEC strains circulating in Bolivia. We found that strains expressing the heat-labile (LT) enterotoxin and colonization factor CS17 were common and belonged to several MLST sequence types but mainly to sequence type-423 and sequence type-443 (Achtman scheme). To further study the LT/CS17 strains we analysed the nucleotide sequence of the CS17 operon and compared the structure to LT/CS17 ETEC isolates from Bangladesh. Sequence analysis confirmed that all sequence type-423 strains from Bolivia had a single nucleotide polymorphism; SNP(bol) in the CS17 operon that was also found in some other MLST sequence types from Bolivia but not in strains recovered from Bangladeshi children. The dominant ETEC clone in Bolivia (sequence type-423/SNP(bol)) was found to persist over multiple years and was associated with severe diarrhoea but these strains were variable with respect to antimicrobial resistance patterns. The results showed that although the LT/CS17 phenotype is common among ETEC strains in Bolivia, multiple clones, as determined by unique MLST sequence types, populate this phenotype. Our data also appear to suggest that acquisition and loss of antimicrobial resistance in LT-expressing CS17 ETEC clones is more dynamic than acquisition or loss of virulence factors.

  6. Clonal Relatedness of Enterotoxigenic Escherichia coli (ETEC) Strains Expressing LT and CS17 Isolated from Children with Diarrhoea in La Paz, Bolivia

    PubMed Central

    Rodas, Claudia; Klena, John D.; Nicklasson, Matilda; Iniguez, Volga; Sjöling, Åsa

    2011-01-01

    Background Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveller's and infantile diarrhoea in the developing world. ETEC produces two toxins, a heat-stable toxin (known as ST) and a heat-labile toxin (LT) and colonization factors that help the bacteria to attach to epithelial cells. Methodology/Principal Findings In this study, we characterized a subset of ETEC clinical isolates recovered from Bolivian children under 5 years of age using a combination of multilocus sequence typing (MLST) analysis, virulence typing, serotyping and antimicrobial resistance test patterns in order to determine the genetic background of ETEC strains circulating in Bolivia. We found that strains expressing the heat-labile (LT) enterotoxin and colonization factor CS17 were common and belonged to several MLST sequence types but mainly to sequence type-423 and sequence type-443 (Achtman scheme). To further study the LT/CS17 strains we analysed the nucleotide sequence of the CS17 operon and compared the structure to LT/CS17 ETEC isolates from Bangladesh. Sequence analysis confirmed that all sequence type-423 strains from Bolivia had a single nucleotide polymorphism; SNPbol in the CS17 operon that was also found in some other MLST sequence types from Bolivia but not in strains recovered from Bangladeshi children. The dominant ETEC clone in Bolivia (sequence type-423/SNPbol) was found to persist over multiple years and was associated with severe diarrhoea but these strains were variable with respect to antimicrobial resistance patterns. Conclusion/Significance The results showed that although the LT/CS17 phenotype is common among ETEC strains in Bolivia, multiple clones, as determined by unique MLST sequence types, populate this phenotype. Our data also appear to suggest that acquisition and loss of antimicrobial resistance in LT-expressing CS17 ETEC clones is more dynamic than acquisition or loss of virulence factors. PMID:22140423

  7. Thermally-Labile Trace Elements in Enstatite Meteorites

    NASA Technical Reports Server (NTRS)

    Wang, M.-S.; Lipschutz, M. E.

    2000-01-01

    RNAA data for Bi, In and Tl in 30 E3-6 chondrites accord well with trends for heated Abee (EH4) suggesting that all EH and EL samples reflect open-system, post-accretionary heating, independent of siderophile content or recovery location.

  8. Thermally-Labile Trace Elements in Enstatite Meteorites

    NASA Technical Reports Server (NTRS)

    Wang, M.-S.; Lipschutz, M. E.

    2000-01-01

    RNAA data for Bi, In and Tl in 30 E3-6 chondrites accord well with trends for heated Abee (EH4) suggesting that all EH and EL samples reflect open-system, post-accretionary heating, independent of siderophile content or recovery location.

  9. Selective trapping of labile S3 in a porous coordination network and the direct X-ray observation.

    PubMed

    Ohtsu, Hiroyoshi; Choi, Wanuk; Islam, Nazrul; Matsushita, Yoshitaka; Kawano, Masaki

    2013-08-07

    S3 is one of the basic allotropes of sulfur but is still a mysterious labile species. We selectively trapped S3 in a pore of a thermally stable coordination network and determined S3 structure by ab initio X-ray powder diffraction analysis. S3 in a pore has a C2v bent structure. The network containing trapped S3 is remarkably stable under ambient conditions and is inert to photoirradiation. S3 in the network could be transformed to S6 by mechanical grinding or heating in the presence of NH4X (X = Cl or Br). S6 could be reverse-transformed to S3 by photoirradiation. We also determined the structure of the network containing S6 by ab initio X-ray powder diffraction analysis.

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

    PubMed

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

    2003-07-30

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

  11. Escherichia Coli

    ERIC Educational Resources Information Center

    Goodsell, David S.

    2009-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

  12. Escherichia Coli

    ERIC Educational Resources Information Center

    Goodsell, David S.

    2009-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

  13. DETERMINATION OF APPARENT QUANTUM YIELD SPECTRA FOR THE FORMATION OF BIOLOGICALLY LABILE PHOTOPRODUCTS

    EPA Science Inventory

    Quantum yield spectra for the photochemical formation of biologically labile photoproducts from dissolved organic matter (DOM) have not been available previously, although they would greatly facilitate attempts to model photoproduct formation rates across latitudinal, seasonal, a...

  14. Construction of hierarchically porous metal–organic frameworks through linker labilization

    DOE PAGES

    Yuan, Shuai; Zou, Lanfang; Qin, Jun-Sheng; ...

    2017-05-25

    One major goal of metal–organic framework (MOF) research is the expansion of pore size and volume. Although many approaches have been attempted to increase the pore size of MOF materials, it is still a challenge to construct MOFs with precisely customized pore apertures for specific applications. W present a new method, namely linker labilization, to increase the MOF porosity and pore size, giving rise to hierarchical-pore architectures. Microporous MOFs with robust metal nodes and pro-labile linkers were initially synthesized. The mesopores were subsequently created as crystal defects through the splitting of a pro-labile-linker and the removal of the linker fragmentsmore » by acid treatment. We also demonstrate that linker labilization method can create controllable hierarchical porous structures in stable MOFs, which facilitates the diffusion and adsorption process of guest molecules to improve the performances of MOFs in adsorption and catalysis.« less

  15. DETERMINATION OF APPARENT QUANTUM YIELD SPECTRA FOR THE FORMATION OF BIOLOGICALLY LABILE PHOTOPRODUCTS

    EPA Science Inventory

    Quantum yield spectra for the photochemical formation of biologically labile photoproducts from dissolved organic matter (DOM) have not been available previously, although they would greatly facilitate attempts to model photoproduct formation rates across latitudinal, seasonal, a...

  16. Construction of hierarchically porous metal-organic frameworks through linker labilization

    NASA Astrophysics Data System (ADS)

    Yuan, Shuai; Zou, Lanfang; Qin, Jun-Sheng; Li, Jialuo; Huang, Lan; Feng, Liang; Wang, Xuan; Bosch, Mathieu; Alsalme, Ali; Cagin, Tahir; Zhou, Hong-Cai

    2017-05-01

    A major goal of metal-organic framework (MOF) research is the expansion of pore size and volume. Although many approaches have been attempted to increase the pore size of MOF materials, it is still a challenge to construct MOFs with precisely customized pore apertures for specific applications. Herein, we present a new method, namely linker labilization, to increase the MOF porosity and pore size, giving rise to hierarchical-pore architectures. Microporous MOFs with robust metal nodes and pro-labile linkers were initially synthesized. The mesopores were subsequently created as crystal defects through the splitting of a pro-labile-linker and the removal of the linker fragments by acid treatment. We demonstrate that linker labilization method can create controllable hierarchical porous structures in stable MOFs, which facilitates the diffusion and adsorption process of guest molecules to improve the performances of MOFs in adsorption and catalysis.

  17. Construction of hierarchically porous metal–organic frameworks through linker labilization

    PubMed Central

    Yuan, Shuai; Zou, Lanfang; Qin, Jun-Sheng; Li, Jialuo; Huang, Lan; Feng, Liang; Wang, Xuan; Bosch, Mathieu; Alsalme, Ali; Cagin, Tahir; Zhou, Hong-Cai

    2017-01-01

    A major goal of metal–organic framework (MOF) research is the expansion of pore size and volume. Although many approaches have been attempted to increase the pore size of MOF materials, it is still a challenge to construct MOFs with precisely customized pore apertures for specific applications. Herein, we present a new method, namely linker labilization, to increase the MOF porosity and pore size, giving rise to hierarchical-pore architectures. Microporous MOFs with robust metal nodes and pro-labile linkers were initially synthesized. The mesopores were subsequently created as crystal defects through the splitting of a pro-labile-linker and the removal of the linker fragments by acid treatment. We demonstrate that linker labilization method can create controllable hierarchical porous structures in stable MOFs, which facilitates the diffusion and adsorption process of guest molecules to improve the performances of MOFs in adsorption and catalysis. PMID:28541301

  18. Modeling the Combined Effect of Pressure and Mild Heat on the Inactivation Kinetics of Escherichia coli, Listeria innocua, and Staphylococcus aureus in Black Tiger Shrimp (Penaeus monodon)

    PubMed Central

    Kaur, Barjinder P.; Rao, P. Srinivasa

    2017-01-01

    The high-pressure inactivation of Escherichia coli, Listeria innocua, and Staphylococcus aureus was studied in black tiger shrimp (Penaeus monodon). The processing parameters examined included pressure (300 to 600 MPa) and temperature (30 to 50°C). In addition, the pressure-hold period (0 to 15 min) was investigated, thus allowing both single-pulse pressure effects (i.e., zero holding time) and pressure-hold effects to be explored. E. coli was found to be the most sensitive strain to single-pulse pressure, followed by L. innocua and lastly S. aureus. Higher pressures and temperatures resulted in higher destruction rates, and the value of the shape parameter (β′) accounted for the downward concavity (β′ > 1) of the survival curves. A simplified Weibull model described the non-linearity of the survival curves for the changes in the pressure-hold period well, and it was comparable to the original Weibull model. The regression coefficients (R2), root mean square error (RMSE), accuracy factor (Af), bias factor (Bf), and residual plots suggested that using linear models to represent the data was not as appropriate as using non-linear models. However, linear models produced good fits for some pressure–temperature combinations. Analogous to their use in thermal death kinetics, activation volume (Va) and activation energy (Ea) can be used to describe the pressure and temperature dependencies of the scale parameter (δ, min), respectively. The Va and Ea values showed that high pressure and temperaturefavored the inactivation process, and S. aureus was the most baro-resistant pathogen. PMID:28790979

  19. Sonication in combination with heat and low pressure as an alternative pasteurization treatment--effect on Escherichia coli K12 inactivation and quality of apple cider.

    PubMed

    Lee, Hyoungill; Kim, Hun; Cadwallader, Keith R; Feng, Hao; Martin, Scoot E

    2013-07-01

    Escherichia coli K12 cells suspended in apple cider were treated by manothermosonication (MTS, 400 kPa/59 °C), thermosonication (TS, 100 kPa/59 °C), and manosonication (MS, 400 kPa/55 °C) for up to 4 min. A 5-log reduction was achieved in 1.4 min by MTS, 3.8 min by TS, and 2.5 min by MS. The inactivation curves of the E. coli exhibited a fast initial reduction followed by a slow inactivation section. The Weibull, log-logistic, and biphasic linear models showed a good fit of the inactivation data. Quality analyses were conducted with raw apple cider (control), thermally-pasteurized (TP), and MTS-, TS-, and MS-treated cider samples over a 3-week period at refrigeration temperature. Titratable acidity and pH did not differ among any of the samples. During storage, the turbidity value of the control was the highest, followed by TP, TS, MTS and MS. All color parameters of the TP sample were significantly different from those receiving the other treatments. The control and sonicated samples showed similar color parameters during storage. In total, 97 aroma compounds were identified in the control, TS-, MS-, and MTS-treated cider samples, while 95 aroma compounds were found in the TP at Week 0. Among all the aroma compounds, 9 key ones were identified in all samples, including ethyl 2-methylbutanoate, butyl acetate, 1-butanol, ethyl hexanoate, 1-hexanol, butanoic acid, β-damascenone, hexanoic acid, and octanoic acid. The profiles of the key aroma compounds in all sonicated samples were more similar to the control than the TP sample at Weeks 0 and 3.

  20. Neuropsychological correlates of emotional lability in children with ADHD

    PubMed Central

    Banaschewski, Tobias; Jennen-Steinmetz, Christine; Brandeis, Daniel; Buitelaar, Jan K.; Kuntsi, Jonna; Poustka, Luise; Sergeant, Joseph A.; Sonuga-Barke, Edmund J.; Frazier-Wood, Alexis C.; Albrecht, Björn; Chen, Wai; Uebel, Henrik; Schlotz, Wolff; van der Meere, Jaap J.; Gill, Michael; Manor, Iris; Miranda, Ana; Mulas, Fernando; Oades, Robert D.; Roeyers, Herbert; Rothenberger, Aribert; Steinhausen, Hans-Christoph; Faraone, Stephen V.; Asherson, Philip

    2012-01-01

    Background Emotional lability (EL) is commonly seen in patients with Attention Deficit/Hyperactivity Disorder (ADHD). The reasons for this association are currently unknown. To address this question we examined the relationship between ADHD and EL symptoms, and performance on a range of neuropsychological tasks to clarify whether EL symptoms are predicted by particular cognitive and/or motivational dysfunctions and whether these associations are mediated by the presence of ADHD symptoms. Methods A large multi-site sample of 424 carefully diagnosed ADHD cases and 564 unaffected siblings and controls aged 6 to 18 years performed a broad neuropsychological test battery, including a Go/No-Go Task, a warned 4-choice Reaction Time task, the Maudsley Index of Childhood Delay Aversion, and Digit span backwards. Neuropsychological variables were aggregated as indices of processing speed, response variability, executive functions, choice impulsivity and the influence of energetic and/or motivational factors. EL and ADHD symptoms were regressed on each neuropsychological variable in separate analyses controlling for age, gender and IQ, and, in subsequent regression analyses, for ADHD and EL symptoms respectively. Results Neuropsychological variables significantly predicted ADHD and EL symptoms with moderate to low regression coefficients. However, the association between neuropsychological parameters on EL disappeared entirely when the effect of ADHD symptoms was taken into account, revealing that the association between the neuropsychological performance measures and EL is completely mediated statistically by variations in ADHD symptoms. Conversely, neuropsychological effects on ADHD symptoms remained after EL symptom severity was taken into account. Conclusions The neuropsychological parameters examined here predict ADHD more strongly than EL. They cannot explain EL symptoms beyond what is already accounted for by ADHD symptom severity. The association between EL and ADHD