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Sample records for ab5-type enterotoxins lt-i

  1. Hydrogen Ordering in Hexagonal Intermetallic AB5 Type Compounds

    NASA Astrophysics Data System (ADS)

    Sikora, W.; Kuna, A.

    2008-04-01

    Intermetallic compounds AB5 type (A = rare-earth atoms, B = transition metal) are known to store reversibly large amounts of hydrogen and as that are discussed in this work. It was shown that the alloy cycling stability can be significantly improved by employing the so-called non-stoichiometric compounds AB5+x and that is why analysis of change of structure turned out to be interesting. A tendency for ordering of hydrogen atoms is one of the most intriguing problems for the unsaturated hydrides. The symmetry analysis method in the frame of the theory of space group and their representation gives opportunity to find all possible transformations of the parent structure. In this work symmetry analysis method was applied for AB5+x structure type (P6/mmm parent symmetry space group). There were investigated all possible ordering types and accompanying atom displacements in positions 1a, 2c, 3g (fully occupied in stoichiometric compounds AB5), in positions 2e, 6l (where atom B could appear in non-stoichiometric compounds) and also 4h, 6m, 6k, 12n, 12o, which could be partly occupied by hydrogen as a result of hydrides. An analysis was carried out of all possible structures of lower symmetry, following from P6/mmm for we k=(0, 0, 0). Also the way of getting the structure described by the P63mc space group with double cell along the z-axiswe k=(0, 0, 0.5), as it is suggested in the work of Latroche et al. is discussed by the symmetry analysis. The analysis was obtained by computer program MODY. The program calculates the so-called basis vectors of irreducible representations of a given symmetry group, which can be used for calculation of possible ordering modes.

  2. Heat-labile Enterotoxins as Adjuvants or Anti-Inflammatory Agents

    PubMed Central

    Liang, Shuang; Hajishengallis, George

    2010-01-01

    Escherichia coli and Vibrio cholerae produce structurally related AB5-type heat-labile enterotoxins which are classified into two major types. The Type I subfamily includes cholera toxin and E. coli LT-I, whereas the Type II subfamily comprises LT-IIa and LT-IIb. In addition to their roles in microbial pathogenesis, the enterotoxins are widely and intensively studied for their exceptionally strong adjuvant and immunomodulatory activities, which are not necessarily dependent upon their abilities to elevate intracellular cAMP levels. Despite general structural similarities, these molecules, in intact or derivative form, display notable differences in their interactions with gangliosides or Toll-like receptors. This divergence results in differential immune response outcomes, the underlying mechanisms of which remain largely uncharacterized. Whereas the study of these molecules has been pivotal in understanding basic mechanisms of immune regulation, a formidable challenge is to dissociate toxicity from useful properties that can be exploited in vaccine development or for the treatment of autoimmune inflammatory diseases. PMID:20461887

  3. Heat-labile enterotoxins as adjuvants or anti-inflammatory agents.

    PubMed

    Liang, Shuang; Hajishengallis, George

    2010-01-01

    Escherichia coli and Vibrio cholerae produce structurally related AB5-type heat-labile enterotoxins, which are classified into two major types. The Type I subfamily includes cholera toxin and E. coli LT-I, whereas the Type II subfamily comprises LT-IIa and LT-IIb. In addition to their roles in microbial pathogenesis, the enterotoxins are widely and intensively studied for their exceptionally strong adjuvant and immunomodulatory activities, which are not necessarily dependent upon their abilities to elevate intracellular cAMP levels. Despite general structural similarities, these molecules, in intact or derivative form, display notable differences in their interactions with gangliosides or Toll-like receptors. This divergence results in differential immune response outcomes, the underlying mechanisms of which remain largely uncharacterized. Whereas the study of these molecules has been pivotal in understanding basic mechanisms of immune regulation, a formidable challenge is to dissociate toxicity from useful properties that can be exploited in vaccine development or for the treatment of autoimmune inflammatory diseases.

  4. Enterotoxins of Staphylococci

    DTIC Science & Technology

    1988-01-01

    staphylococcal enterotoxin B in monkeys. Appl . Microbial. 16:187-192. Huang, I.-Y. and Bergdoll. M. S. (1970). The primary structure of staphylococcal enterotoxin...EPIDEMIOLOGY 132 III. PRODUCTION AND ISOLATION 132 A. Production 132 B. Purification 134 C. Purity 135 IV. STRUCTURE AND FUNCTION 138 A. Basic Structure 138 B...Primary Structure and Active Site 138 C. Modification Studies 142 D. Conformation 143 V. DETECTION METHODS 146 VI. SYNTHESIS 148 A. Cloning of

  5. Improvement in low-temperature and instantaneous high-rate output performance of Al-free AB5-type hydrogen storage alloy for negative electrode in Ni/MH battery: Effect of thermodynamic and kinetic regulation via partial Mn substituting

    NASA Astrophysics Data System (ADS)

    Zhou, Wanhai; Zhu, Ding; Tang, Zhengyao; Wu, Chaoling; Huang, Liwu; Ma, Zhewen; Chen, Yungui

    2017-03-01

    A series of Al-free Mn-modified AB5-type hydrogen storage alloys have been designed and the effects of thermodynamic stability and electrochemical kinetics on electrochemical performance via Mn substituting have been investigated. Compared with high-Al alloys, the Al-free alloys in this study have better low-temperature performance and instantaneous high-rate output because of the higher surface catalytic ability. After partial substitution of Ni by Mn, both the hydrogen desorption capacity and plateau pressure decrease, and correspondingly results in an improved thermodynamic stability which is adverse to low-temperature delivery. Additionally, with the improvement of charge acceptance ability and anti-corrosion property via Mn substitution, the room-temperature discharge capacity and cycling stability increase slightly. However, Mn adversely affects the electrochemical kinetics and deteriorates both the surface catalytic ability and the bulk hydrogen diffusion ability, leading to the drop of low-temperature dischargeability, high-rate dischargeability and peak power (Ppeak). Based on the thermodynamic and kinetic regulation and overall electrochemical properties, the optimal composition is obtained when x = 0.2, the discharge capacity is 243.6 mAh g-1 at -40 °C with 60 mA g-1, and the Ppeak attains to 969.6 W kg-1 at -40 °C.

  6. Detection of Staphylococcal Enterotoxin in Food

    PubMed Central

    Casman, Ezra P.; Bennett, Reginald W.

    1965-01-01

    Methods are described for the extraction and serological detection of trace amounts of enterotoxins A and B in foods incriminated in outbreaks of staphylococcal food poisoning. Evidence is presented for the probable applicability of the methods for the detection of unidentified enterotoxins. PMID:14325876

  7. The staphylococcal enterotoxin (SE) family

    PubMed Central

    Krakauer, Teresa; Stiles, Bradley G

    2013-01-01

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

  8. Bacterial Toxins—Staphylococcal Enterotoxin B

    PubMed Central

    FRIES, BETTINA C.; VARSHNEY, AVANISH K.

    2015-01-01

    Staphylococcal enterotoxin B is one of the most potent bacterial superantigens that exerts profound toxic effects upon the immune system, leading to stimulation of cytokine release and inflammation. It is associated with food poisoning, nonmenstrual toxic shock, atopic dermatitis, asthma, and nasal polyps in humans. Currently, there is no treatment or vaccine available. Passive immunotherapy using monoclonal antibodies made in several different species has shown significant inhibition in in vitro studies and reduction in staphylococcal enterotoxin B-induced lethal shock in in vivo studies. This should encourage future endeavors to develop these antibodies as therapeutic reagents. PMID:26184960

  9. The Systemic and Pulmonary Immune Response to Staphylococcal Enterotoxins

    PubMed Central

    Kumar, Sanjeev; Ménoret, Antoine; Ngoi, Soo-Mun; Vella, Anthony T.

    2010-01-01

    In response to environmental cues the human pathogen Staphylococcus aureus synthesizes and releases proteinaceous enterotoxins. These enterotoxins are natural etiologic entities of severe food poisoning, toxic shock syndrome, and acute diseases. Staphylococcal enterotoxins are currently listed as Category B Bioterrorism Agents by the Center for Disease Control and Prevention. They are associated with respiratory illnesses, and may contribute to exacerbation of pulmonary disease. This likely stems from the ability of Staphylococcal enterotoxins to elicit powerful episodes of T cell stimulation resulting in release of pro-inflammatory cytokines. Here, we discuss the role of the immune system and potential mechanisms of disease initiation and progression. PMID:22069664

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

    PubMed Central

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

    1984-01-01

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

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

    PubMed Central

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

    2015-01-01

    The epidemiological and bacteriological investigations on four foodborne outbreaks caused by a new type of enterotoxin-producing Clostridium perfringens are described. C. perfringens isolated from patients of these outbreaks did not produce any known enterotoxin and did not carry the C. perfringens enterotoxin gene. However, the culture filtrates of these isolates induced the accumulation of fluid in rabbit ileal loop tests. The molecular weight of the new enterotoxin may be between 50,000 and 100,000, although the known C. perfringens enterotoxin is ca. 35,000. This new enterotoxin was heat labile, and its biological activities were inactivated by heating for 5 min at 60°C. The new enterotoxin was sensitive to pH values higher than 11.0 and protease treatment but was resistant to trypsin treatment. These results suggest that the new enterotoxin may be a protein. Although C. perfringens enterotoxin induced morphological changes in Vero cells, the changes induced by the new enterotoxin differed from those by the known C. perfringens enterotoxin. The new enterotoxin also induced morphological changes in L929 cells, whereas the known C. perfringens enterotoxin did not, because L929 cells lacked an appropriate enterotoxin receptor. Although C. perfringens enterotoxin is recognized as the only diarrheagenic toxin responsible for C. perfringens foodborne outbreaks, the results of the present study indicate that C. perfringens isolated from these four outbreaks produced a new type of enterotoxin. PMID:25568432

  12. Recombinant Staphylococcal Enterotoxin Type A Stimulate Antitumoral Cytokines.

    PubMed

    Agheli, Reza; Emkanian, Bijan; Halabian, Raheleh; Fallah Mehrabadi, Jalil; Imani Fooladi, Abbas Ali

    2017-02-01

    About 20 different types of staphylococcal enterotoxins are produced by Staphylococcus aureus, in which type A is more common in food poisoning syndrome. Also staphylococcal enterotoxin A superantigen is a potent inducer of cytotoxic T lymphocyte activity and cytokine production and could stimulate T cells containing T-cell receptor beta chain domains when binding to major histocompatibility complex class II molecules. Hence, it is an important reagent in cancer immunotherapy. For the construction of pET-21a/ entA cassette, the staphylococcal enterotoxin type A gene was isolated from S aureus strain HN2, cloned into pET-21a, and introduced into Escherichia coli strain BL-21(DE3). Consequently, Western blot analysis showed pET-21a/ entA cassette expression inserted entA gene successfully. It is the first prompt using a pET-21a as a cloning vector for entA gene and expression of construct in BL-21(DE3). In addition, this study examined the ability of standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A to activate T cells in vitro. Lymphocyte cells derived from lymph node BALB/c mice were exposed to standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin (1.10, 102,103, and 104 ng/µL) in order to evaluate the magnitude of proliferation, activation, and apoptosis of lymphocyte cells based on MTT and apoptosis assays, respectively. Our investigation showed that the function of cloned staphylococcal enterotoxin A was same as standard staphylococcal enterotoxin A, and the optimal concentration for the activation of lymphocyte cells and induction of apoptosis was 100 ng/µL and 1000 ng/µL ( P < .05), respectively. Quantification of cytokines clearly showed that lymphocyte cells exposed to standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A significantly secreted higher interferon γ and tumor necrosis factor α compared to control. According to our results, the biological activity of standard

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

    PubMed

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

    1995-07-01

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

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

    USDA-ARS?s Scientific Manuscript database

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

  15. Fusion of Escherichia coli heat-stable enterotoxin and heat-labile enterotoxin B subunit.

    PubMed

    Guzman-Verduzco, L M; Kupersztoch, Y M

    1987-11-01

    The 3' terminus of the DNA coding for the extracellular Escherichia coli heat-stable enterotoxin (ST) devoid of transcription and translation stop signals was fused to the 5' terminus of the DNA coding for the periplasmic B subunit of the heat-labile enterotoxin (LTB) deleted of ribosomal binding sites and leader peptide. By RNA-DNA hybridization analysis, it was shown that the fused DNA was transcribed in vivo into an RNA species in close agreement with the expected molecular weight inferred from the nucleotide sequence. The translation products of the fused DNA resulted in a hybrid molecule recognized in Western blots (immunoblots) with antibodies directed against the heat-labile moiety. Anti-LTB antibodies coupled to a solid support bound ST and LTB simultaneously when incubated with ST-LTB cellular extracts. By [35S]cysteine pulse-chase experiments, it was shown that the fused ST-LTB polypeptide was converted from a precursor with an equivalent electrophoretic mobility of 20,800 daltons to an approximately 18,500-dalton species, which accumulated within the cell. The data suggest that wild-type ST undergoes at least two processing steps during its export to the culture supernatant. Blocking the natural carboxy terminus of ST inhibited the second proteolytic step and extracellular delivery of the hybrid molecule.

  16. [Assessment of the probability of encountering staphylococcal enterotoxins in lactic acid cheese packaged in laminates].

    PubMed

    Steinka, Izabela

    2004-01-01

    Immunoassay methods were used to identify the presence of staphylococcal enterotoxins in lactic acid cheese vacuum and non-vacuum packed. There was assessed the probability of encountering staphylococcal enterotoxin in cheese dependent on different systems of packaging, count of staphylococcal cells, intensiveness of coagulase synthesis and tightness of packaging. The presence of enterotoxin was identified in 5% of researched samples of products stored for 14 days. The influence of packaging system and tightness on presence of enterotoxin was observed. The probability of presence of staphylococcal and enterotoxin in relation to researched factors was presented by the mathematical models.

  17. Evaluation of an alternative extraction procedure for enterotoxin determination in dairy products.

    PubMed

    Meyrand, A; Atrache, V; Bavai, C; Montet, M P; Vernozy-Rozand, C

    1999-06-01

    A concentration protocol based on trichloroacetic acid precipitation was evaluated and compared with the reference method using dialysis concentration. Different quantities of purified staphylococcal enterotoxins were added to pasteurized Camembert-type cheeses. Detection of enterotoxins in these cheeses was performed using an automated detection system. Raw goat milk Camembert-type cheeses involved in a staphylococcal food poisoning were also tested. Both enterotoxin extraction methods allowed detection of the lowest enterotoxin concentration level used in this study (0.5 ng g-1). Compared with the dialysis concentration method, TCA precipitation of staphylococcal enterotoxins was 'user-friendly' and less time-consuming. These results suggest that TCA precipitation is a rapid (1 h), simple and reliable method of extracting enterotoxin from food which gives excellent recovery from dairy products.

  18. 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...Immun 18:110, 1977 2. Klipstein FA, Engert RF: Immunological relationship of different preparations of coliform enterotoxins. Infec Immun 21:771, 1978...3. Klipstein FA, Engert RF: Reversal of jejunal water secretion by glucose in rats exposed to coliform enterotoxins. Gastroenteroloj y 75:255, 1978 4

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

    PubMed

    Seo, Keun Seok

    2016-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1995-01-01

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

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

    USDA-ARS?s Scientific Manuscript database

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

  2. Crystal structure and electrochemical characteristics of non-AB 5 type La-Ni system alloys

    NASA Astrophysics Data System (ADS)

    Shi, Siqi; Ouyang, Chuying; Lei, Minsheng

    The La-Ni system compounds have been prepared by arc-melting method under Ar atmosphere. X-ray diffraction analysis reveals that the as-prepared alloys consist of different phases. The electrochemical properties, including activation, maximum discharge capacity, high rate chargeability (HRC), and high rate dischargeability (HRD) of these alloy electrodes have been studied through the charge-discharge recycle testing at different temperatures and charge (or discharge) currents. Among the La-Ni alloy electrodes studied, LaNi 2.28 alloy has the most excellent high rate charging performance, and La 2Ni 7 alloy exhibit the highest high rate dischargeability, while La 7Ni 3 alloy is capable of discharging at low temperature.

  3. Analysis of the VIDAS® Staph Enterotoxin III (SET3) for Detection of Staphylococcal Enterotoxins G, H, and I in Foods.

    PubMed

    Hait, Jennifer M; Nguyen, Angela T; Tallent, Sandra M

    2018-04-20

    Background : Staphylococcal food poisoning (SFP) frequently causes illnesses worldwide. SFP occurs from the ingestion of staphylococcal enterotoxins (SEs) preformed in foods by enterotoxigenic strains of Staphylococcus species, primarily S. aureus . SEG, SEH, and SEI induce emesis and have been implicated in outbreaks. Immunological-based methods are deemed the most practical methods for the routine analysis of SEs in foods given their ease of use, sensitivity, specificity, and commercial availability. These kits are routinely used to test for SEA-SEE. However, only recently has a kit been developed to detect SEG, SEH, and SEI. Objective: Our research examined the performance of the novel VIDAS ® Staph Enterotoxin III (SET3) for the detection of staphylococcal enterotoxins SEG, SEH, and SEI in foods. Methods : Here we assess the sensitivity and specificity of SET3 using duplicate test portions of six foods at varying concentrations of inclusivity and exclusivity inocula: pure SEG, SEH, SEI, S. aureus strain extracts positive for seg, seh , and sei , as well as SEA, SEB, SEC, SED, and SEE. Results : The overall detection limit was less than 2.09 ng/mL for foods inoculated with SEG, SEH, and SEI, with no cross reactivity observed. Highlights : Integrating concurrent testing to detect the presence of SEA-SEE and SEG-SEI utilizing the SET3 along with the VIDAS SET2, Ridascreen ® SET total, or other comparable kits will be instrumental for the future food assessments in our laboratory and may become the new standard for SE analysis of foods.

  4. Nasal carriage of enterotoxin-producing Staphylococcus aureus among restaurant workers in Kuwait City.

    PubMed Central

    al Bustan, M. A.; Udo, E. E.; Chugh, T. D.

    1996-01-01

    Enterotoxin-producing Staphylococcus aureus is a common cause of staphylococcal food poisoning. To determine the incidence of carriage of enterotoxin-producing S. aureus in a sample of the healthy population in Kuwait city, restaurant workers in the city were screened for nasal carriage of S. aureus. 26.6% of 500 workers studied carried S. aureus and 86.6% of the S. aureus produced staphylococcal enterotoxins. 28% produced enterotoxin A, 28.5% produced enterotoxin B, 16.4% produced enterotoxin C and 3.5% produced enterotoxin D. Ten isolates produced both enterotoxins A and B or A and C. 73% of the isolates were untypeable with standard phages. However, 17.1%, 3% and 6% belonged to phage groups I, II and III respectively. The results demonstrated a high level of enterotoxigenic S. aureus carriage among restaurant workers which although lower than that reported for the general population and hospital workers may be important in the restaurant industry. PMID:8666076

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

    USDA-ARS?s Scientific Manuscript database

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

  6. Action of Escherichia coli Enterotoxin: Adenylate Cyclase Behavior of Intestinal Epithelial Cells in Culture

    PubMed Central

    Kantor, Harvey S.; Tao, Pearl; Wisdom, Charlene

    1974-01-01

    Heat-labile enterotoxin preparations obtained from two enteropathogenic strains of Escherichia coli of porcine and human origin were shown to stimulate adenylate cyclase activity of human embryonic intestinal epithelial cells in culture. Comparable results were also obtained when cholera toxin was used. The degree of enzyme stimulation was proportional to the concentration of enterotoxin. Similar preparations from two strains of non-enterotoxigenic E. coli had no effect on adenylate cyclase activity. Cells exposed to enterotoxin could be washed after 1 min of contact time without altering the subsequent course of maximum adenylate cyclase activity, which was maintained for at least 18 h at 37 C. During long periods (18 h) of tissue culture incubation, the determination of adenylate cyclase activity was 200- to 300-fold more sensitive than quantitating fluid accumulation in the adult rabbit ileal loop model. Decreasing the incubation time appreciably reduced the sensitivity of the epithelial cells to enterotoxin. E. coli enterotoxin is an effective activator of nonintestinal adenylate cyclase systems. Treatment of KB and HEp-2 cell lines with enterotoxin also resulted in significant enzyme stimulation. The intestinal epithelial cell tissue culture model provides a sensitive homogenous biological system for studying the response of intestinal adenylate cyclase to enterotoxin while eliminating the numerous cellular and tissue components present in the ligated ileal loop model. PMID:4364505

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

    PubMed Central

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

    2016-01-01

    Clostridium perfringens enterotoxin (CPE) is responsible for causing the gastrointestinal symptoms of several C. perfringens food- and nonfood-borne human gastrointestinal diseases. The enterotoxin gene (cpe) is located on either the chromosome (for most C. perfringens type A food poisoning strains) or large conjugative plasmids (for the remaining type A food poisoning and most, if not all, other CPE-producing strains). In all CPE-positive strains, the cpe gene is strongly associated with insertion sequences that may help to assist its mobilization and spread. During disease, CPE is produced when C. perfringens sporulates in the intestines, a process involving several sporulation-specific alternative sigma factors. The action of CPE starts with its binding to claudin receptors to form a small complex; those small complexes then oligomerize to create a hexameric prepore on the membrane surface. Beta hairpin loops from the CPE molecules in the prepore assemble into a beta barrel that inserts into the membrane to form an active pore that enhances calcium influx, causing cell death. This cell death results in intestinal damage that causes fluid and electrolyte loss. CPE is now being explored for translational applications including cancer therapy/diagnosis, drug delivery, and vaccination. PMID:26999202

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

    PubMed

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

    2007-09-20

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

  9. A cytotoxicity assay for Clostridium spiroforme enterotoxin in cecal fluid of rabbits.

    PubMed

    Butt, M T; Papendick, R E; Carbone, L G; Quimby, F W

    1994-02-01

    Clostridium spiroforme enterotoxin-mediated diarrhea can be a common cause of mortality among weanling age rabbits. Definitive diagnosis of this disorder requires detection of the causative enterotoxin. Using filtered cecal supernatant from necropsy specimens, antibodies to C. spiroforme and its products and Vero cells, a cytotoxicity assay was performed on 22 rabbits with clinical signs and lesions consistent with C. spiroforme enterotoxin-mediated diarrhea. A cytotoxic effect was detected, generally within 4 h, in 18 of 22 rabbits. The cytotoxic effect was blocked by preincubation of the cecal material with antibodies to C. spiroforme and its products. Culture of cecal contents and smears of cecal contents identified C. spiroforme in 10/22 and 12/22 cases, respectively. This cytotoxicity assay provided a rapid and sensitive method for diagnosing C. spiroforme enterotoxin-mediated diarrhea.

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

    DTIC Science & Technology

    1980-01-15

    PUBLICATIONS Supported by Contract No. DAMD 17-77-C-7032 1. Klipstein FA, Engert RF: Immunological interrelationships between cholera toxin and the...heat-labile and heat-stable enterotoxins of coliform bacteria. Infec Immun 18:110, 1977 2. Klipstein FA, Engert RF: Immunological relationship of...different preparations of coliform enterotoxins. Infec Immun 21:771, 1978 3. Klipstein FA, Engert RF: Reversal of jejunal water secretion by glucose in

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

    DTIC Science & Technology

    1981-01-01

    FA, Engert RF: Immunological interrelationships between cholera toxin and the heat-labile and heat-stable enterotoxins of coliforn, bacteria. Infec...Irnmun 18:110, 1977 2. Klipstein FA, Engert RF: Immunological relaticnsh~p of different preparations of coliform enterotoxins. Infec Immun 21:771, 1918...3 Klipstein FA, Engert RF: Reversal of jejunal water secretion by glucose in rats exposed to coliform enterotoxcins. Gastroenterology 75:255, 1978 4

  12. Peroral Immunization of Rats with Escherichia coli Heat-Labile Enterotoxin Delivered by Microspheres

    PubMed Central

    Klipstein, Frederick A.; Engert, Richard F.; Sherman, William T.

    1983-01-01

    The antigenicity of the Escherichia coli heat-labile enterotoxin was not protected against the adverse effect of gastric acidity when the toxin was given together with bicarbonate for peroral immunization to rats, but immunization with the heat-labile enterotoxin encapsulated in pH-dependent microspheres aroused the same strong degree of serum and mucosal antitoxin responses and of protection against challenge as was achieved by peroral immunization after ablation of gastric secretions by pretreatment with cimetidine. PMID:6339378

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

    DTIC Science & Technology

    1979-01-15

    Animal, Resources, National Academy of Sciences National Research Council. -. - L S~- A - - -7- PUBLICATIONS 1. Klipstein FA, Engert RF: Immunological... Engert RF: Immunological relationship of different preparations of coliform enterotoxins. Infec Immun 21:771, 1978 3. Klipstein FA, Engert RF...Reversal of jejunal water secretion by glucose in rats exposed to coliform enterotoxins. Gastroenterology 75:255, 1978 4. Klipstein FA, Rowe B, Engert RF

  14. The staphylococcal enterotoxin (SE) family: SEB and siblings.

    PubMed

    Krakauer, Teresa; Stiles, Bradley G

    2013-11-15

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

  15. Neutralization of Staphylococcal Enterotoxin B by an Aptamer Antagonist

    PubMed Central

    Wang, Kaiyu; Gan, Longjie; Jiang, Li; Zhang, Xianhui; Yang, Xiangyue; Chen, Min

    2015-01-01

    Staphylococcal enterotoxin B (SEB) is a major virulence factor for staphylococcal toxic shock syndrome (TSS). SEB activates a large subset of the T lymphocytic population, releasing proinflammatory cytokines. Blocking SEB-initiated toxicity may be an effective strategy for treating TSS. Using a process known as systematic evolution of ligands by exponential enrichment (SELEX), we identified an aptamer that can antagonize SEB with nanomolar binding affinity (Kd = 64 nM). The aptamer antagonist effectively inhibits SEB-mediated proliferation and cytokine secretion in human peripheral blood mononuclear cells. Moreover, a PEGylated aptamer antagonist significantly reduced mortality in a “double-hit” mouse model of SEB-induced TSS, established via sensitization with d-galactosamine followed by SEB challenge. Therefore, our novel aptamer antagonist may offer potential therapeutic efficacy against SEB-mediated TSS. PMID:25624325

  16. Crystallization and preliminary crystallographic analysis of the Clostridium perfringens enterotoxin

    PubMed Central

    Briggs, David C.; Smedley, James G.; McClane, Bruce A.; Basak, Ajit K.

    2010-01-01

    Clostridium perfringens is a Gram-positive anaerobic species of bacterium that is notable for its ability to produce a plethora of toxins, including membrane-active toxins (α-toxins), pore-forming toxins (∊-toxins) and binary toxins (ι-toxins). Here, the crystallization of the full-length wild-type C. perfringens enterotoxin is reported, which is the causative agent of the second most prevalent food-borne illness in the United States and has been implicated in many other gastrointestinal pathologies. Several crystal forms were obtained. However, only two of these optimized crystal forms (I and II) were useable for X-ray diffraction data collection. The form I crystals diffracted to d min = 2.7 Å and belonged to space group C2, while the form II crystals diffracted to d min = 4 Å and belonged to space group P213. PMID:20606275

  17. 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

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

    PubMed Central

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

    1971-01-01

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

  19. Campylobacter jejuni chromosomal sequences that hybridize to Vibrio cholerae and Escherichia coli LT enterotoxin genes.

    PubMed

    Calva, E; Torres, J; Vázquez, M; Angeles, V; de la Vega, H; Ruíz-Palacios, G M

    1989-02-20

    Campylobacter jejuni is one of the main etiologic agents of gastrointestinal illness in developing and developed areas throughout the world. Isolation of enterotoxin-producing C. jejuni has been associated with clinical symptoms of a watery-secretory type of diarrhea. Although physiological and immunological relatedness has been demonstrated between the C. jejuni enterotoxin (CJT), the Vibrio cholerae enterotoxin (CT), and the heat-labile cholera-like Escherichia coli enterotoxin (LT), nucleotide sequence similarity between C. jejuni DNA and either the toxA, toxB, eltA or eltB genes remained to be shown. We found that binding to ganglioside GM1 prevented recognition of CJT by monoclonal antibodies directed to either CT or LT. This indicates antigenic similarity between the three enterotoxins in the ganglioside GM1-binding site. Therefore we searched for corresponding similarities at the DNA level and found, by oligodeoxynucleotide hybridization, C. jejuni chromosomal nucleotide sequences similar to the coding region for a postulated ganglioside GM1-binding site on toxB and eltB.

  20. Influence of Food Microorganisms on Staphylococcal Growth and Enterotoxin Production in Meat

    PubMed Central

    McCoy, D. W.; Faber, J. E.

    1966-01-01

    Forty-four microorganisms were studied for their influence on staphylococcal growth and enterotoxin production. Inhibition was found to be more common than stimulation. Two types of inhibition were observed: inhibition of staphylococcal growth, and inhibition of enterotoxin formation with no apparent effect on growth. By use of a plate test, 12 of the 44 food microorganisms were found to inhibit staphylococcal growth at 35 C. Of the 12, 3 also inhibited growth at 25 C. No significant differences in inhibition were observed with the 15 strains of enterotoxigenic staphylococci. In meat slurries, inhibition of staphylococcal growth was found to be greater at 25 C than at 35 C. Results on inhibition obtained from the plate test could not be correlated with the effect of the organisms in slurries. Environmental conditions were found to affect markedly the influence of food microorganisms on staphylococci. Of the 44 food microorganisms studied, only Bacillus cereus was observed to stimulate significantly staphylococcal growth and enterotoxin formation. Stimulation was more pronounced with Staphylococcus aureus 196E than with other strains of enterotoxigenic staphylococci. Bacillus megaterium and Brevibacterium linens were inhibited by staphylococci. These organisms were completely inhibited when inoculated in mixed cultures with staphylococci. In pure cultures, good staphylococcal growth was found to be accompanied by enterotoxin production; however, in the presence of food microorganisms, good staphylococcal growth occurred without the formation of detectable levels of enterotoxin A. PMID:5970822

  1. Quantitative detection of type A staphylococcal enterotoxin by Laurell electroimmunodiffusion.

    PubMed

    Gasper, E; Heimsch, R C; Anderson, A W

    1973-03-01

    The detection of staphylococcal enterotoxin A by the quantitative technique of electroimmunodiffusion is described. High dilutions of type-specific rabbit antiserum were used in 1% agarose gels, 1 mm thick, and prepared in 0.05-mug barbital buffer, pH 8.6. Volumes of 10 muliters containing 1.5 to 10 ng of toxin were electrophoresed out of 4-mm diameter wells at 5 mA/cm width of gel. The precipitin cones formed were made visible by first immersing the agarose gels in 0.2 M NaCl and then overlaying the surface with the purified globulin fraction of sheep serum against rabbit globulin, followed by soaking of the gels in 1% aqueous cadmium acetate and staining with 0.1% thiazine red in 1% glacial acetic acid. Fully extended cones, 4 to 23 mm in length depending on toxin concentration and antiserum dilution, were developed in 2 to 5 h of electrophoresis, and visualization was achieved within 2 to 3 h. Because the method is qualitative, quantitative, simple, rapid, and sensitive, it offers a practical tool for the detection of small amounts of bacterial toxins in contaminated foods. The method should also qualify as a sensitive detection device in biochemical procedures which attempt to trace, detect, and identify biological substances in nanogram quantities, provided these substances are antigenic and capable of forming a precipitate with their specific antibodies.

  2. The interaction of Clostridium perfringens enterotoxin with receptor claudins

    PubMed Central

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

    2016-01-01

    Clostridium perfringens enterotoxin (CPE) has significant medical importance due to its involvement in several common human gastrointestinal diseases. This 35 kDa single polypeptide toxin consists of two domains: a C-terminal domain involved in receptor binding and an N-terminal domain involved in oligomerization, membrane insertion and pore formation. The action of CPE starts with its binding to receptors, which include certain members of the claudin tight junction protein family; bound CPE then forms a series of complexes, one of which is a pore that causes the calcium influx responsible for host cell death. Recent studies have revealed that CPE binding to claudin receptors involves interactions between the C-terminal CPE domain and both the 1st and 2nd extracellular loops (ECL-1 and ECL-2) of claudin receptors. Of particular importance for this binding is the docking of ECL-2 into a pocket present in the C-terminal domain of the toxin. This increased understanding of CPE interactions with claudin receptors is now fostering the development of receptor decoy therapeutics for CPE-mediated gastrointestinal disease, reagents for cancer therapy/diagnoses and enhancers of drug delivery. PMID:27090847

  3. Clostridium perfringens Enterotoxin Interacts with Claudins via Electrostatic Attraction*

    PubMed Central

    Kimura, Jun; Abe, Hiroyuki; Kamitani, Shigeki; Toshima, Hirono; Fukui, Aya; Miyake, Masami; Kamata, Yoichi; Sugita-Konishi, Yoshiko; Yamamoto, Shigeki; Horiguchi, Yasuhiko

    2010-01-01

    Clostridium perfringens enterotoxin (CPE), a causative agent of food poisoning, is a pore-forming toxin disrupting the selective permeability of the plasma membrane of target cells, resulting in cell death. We previously identified claudin as the cell surface receptor for CPE. Claudin, a component of tight junctions, is a tetratransmembrane protein and constitutes a large family of more than 20 members, not all of which serve as the receptor for CPE. The mechanism by which the toxin distinguishes the sensitive claudins is unknown. In this study, we localized the region of claudin responsible for interaction with CPE to the C-terminal part of the second extracellular loop and found that the isoelectric point of this region in sensitive claudins was higher than insensitive claudins. Amino acid substitutions to lower the pI resulted in reduced sensitivity to CPE among sensitive claudins, whereas substitutions to raise the pI endowed CPE-insensitive claudins with sensitivity. The steric structure of the claudin-binding domain of CPE reveals an acidic cleft surrounded by Tyr306, Tyr310, Tyr312, and Leu315, which were reported to be essential for interaction with the sensitive claudins. These results imply that an electrostatic attraction between the basic claudin region and the acidic CPE cleft is involved in their interaction. PMID:19903817

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

    USDA-ARS?s Scientific Manuscript database

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

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

    PubMed

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

    2018-03-01

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

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

    PubMed Central

    Stiffler-Rosenberg, G; Fey, H

    1978-01-01

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

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

    PubMed

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

    2007-01-01

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

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2013-02-01

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

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

    DTIC Science & Technology

    1980-03-01

    Purification of staphylococcal enterotoxin 2. Liu CT, DeLauter RD. Faulkner RT: cation of monkeys by intravenous staphylo- B. Biochemistry 4:1011...nteotoinB (EB)ata ds- to produce death within 24 hours in Staphylococcal enterotoxin B is a age level of 50 pg/kg of body weight monkeys , 13 dogs...8KB accumulates in the renal proximal peatbyrod&-tomy and Mesm R. D. DeaII15 and caq1 G. 0Croe for tehela sitace. the monkey and rabbit is still of much

  11. Effects of Meat-curing Salts and Temperature on Production of Staphylococcal Enterotoxin B1

    PubMed Central

    McLean, Ruth A.; Lilly, Helen D.; Alford, John A.

    1968-01-01

    We investigated the effect of time, temperature, and the presence of sodium chloride, nitrates, and nitrites in the medium on the growth and production of enterotoxin B by Staphylococcus aureus. Assays by the double gel-diffusion method showed that maximal enterotoxin B production occurs at the beginning of the stationary phase of growth. Lowering the temperature of incubation decreased the amount of toxin produced without affecting the total amount of growth. Increases in concentration of curing salts reduced toxin production more rapidly than cell growth. The relationship of these observations to food-poisoning outbreaks is briefly discussed. PMID:4967190

  12. Effects of Bile Acids and Nisin on the Production of Enterotoxin by Clostridium perfringens in a Nutrient-Rich Medium.

    PubMed

    Park, Miseon; Rafii, Fatemeh

    2018-01-01

    Clostridium perfringens is the second most common cause of bacterial foodborne illness in the United States, with nearly a million cases each year. C. perfringens enterotoxin (CPE), produced during sporulation, damages intestinal epithelial cells by pore formation, which results in watery diarrhea. The effects of low concentrations of nisin and bile acids on sporulation and toxin production were investigated in C. perfringens SM101, which carries an enterotoxin gene on the chromosome, in a nutrient-rich medium. Bile acids and nisin increased production of enterotoxin in cultures; bile acids had the highest effect. Both compounds stimulated the transcription of enterotoxin and sporulation-related genes and production of spores during the early growth phase. They also delayed spore outgrowth and nisin was more inhibitory. Bile acids and nisin enhanced enterotoxin production in some but not all other C. perfringens isolates tested. Low concentrations of bile acids and nisin may act as a stress signal for the initiation of sporulation and the early transcription of sporulation-related genes in some strains of C. perfringens , which may result in increased strain-specific production of enterotoxin in those strains. This is the first report showing that nisin and bile acids stimulated the transcription of enterotoxin and sporulation-related genes in a nutrient-rich bacterial culture medium.

  13. Effects of Bile Acids and Nisin on the Production of Enterotoxin by Clostridium perfringens in a Nutrient-Rich Medium

    PubMed Central

    Park, Miseon

    2018-01-01

    Clostridium perfringens is the second most common cause of bacterial foodborne illness in the United States, with nearly a million cases each year. C. perfringens enterotoxin (CPE), produced during sporulation, damages intestinal epithelial cells by pore formation, which results in watery diarrhea. The effects of low concentrations of nisin and bile acids on sporulation and toxin production were investigated in C. perfringens SM101, which carries an enterotoxin gene on the chromosome, in a nutrient-rich medium. Bile acids and nisin increased production of enterotoxin in cultures; bile acids had the highest effect. Both compounds stimulated the transcription of enterotoxin and sporulation-related genes and production of spores during the early growth phase. They also delayed spore outgrowth and nisin was more inhibitory. Bile acids and nisin enhanced enterotoxin production in some but not all other C. perfringens isolates tested. Low concentrations of bile acids and nisin may act as a stress signal for the initiation of sporulation and the early transcription of sporulation-related genes in some strains of C. perfringens, which may result in increased strain-specific production of enterotoxin in those strains. This is the first report showing that nisin and bile acids stimulated the transcription of enterotoxin and sporulation-related genes in a nutrient-rich bacterial culture medium. PMID:29675044

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

    USDA-ARS?s Scientific Manuscript database

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

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

    USDA-ARS?s Scientific Manuscript database

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2015-10-01

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

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

    PubMed

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

    2011-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-09-14

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

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

    EPA Science Inventory

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

  2. Interleukin 2 secretion by T cells for detection of biologically active Staphylococcal enterotoxin type E

    USDA-ARS?s Scientific Manuscript database

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

  3. Expression of Staphylococcal Enterotoxins under Stress Encountered during Food Production and Preservation.

    PubMed

    Schelin, Jenny; Susilo, Yusak Budi; Johler, Sophia

    2017-12-15

    Staphylococcal food poisoning (SFP) is the most prevalent cause of food-borne intoxications worldwide. Consumption of enterotoxins preformed in food causes violent vomiting and can be fatal in children and the elderly. While being repressed by competing bacteria in most matrices, Staphylococcus aureus benefits from crucial competitive advantages in foods with high osmolarity or low pH. During recent years, the long-standing belief in the feasibility of assessing SFP risk based on colony-forming units of S. aureus present in food products has been disproven. Instead, researchers and food business operators are acutely aware of the imminent threat arising from unforeseeable enterotoxin production under stress conditions. This paradigm shift led to a variety of new publications enabling an improved understanding of enterotoxin expression under stress conditions encountered in food. The wealth of data provided by these studies is extremely diverse, as it is based on different methodological approaches, staphylococcal strains, stressors, and enterotoxins. Therefore, in this review, we aggregated and critically evaluated the complex findings of these studies, to provide readers with a current overview of the state of research in the field.

  4. Rauvolfia Grandiflora (Apocynaceae) Extract Interferes With Staphylococcal Density, Enterotoxin Production And Antimicrobial Activity

    PubMed Central

    de Almeida Carlos, Lanamar; da Silva Amaral, Kenas Aguiar; Curcino Vieira, Ivo José; Mathias, Leda; Braz-Filho, Raimundo; Silva Samarão, Solange; Vieira-da-Motta, Olney

    2010-01-01

    Staphylococci bacteria are involved in many human and animal infections and development of alternative antimicrobial drugs against pathogenic bacteria is of great interest to the pharmaceutical industry. This study investigated the in vitro effect of Rauvolfia grandiflora methanol extract (root bark fraction) (RGE) on the density of ATCC strains of Staphylococcus aureus and Staphylococcus epidermidis, and a clinical enterotoxin-producer, S. aureus bovine strain. The alkaloid, isoreserpiline, obtained from dichloromethane extract of R. grandiflora was ineffective against the strains tested. After incubation of staphylococci strains in the presence of 1.2 μg.mL-1 RGE, a significant inhibition of cell growth was observed using both spectrophotometry and ELISA assays. Twelve drugs were evaluated for their antimicrobial effects on culture RGE-treated cells using the disk diffusion method. Penicillin resistant strains became sensitive to the drug after RGE treatment. Furthermore, enterotoxin production by RGE-treated S. aureus was evaluated using a standardized ELISA method. Although staphylococcal LSA 88 bovine strain cells remained viable after exposure to the extract, enterotoxin production was precluded in 20% after RGE treatment. Significant interference in staphylococci cell density, drug sensitivity and enterotoxin secretion was observed after treatment. The study highlights the necessity to find new methods of disease prevention and new antibiotic therapies against staphylococcal infections. PMID:24031536

  5. Rauvolfia grandiflora (apocynaceae) extract interferes with staphylococcal density, enterotoxin production and antimicrobial activity.

    PubMed

    de Almeida Carlos, Lanamar; da Silva Amaral, Kenas Aguiar; Curcino Vieira, Ivo José; Mathias, Leda; Braz-Filho, Raimundo; Silva Samarão, Solange; Vieira-da-Motta, Olney

    2010-07-01

    Staphylococci bacteria are involved in many human and animal infections and development of alternative antimicrobial drugs against pathogenic bacteria is of great interest to the pharmaceutical industry. This study investigated the in vitro effect of Rauvolfia grandiflora methanol extract (root bark fraction) (RGE) on the density of ATCC strains of Staphylococcus aureus and Staphylococcus epidermidis, and a clinical enterotoxin-producer, S. aureus bovine strain. The alkaloid, isoreserpiline, obtained from dichloromethane extract of R. grandiflora was ineffective against the strains tested. After incubation of staphylococci strains in the presence of 1.2 μg.mL(-1) RGE, a significant inhibition of cell growth was observed using both spectrophotometry and ELISA assays. Twelve drugs were evaluated for their antimicrobial effects on culture RGE-treated cells using the disk diffusion method. Penicillin resistant strains became sensitive to the drug after RGE treatment. Furthermore, enterotoxin production by RGE-treated S. aureus was evaluated using a standardized ELISA method. Although staphylococcal LSA 88 bovine strain cells remained viable after exposure to the extract, enterotoxin production was precluded in 20% after RGE treatment. Significant interference in staphylococci cell density, drug sensitivity and enterotoxin secretion was observed after treatment. The study highlights the necessity to find new methods of disease prevention and new antibiotic therapies against staphylococcal infections.

  6. Effect of Sodium Chloride and pH on Enterotoxin B Production

    PubMed Central

    Genigeorgis, Constantin; Sadler, Walter W.

    1966-01-01

    Genigeorgis, Constantin (University of California, Davis), and Walter W. Sadler. Effect of sodium chloride and pH on enterotoxin B production. J. Bacteriol. 92:1383–1387. 1966.—The growth and production of enterotoxin B by Staphylococcus aureus strain S-6 in Brain Heart Infusion broth with 2 to 16% sodium chloride and an initial pH of 5.1 to 6.9 was studied during a 10-day incubation period at 37 C. Growth was good at pH 6.9 and with a 16% concentration of salt, but no cells survived after 10 days of incubation at pH 5.1 and with a 16% concentration of salt. With geldiffusion technique, enterotoxin B was detected in broth with pH 6.9 and up to 10% salt or pH 5.1 and up to 4% salt. Growth and enterotoxin production were better when pH was increased and salt concentration was decreased. The dependence of toxin production on the interaction of these two factors was demonstrated. PMID:5924269

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

    USDA-ARS?s Scientific Manuscript database

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

  8. Lung and pharyngeal abscess caused by enterotoxin G- and I-producing Staphylococcus aureus.

    PubMed

    Barnett, S Y; Hattotuwa, K L; Teare, L

    2012-05-01

    We report a particularly serious case of extensive meticillin sensitive Staphylococcal lung and pharyngeal abscess. Our patient had no significant risk factors for severe infection. The detection of enterotoxin G and I here suggest that when present together, these toxins work synergistically to produce a more virulent strain of Staphylococcus aureus. Copyright © 2011. Published by Elsevier Ltd.

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

    PubMed

    Wu, Xulei; Su, Yi-Cheng

    2014-08-01

    This study investigated the survival of Staphylococcus aureus in precooked tuna meat for producing canned products during frozen storage (-20 ± 2 °C) as well as its growth and enterotoxin production at 35 to 37 °C after the storage. Samples (50 ± 5 g) of precooked albacore (loin, chunk, and flake) and skipjack (chunk and flake) tuna were inoculated with 5 enterotoxin-producing strains of S. aureus at a level of approximately 3.5 log CFU/g and individually packed in a vacuum bag after 3 h incubation at 35 to 37 °C. Vacuum-packed samples were stored in a freezer (-20 ± 2 °C) for 4 wk. The frozen samples were then thawed in 37 °C circulating water for 2 h and incubated at 35 to 37 °C for 22 h. Populations of S. aureus in all precooked tuna samples decreased slightly (<0.7 log CFU/g) after 4 wk of storage at -20 ± 2 °C, but increased rapidly once the samples were thawed and held at 35 to 37 °C. Total S. aureus counts in albacore and skipjack samples increased by greater than 3 log CFU/g after 6 and 8 h of exposure to 35 to 37 °C, respectively. All samples became spoiled after 10 h of exposure to 35 to 37 °C, while no enterotoxin was detected in any samples. However, enterotoxins were detected in albacore loin and other samples after 12 and 24 h of incubation at 35 to 37 °C, respectively. Frozen precooked tuna meat should be used for producing canned tuna within 6 to 8 h of thawing to avoid product spoilage and potential enterotoxin production by S. aureus in contaminated precooked tuna meat. © 2014 Institute of Food Technologists®

  10. [Enterotoxin genes occurance among S. aureus strains isolated from inpatients and carriers].

    PubMed

    Lawrynowicz-Paciorek, Maja; Kochman, Maria; Piekarska, Katarzyna; Wyrebiak, Agata; Potracka, Ewa; Leniak-Chmiel, Urszula; Magdziak, Agnieszka

    2006-01-01

    We examined 44 inpatients and 66 carriers Staphylococcus aureus strains, isolated in years 2002-2005, for the presence of 18 enterotoxin genes (se/sel) (by PCR), the ability for A-D enterotoxin production (by SET-RPLA) and antibiotic resistance distribution (by disc diffusion method). se/sel genes were detected in 90,9% of all strains, sea (70,5%) and selk and selq (52,3%) - among inpatients strains and egc (65,2%) - among carriers strains were the most frequently se/sel genes found. Positive results of SET-RPLA were consistent with PCR results. There was no correlation observed between antibiotic resistance and se/sel genes distribution among tested S. aureus strains.

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

    PubMed

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

    2014-09-05

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

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

    PubMed

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

    2015-01-01

    Enterotoxins from pathogenic bacteria are known as the main reason that can cause the bacterial foodborne diseases. In this study, aptamers that bound to Staphylococcus aureus enterotoxin C1 (SEC1) with high affinity and selectivity were generated in vitro by twelve rounds of selection based on magnetic separation technology, with a low-level dissociation constant (Kd) value of 65.14 ± 11.64 nmol/L of aptamer C10. Aptamer-based quantification of SEC1 in the food sample by a graphene oxide (GO)-based method was implemented to investigate the potential of the aptamer against SEC1 with a limit of detection of 6 ng/mL. On the basis of this work, biosensors using the selected SEC1 aptamers as new molecular recognition elements could be applied for innovative determinations of SEC1. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Intrinsic and chemically produced microheterogeneity of Staphylococcus aureus enterotoxin type C.

    PubMed Central

    Metzger, J F; Johnson, A D; Spero, L

    1975-01-01

    Staphylococcus aureus enterotoxins C1 (SEC1) and C2 (SEC2) produced from 50-liter quantities of crude culture supernatants were purified chromatographically in a neutral or acid milieu. Microheterogenity of SEC1 was markedly increased by treatment of the purified toxin with alkali, and new more acidic charged species appeared. SEC2 was more heterogenous than any of the other S. aureus enterotoxins and was affected only slightly by treatment with alkali. Prolonged incubation of the organism during production of the SEC2 produced changes in charged species that may be related to a bacterial deamidase, since similar changes were not seen with alkaline treatment of the purified toxin. Although SEC1 and SEC2 showed complete identity immunologically, they are separate, distinct toxins, and alkali treatment of SEC1 did not produce SEC2. Images PMID:237837

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

    PubMed Central

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

    2014-01-01

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

  15. Prevalence of Staphylococcal Enterotoxins in Ready-to-Eat Foods Sold in Istanbul.

    PubMed

    Ulusoy, Beyza H; Çakmak Sancar, Burcu; Öztürk, Muhsin

    2017-10-01

    The aim of this study was to investigate the prevalence of staphylococcal enterotoxins (SEs) in ready-to-eat (RTE) foods sold in Istanbul, Turkey. A total of 5,241 samples were randomly collected from various caterers, hotels, and restaurants from 2014 to 2016. The samples were classified into four groups: (i) various cooked RTE meat and vegetable meals, (ii) various RTE salads, charcuterie, and cold appetizers, (iii) various cooked RTE bakery products (pasta, pastries, pizza, pita, ravioli, etc.), and (iv) any cooked RTE sweets and desserts (pudding, custard, cream, ashura, etc.). The samples were examined for the presence of SEs by 3M Tecra Staph Enterotoxin Visual Immunoassay method, which is a manual enzyme-linked immunosorbent assay method. Among all samples, only 1 (0.019%) RTE meal (vegetable meal with meat) was found to be contaminated with SEs, a good result in terms of staphylococcal food poisoning risk and public health.

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

    PubMed Central

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

    2011-01-01

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

  17. Lack of Evidence of Enterotoxin Involvement in Pathogenesis of Campylobacter Diarrhea

    DTIC Science & Technology

    1992-01-01

    co/i or 1: cholerae genes encod- diarrheal disease (7, 8). but specific virulence ing enterotoxin production (29); (ii) several mechanisms are not...or watery trast to the secretory diarrhea caused by enter- stools and the absence of fever, consistent with otoxigenic E. co/i or V cholerae (21...potential of enteric host response to the toxin. Ifpresent. these find- pathogens, for many organisms, including Vibrio ings would indicate that toxinogenesis

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

    PubMed Central

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

    2014-01-01

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

  19. Pilot Scale Production and Testing of a Recombinant Staphylococcal Enterotoxin (SEB) Triple Mutant

    DTIC Science & Technology

    2017-09-01

    1 PILOT-SCALE PRODUCTION AND TESTING OF A RECOMBINANT STAPHYLOCOCCAL ENTEROTOXIN (SEB) TRIPLE MUTANT ECBC...Disclaimer The findings in this report are not to be construed as an official Department of the Army position unless so designated by other authorizing...TYPE Final 3. DATES COVERED (From - To) Mar 2010 – Dec 2011 4. TITLE AND SUBTITLE Pilot-Scale Production and Testing of a Recombinant

  20. Effect of pH, Sodium Chloride, and Sodium Nitrite on Enterotoxin A Production

    PubMed Central

    Tompkin, R. B.; Ambrosino, J. M.; Stozek, S. K.

    1973-01-01

    The combined effects of pH, sodium chloride, and sodium nitrite were studied by using a dialysis sac technique in brain heart infusion broth. Growth and enterotoxin A production by Staphylococcus aureus strain 100 were found to decrease with the addition of sodium nitrite, with a decrease in pH from 7.0, and with an increase in sodium chloride concentration. The significance of these results is discussed in relation to cured meats. PMID:4203331

  1. The Hemolytic Enterotoxin HBL Is Broadly Distributed among Species of the Bacillus cereus Group

    PubMed Central

    Prüß, Birgit M.; Dietrich, Richard; Nibler, Birgit; Märtlbauer, Erwin; Scherer, Siegfried

    1999-01-01

    The prevalence of the hemolytic enterotoxin complex HBL was determined in all species of the Bacillus cereus group with the exception of Bacillus anthracis. hblA, encoding the binding subunit B, was detected by PCR and Southern analysis and was confirmed by partial sequencing of 18 strains. The sequences formed two clusters, one including B. cereus and Bacillus thuringiensis strains and the other one consisting of Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis strains. From eight B. thuringiensis strains, the enterotoxin gene hblA could be amplified. Seven of them also expressed the complete HBL complex as determined with specific antibodies against the L1, L2, and B components. Eleven of 16 B. mycoides strains, all 3 B. pseudomyoides strains, 9 of 15 B. weihenstephanensis strains, and 10 of 23 B. cereus strains carried hblA. While HBL was not expressed in the B. pseudomycoides strains, the molecular assays were in accordance with the immunological assays for the majority of the remaining strains. In summary, the hemolytic enterotoxin HBL seems to be broadly distributed among strains of the B. cereus group and relates neither to a certain species nor to a specific environment. The consequences of this finding for food safety considerations need to be evaluated. PMID:10584001

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

  3. Staphylococcal food poisoning caused by Staphylococcus argenteus harboring staphylococcal enterotoxin genes.

    PubMed

    Wakabayashi, Yuki; Umeda, Kaoru; Yonogi, Shinya; Nakamura, Hiromi; Yamamoto, Kaori; Kumeda, Yuko; Kawatsu, Kentaro

    2018-01-16

    Staphylococcal food poisoning (SFP) is caused by staphylococcal enterotoxins (SEs) preformed in food materials. SE genes are encoded on mobile genetic elements and are widely found across Staphylococcus species including S. argenteus, although most SFP cases are caused by S. aureus. S. argenteus, recently discriminated from S. aureus as a novel species, are non-pigmented staphylococci phenotypically related to S. aureus. In 2014 and 2015, two independent food poisoning cases occurred in Osaka, Japan, in which non-pigmented staphylococci were predominantly isolated. Several enterotoxin genes (seb, seg, sei, sem, sen, seo, and selu2) were found in their genome and the production of SEB was confirmed by reverse passive agglutination tests. The non-pigmented isolates from patients, food handlers, food, and cooking utensils all produced the same pulsed-field gel electrophoresis pattern. These non-pigmented isolates were coagulase-positive and biochemically identical to S. aureus. We performed further genetic analysis using nucA sequencing and multi-locus sequence typing, and identified these isolates as S. argenteus. We also found that seb was encoded on the Staphylococcus aureus pathogenicity island, while seg, sei, sem, sen, seo, and selu2 were encoded on the enterotoxin gene cluster. From these results, we concluded that the two food poisoning outbreaks were SFP cases caused by S. argenteus harboring SE genes. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Production of Staphylococcal Enterotoxins D and R in Milk and Meat Juice by Staphylococcus aureus Strains.

    PubMed

    Schubert, Justyna; Podkowik, Magdalena; Bystroń, Jarosław; Bania, Jacek

    2017-04-01

    Seventeen Staphylococcus aureus strains were tested for production of staphylococcal enterotoxin D (SED) and staphylococcal enterotoxin R (SER) in milk and meat juice. SED was secreted in milk by 12 S. aureus strains at 6-54 ng/mL at 24 h and 9-98 ng/mL at 48 h. Another five strains secreted SED at 0.9-1.9 μg/mL at 24 h and at 1.2-2.4 μg/mL at 48 h. Strains producing high levels of SED in milk secreted 77-666 μg/mL of SED in meat juice at 24 h and 132-1225 μg/mL at 48 h. Strains producing lower amounts of SED in milk secreted 228-1109 ng/mL of SED at 24 h and 377-1782 ng/mL at 48 h in meat juice. Tested S. aureus strains produced SER in milk at 33-183 ng/mL at 24 h and 41-832 ng/mL at 48 h. Fourteen strains produced more SER in meat juice than in milk (17- to 232-fold and 15- to 269-fold more at 24 and 48 h, respectively). Three S. aureus strains secreted less than 74 ng/mL of SER in meat juice. Expression pattern of known enterotoxin regulators, that is, agrA, sarA, hld, rot, and sigB, was similar in selected strong and weak SED producers grown in both food matrices and could not explain differences in enterotoxin protein level. This suggests that enterotoxin regulation is more complex than previously thought. We demonstrated that in a number of tested S. aureus strains, production of SED and SER was significantly decreased in milk when compared with meat juice, supporting previous reports. However, certain strains secreted high amounts of SED and SER, irrespective of environment, likely contributing to higher food safety risk.

  5. First proficiency testing to evaluate the ability of European Union National Reference Laboratories to detect staphylococcal enterotoxins in milk products.

    PubMed

    Hennekinne, Jacques-Antoine; Gohier, Martine; Maire, Tiphaine; Lapeyre, Christiane; Lombard, Bertrand; Dragacci, Sylviane

    2003-01-01

    The European Commission has designed a network of European Union-National Reference Laboratories (EU-NRLs), coordinated by a Community Reference Laboratory (CRL), for control of hygiene of milk and milk products (Council Directive 92/46/ECC). As a common contaminant of milk and milk products such as cheese, staphylococcal enterotoxins are often involved in human outbreaks and should be monitored regularly. The main tasks of the EU-CRLs were to select and transfer to the EU-NRLs a reference method for detection of enterotoxins, and to set up proficiency testing to evaluate the competency of the European laboratory network. The first interlaboratory exercise was performed on samples of freeze-dried cheese inoculated with 2 levels of staphylococcal enterotoxins (0.1 and 0.25 ng/g) and on an uninoculated control. These levels were chosen considering the EU regulation for staphylococcal enterotoxins in milk and milk products and the limit of detection of the enzyme-linked immunosorbent assay test recommended in the reference method. The trial was conducted according to the recommendations of ISO Guide 43. Results produced by laboratories were compiled and compared through statistical analysis. Except for data from 2 laboratories for the uninoculated control and cheese inoculated at 0.1 ng/g, all laboratories produced satisfactory results, showing the ability of the EU-NRL network to monitor the enterotoxin contaminant.

  6. 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

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

    PubMed

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

    2014-11-01

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

  8. Staphylococcal enterotoxin C2 promotes osteogenesis of mesenchymal stem cells and accelerates fracture healing

    PubMed Central

    Wu, T.; Wang, B.; Sun, Y.; Liu, Y.; Li, G.

    2018-01-01

    Objectives As one of the heat-stable enterotoxins, Staphylococcal enterotoxin C2 (SEC2) is synthesized by Staphylococcus aureus, which has been proved to inhibit the growth of tumour cells, and is used as an antitumour agent in cancer immunotherapy. Although SEC2 has been reported to promote osteogenic differentiation of human mesenchymal stem cells (MSCs), the in vivo function of SCE2 in animal model remains elusive. The aim of this study was to further elucidate the in vivo effect of SCE2 on fracture healing. Materials and Methods Rat MSCs were used to test the effects of SEC2 on their proliferation and osteogenic differentiation potentials. A rat femoral fracture model was used to examine the effect of local administration of SEC2 on fracture healing using radiographic analyses, micro-CT analyses, biomechanical testing, and histological analyses. Results While SEC2 was found to have no effect on rat MSCs proliferation, it promoted the osteoblast differentiation of rat MSCs. In the rat femoral fracture model, the local administration of SEC2 accelerated fracture healing by increasing fracture callus volumes, bone volume over total volume (BV/TV), and biomechanical recovery. The SEC2 treatment group has superior histological appearance compared with the control group. Conclusion These data suggest that local administration of SEC2 may be a novel therapeutic approach to enhancing bone repair such as fracture healing. Cite this article: T. Wu, J. Zhang, B. Wang, Y. Sun, Y. Liu, G. Li. Staphylococcal enterotoxin C2 promotes osteogenesis of mesenchymal stem cells and accelerates fracture healing. Bone Joint Res 2018;7:179–186. DOI: 10.1302/2046-3758.72.BJR-2017-0229.R1. PMID:29682284

  9. Enzyme-linked immunosorbent assay for Escherichia coli heat-stable enterotoxin type II.

    PubMed Central

    Handl, C; Rönnberg, B; Nilsson, B; Olsson, E; Jonsson, H; Flock, J I

    1988-01-01

    The gene for Escherichia coli heat-stable enterotoxin type II (STII) was fused to the genes for protein A from Staphylococcus aureus and beta-galactosidase in two different expression systems. Antibodies raised in rabbits against the protein A-STII fusion protein recognized the beta-galactosidase-STII fusion protein. The latter fusion protein was used as the immobilized antigen in an enzyme-linked immunosorbent assay (ELISA) for detection of STII. The correlation between the results of the ELISA and the intestinal loop test in piglets was 95%, suggesting that the ELISA can be used to reliably detect STII. Images PMID:3049659

  10. Cloning Sequencing and Structural Manipulation of the Enterotoxin D and E Genes from Staphylococcus aureus

    DTIC Science & Technology

    1990-07-01

    Iandolo, J.J. and R.K. Tweten. 1988. Purification of staphylococcal enterotoxin. In. Methods of Enzymology, (N.O. Kaplan , S. Harshman, Eds.) volume 165...N C V 9 L G 0 K 1 S P A K I C T S N 0 149 "T7AACTGG&&C&hICG&clhht:7caflCATGTAATAa~aI ACca CAACaCAA 942 L K D G D K L Z L I G T P r D H K V N 0 H L..L

  11. An Enterotoxin-Like Binary Protein from Pseudomonas protegens with Potent Nematicidal Activity.

    PubMed

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

    2017-10-01

    Soil microbes are a major food source for free-living soil nematodes. It is known that certain soil bacteria have evolved systems to combat predation. We identified the nematode-antagonistic Pseudomonas protegens strain 15G2 from screening of microbes. Through protein purification we identified a binary protein, designated Pp-ANP, which is responsible for the nematicidal activity. This binary protein inhibits Caenorhabditis elegans growth and development by arresting larvae at the L1 stage and killing older-staged worms. The two subunits, Pp-ANP1a and Pp-ANP2a, are active when reconstituted from separate expression in Escherichia coli The binary toxin also shows strong nematicidal activity against three other free-living nematodes ( Pristionchus pacificus , Panagrellus redivivus , and Acrobeloides sp.), but we did not find any activity against insects and fungi under test conditions, indicating specificity for nematodes. Pp-ANP1a has no significant identity to any known proteins, while Pp-ANP2a shows ∼30% identity to E. coli heat-labile enterotoxin (LT) subunit A and cholera toxin (CT) subunit A. Protein modeling indicates that Pp-ANP2a is structurally similar to CT/LT and likely acts as an ADP-ribosyltransferase. Despite the similarity, Pp-ANP shows several characteristics distinct from CT/LT toxins. Our results indicate that Pp-ANP is a new enterotoxin-like binary toxin with potent and specific activity to nematodes. The potency and specificity of Pp-ANP suggest applications in controlling parasitic nematodes and open an avenue for further research on its mechanism of action and role in bacterium-nematode interaction. IMPORTANCE This study reports the discovery of a new enterotoxin-like binary protein, Pp-ANP, from a Pseudomonas protegens strain. Pp-ANP shows strong nematicidal activity against Caenorhabditis elegans larvae and older-staged worms. It also shows strong activity on other free-living nematodes ( Pristionchus pacificus , Panagrellus redivivus , and

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

    PubMed

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

    2015-01-01

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

  13. The aggregational status of cholera enterotoxin fragment A following biochemical fractionation.

    PubMed

    Knoop, F C

    1978-01-01

    Aggregates of frabment A of Vibrio cholerae enterotoxin were revealed following isoelectric focusing in 8 M urea of molecular sieve chromatography in 4% (v/v) formic acid. These aggregates consisted of dimers which required the presence of 10 M urea, 1% sodium dodecyl sulfate (SDS), 2 mM ethylenediaminetetracetic acid (EDTA) and heat (60 degrees C for 1 h) for complete dissociation. All aggregates were homogeneous when tested by standard analytical and SDS polyacrylamide gel electrophoresis or immunodiffusion analysis. Aggregates of fragment A were biologically active in the mouse Y1 adrenal cell assay.

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

    PubMed

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

    2012-02-01

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

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

    PubMed

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

    2017-07-15

    Bacillus cereus sensu lato species, as well as Staphylococcus aureus , are important pathogenic bacteria which can cause foodborne illness through the production of enterotoxins. This study characterised enterotoxin genes of these species and examined growth and enterotoxin production dynamics of isolates when grown in milk or meat-based broth. All B. cereus s. l. isolates harboured nheA , hblA and entFM toxin genes, with lower prevalence of bceT and hlyII . When grown at 16 °C, toxin production by individual B. cereus s. l. isolates varied depending on the food matrix; toxin was detected at cell densities below 5 log 10 (CFU/mL). At 16 °C no staphylococcal enterotoxin C (SEC) production was detected by S. aureus isolates, although low levels of SED production was noted. At 30 °C all S. aureus isolates produced detectable enterotoxin in the simulated meat matrix, whereas SEC production was significantly reduced in milk. Relative to B. cereus s. l. toxin production, S. aureus typically required reaching higher cell numbers to produce detectable levels of enterotoxin. Phylogenetic analysis of the sec and sel genes suggested population evolution which correlated with animal host adaptation, with subgroups of bovine isolates or caprine/ovine isolates noted, which were distinct from human isolates. Taken together, this study highlights the marked differences in the production of enterotoxins both associated with different growth matrices themselves, but also in the behaviour of individual strains when exposed to different food matrices.

  16. 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

  17. Some properties of purified Escherichia coli heat-stable enterotoxin II.

    PubMed Central

    Hitotsubashi, S; Fujii, Y; Yamanaka, H; Okamoto, K

    1992-01-01

    We examined the biological properties of purified Escherichia coli heat-stable enterotoxin II (STII) using mouse intestinal loop assays and compared these properties with those of heat-stable enterotoxin I (STI) and cholera toxin (CT). The action of STII over time differed from those of STI and CT. STII did not alter cyclic GMP or cyclic AMP levels in intestinal mucosal cells. Our results supported the idea that the mechanism of action of STII in inducing fluid secretion is different from the mechanisms of action of STI and CT. This hypothesis was further supported by the fact that an anti-STII neutralizing serum did not neutralize the activities of STI and CT. Subsequently, we examined the involvement of prostaglandins in the action of STII. The level of prostaglandin E2 in the fluid accumulated as a result of the action of STII increased, and the prostaglandin synthesis inhibitors aspirin and indomethacin significantly reduced the response to STII. These results implicate prostaglandin E2 in the mechanism of action of STII. Images PMID:1398961

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

    PubMed Central

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

    2010-01-01

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

  19. Effect of beef broth protein on the thermal inactivation of staphylococcal enterotoxin B1.

    PubMed Central

    Lee, I C; Stevenson, K E; Harmon, L G

    1977-01-01

    Enterotoxin B produced by Staphylococus aureus 243 in brain heart infusion broth was concentrated by dialysis against 40% polyethylene glycol (20 M), partially purified on a Sephadex G-100 column and heated at 110 degrees C in thermal death time cans. Various heating menstrua included 0.04 M Veronal buffer (pH 7.4), beef broth, and fractions of beef broth obtained by ultrafiltration or precipitation with ammonium sulfate. The toxin was assayed serologically using the microslide gel double-diffusion method. The time requiring for 90% inactivation at 110 degrees C (D110 value) obtained in buffer and in beef broth was 18 and 60 min, respectively. When the concentration of beef broth was increased fivefold, the D110 increased to 78 min. The apparent protective effect or protein was further investigated using beef broth protein obtained by precipitation with (NH4)2SO4. The D110 values were 51 and 70 min when the protein concentration in the heating menstruum was 3.8 and 7.7 mg/ml, respectively. However, when the beef broth protein was dialyzed against buffer before use as a heating menstrum, the D110 was only 39 or 41 min at comparable protein concentrations. Results indicated a dialyzable factor, whose protective effect was partially destroyed by trypsin and chymotrypsin but did not by disodium ethylenediaminetetraacetate, was involved in the protection of enterotoxin B during heating. PMID:403860

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

    NASA Astrophysics Data System (ADS)

    Gordon, Geoffrey; Lo, Chun-Min

    2007-03-01

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

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

    PubMed Central

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

    1989-01-01

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

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

    USDA-ARS?s Scientific Manuscript database

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

  3. Low cost bioluminescence imaging as an alternative to in vivo bioassays for quantifying biologically active staphylococcal enterotoxin type E

    USDA-ARS?s Scientific Manuscript database

    Staphylococcus aureus is a major causative agent implicated in outbreaks of food poisoning, acting through the production of a range of toxins including staphylococcal enterotoxin type E (SEE). While tests such as enzyme-linked immunosorbent (ELISA) exist to detect the toxin molecules, existing meth...

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

    PubMed

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

    2014-02-01

    The objectives of this study were to determine the occurrence and diversity of Staphylococcus spp. in milk from healthy cows and cows with subclinical mastitis in Brazil and to examine the profile of enterotoxin genes and some enterotoxins produced by Staphylococcus spp. A total of 280 individual mammary quarter milk samples from 70 healthy cows and 292 samples from 73 cows with subclinical mastitis were collected from 11 farms in the state of São Paulo, Brazil. Staphylococcus spp. were recovered from 63 (22.5%) samples from healthy cows and from 80 samples (27.4%) from cows with mastitis. The presence of Staphylococcus aureus was significantly different between these 2 groups and was more prevalent in the cows with mastitis. The presence of Staphylococcus saprophyticus was also significantly different between these 2 groups, but this organism was more prevalent in healthy cows. No statistically significant differences were observed in the numbers of other staphylococci in milk samples from the 2 groups. The sea gene was the most prevalent enterotoxin gene in both groups. Eight of 15 (53.3%) Staph. aureus carried this gene and all produced the SEA toxin. In the coagulase-negative staphylococci (CNS) group, 61 of 128 (47.5%) had the same gene and just 1 (1.6%) Staphylococcus epidermidis strain produced the enterotoxin in vitro. Because CNS were isolated from both groups of cows and most CNS contained enterotoxin genes but did not produce toxins, the role of CNS in mastitis should be carefully defined. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  5. Nanoshell-Enhanced Raman Spectroscopy on a Microplate for Staphylococcal Enterotoxin B Sensing.

    PubMed

    Wang, Wenbin; Wang, Weiwei; Liu, Liqiang; Xu, Liguang; Kuang, Hua; Zhu, Jianping; Xu, Chuanlai

    2016-06-22

    A sensitive surface-enhanced Raman scattering (SERS) immunosensor based on the Au nanoparticle (Au NP) shell structure was developed to detect staphylococcal enterotoxin B (SEB) on a microplate. Au NPs modified with 4-nitrothiophenol (4-NTP) and coated with Ag shell of controlled thickness at 6.6 nm exhibited excellent SERS intensity and were used as signal reporters in the detection of SEB. The engaged 4-NTP allowed the significant electromagnetic enhancement between Au NPs and the Ag shell and prevented the dissociation of the Raman reporter. More importantly, 4-NTP-differentiated SERS signals between the sample and microplate. The SERS-based immunosensor had a limit of detection of 1.3 pg/mL SEB. Analysis of SEB-spiked milk samples revealed that the developed method had high accuracy. Therefore, the SERS-encoded Au@Ag core-shell structure-based immunosensor is promising for the detection of biotoxins, pathogens, and environmental pollutants.

  6. Claudins Overexpression in Ovarian Cancer: Potential Targets for Clostridium Perfringens Enterotoxin (CPE) Based Diagnosis and Therapy

    PubMed Central

    English, Diana P.; Santin, Alessandro D.

    2013-01-01

    Claudins are a family of tight junction proteins regulating paracellular permeability and cell polarity with different patterns of expression in benign and malignant human tissues. There are approximately 27 members of the claudin family identified to date with varying cell and tissue-specific expression. Claudins-3, -4 and -7 represent the most highly differentially expressed claudins in ovarian cancer. While their exact role in ovarian tumors is still being elucidated, these proteins are thought to be critical for ovarian cancer cell invasion/dissemination and resistance to chemotherapy. Claudin-3 and claudin-4 are the natural receptors for the Clostridium perfringens enterotoxin (CPE), a potent cytolytic toxin. These surface proteins may therefore represent attractive targets for the detection and treatment of chemotherapy-resistant ovarian cancer and other aggressive solid tumors overexpressing claudin-3 and -4 using CPE-based theranostic agents. PMID:23685873

  7. Spectral surface plasmon resonance biosensor for detection of staphylococcal enterotoxin B in milk.

    PubMed

    Homola, Jirí; Dostálek, Jakub; Chen, Shengfu; Rasooly, Avraham; Jiang, Shaoyi; Yee, Sinclair S

    2002-05-05

    This work evaluates a newly developed wavelength modulation-based SPR biosensor for the detection of staphylococcal enterotoxin B (SEB) in milk. Two modes of operation of the SPR biosensor are described: direct detection of SEB and sandwich assay. In the sandwich assay detection mode, secondary antibodies are bound to the already captured toxin to amplify sensor response. Samples including SEB in buffer and SEB in milk were analyzed in this work. The SPR biosensor has been shown to be capable of directly detecting concentrations of SEB in buffer as low as 5 ng/ml. In sandwich detection mode, the lowest detection limit was determined to be 0.5 ng/ml for both buffer and milk samples. The reported wavelength modulation-based SPR sensor provides a generic platform which can be tailored for detection of various foodborne pathogens and agents for food analysis and testing.

  8. 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.

  9. Type II Heat-labile Enterotoxins: Structure, Function, and Immunomofdulatory Properties

    PubMed Central

    Hajishengallis, George; Connell, Terry D.

    2012-01-01

    The heat-labile enterotoxins (HLTs) of Escherichia coli and Vibrio cholerae are classified into two major types on the basis of genetic, biochemical, and immunological properties. Type I and Type II HLT have been intensively studied for their exceptionally strong adjuvant activities. Despite general structural similarities, these molecules, in intact or derivative (non-toxic) forms, display notable differences in their mode of immunomodulatory action. The molecular basis of these differences has remained largely uncharacterized until recently. This review focuses on the Type II HLTs and their immunomodulatory properties which depend largely on interactions with unique gangliosides and Toll-like receptors that are not utilized by the Type I HLTs. PMID:23137790

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

    PubMed

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

    2009-08-15

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2016-11-01

    Our aim was to assess the effect of pasteurization temperature on inactivation of staphylococcal enterotoxins (SE). Milk samples were inoculated with log 4.38 to 5.18cfu/mL of 40 different Staphylococcus aureus strains having the ability to produce types A, B, or C SE and incubated at 37°C for 24h to develop SE. This incubation was followed by heat treatment for 15 s at 72, 85, and 92°C. Samples were analyzed for Staph. aureus count by plate method and, specifically, for SE presence. An enzyme-linked immunofluorescent assay on a MiniVIDAS analyzer (bioMérieux, Marcy l'Étoile, France) was used to detect SE, which were determined semiquantitatively based on test values. The Staph. aureus count in milk before pasteurization did not affect the amount of SE. Before pasteurization, SEB was detected in the lowest amount compared with other SE types. Staphylococcal enterotoxins were markedly reduced with pasteurization and inactivated at pasteurization temperatures to an extent depending on the amount in the sample before pasteurization. After pasteurization at 72°C, SE were detected in 87.5% of samples (35/40), after pasteurization at 85°C in 52.5% of samples (21/40), and after pasteurization at 92°C in 45.0% of samples (18/40). We determined that SE may still persist in milk even when Staph. aureus bacteria are inactivated through pasteurization. Although pasteurization may partially inactivate SE in milk, a key measure in the prevention of staphylococcal enterotoxicosis linked to pasteurized milk consumption is to avoid any cold chain disruption during milk production and processing. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  15. A Single-Stranded DNA Aptamer That Selectively Binds to Staphylococcus aureus Enterotoxin B

    PubMed Central

    DeGrasse, Jeffrey A.

    2012-01-01

    The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APTSEB1, successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide. PMID:22438927

  16. A single-stranded DNA aptamer that selectively binds to Staphylococcus aureus enterotoxin B.

    PubMed

    DeGrasse, Jeffrey A

    2012-01-01

    The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APT(SEB1), successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide.

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

    PubMed Central

    Li, Jihong; Freedman, John C.; Evans, Daniel R.

    2017-01-01

    ABSTRACT Clostridium perfringens type D strains cause enterotoxemia and enteritis in livestock via epsilon toxin production. In type D strain CN3718, CodY was previously shown to increase the level of epsilon toxin production and repress sporulation. C. perfringens type A strains producing C. perfringens enterotoxin (CPE) cause human food poisoning and antibiotic-associated diarrhea. Sporulation is critical for C. perfringens type A food poisoning since spores contribute to transmission and resistance in the harsh food environment and sporulation is essential for CPE production. Therefore, the current study asked whether CodY also regulates sporulation and CPE production in SM101, a derivative of C. perfringens type A food-poisoning strain NCTC8798. An isogenic codY-null mutant of SM101 showed decreased levels of spore formation, along with lower levels of CPE production. A complemented strain recovered wild-type levels of both sporulation and CPE production. When this result was coupled with the earlier results obtained with CN3718, it became apparent that CodY regulation of sporulation varies among different C. perfringens strains. Results from quantitative reverse transcriptase PCR analysis clearly demonstrated that, during sporulation, codY transcript levels remained high in SM101 but rapidly declined in CN3718. In addition, abrB gene expression patterns varied significantly between codY-null mutants of SM101 and CN3718. Compared to the levels in their wild-type parents, the level of abrB gene expression decreased in the CN3718 codY-null mutant strain but significantly increased in the SM101 codY-null mutant strain, demonstrating CodY-dependent regulation differences in abrB expression between these two strains. This difference appears to be important since overexpression of the abrB gene in SM101 reduced the levels of sporulation and enterotoxin production, supporting the involvement of AbrB repression in regulating C. perfringens sporulation. PMID:28052992

  18. Nutritional Requirements for Synthesis of Heat-Labile Enterotoxin by Enterotoxigenic Strains of Escherichia coli

    PubMed Central

    Gilligan, Peter H.; Robertson, Donald 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 Fe3+ was slightly stimulatory for porcine strains. The stimulation by Fe3+ 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. PMID:33900

  19. Detection of Staphylococcus aureus enterotoxins in sheep cheese and dairy desserts by multiplex PCR technique.

    PubMed

    Ertas, Nurhan; Gonulalan, Zafer; Yildirim, Yeliz; Kum, Erhan

    2010-08-15

    The aim of this study was to investigate the presence of Staphylococcus aureus (S. aureus) and staphylococcal enterotoxins (SEs) genes in sheep cheese and dairy dessert samples by multiplex PCR (mPCR) technique. A total of 150 samples were analyzed consisting of 50 dairy dessert samples and 100 sheep cheese. Coagulase positive staphylococci (CPS) were found in 86 (57.3%) out of 150 analyzed samples. S. aureus were isolated from 60 (60%), 26 (52%) of sheep cheese and from of dairy desserts, respectively. Five suspected colonies were tested from each sheep cheese and dairy dessert samples for phenotypic and genotypic characterizations. A total of 430 isolates from the 86 positive samples were investigated in this study. Eighty (18.6%) isolates were characterized as S. aureus. The enterotoxin genes (sea, seb, sec, sed) were found in 13 (3.02%) out of 80 isolates. From cheese isolates, sea, seb and sed were detected in 5 (1.6%), 2 (0.6%), 1 (0.3%), respectively. From dairy dessert isolates, sea, sec and sed were detected in 3 (2.3%), 1 (0.76%), 1 (0.76%), respectively. The presence of SEs was identified in 12 (2.8%) out of 80 isolates by using ELISA technique. It was determined that these SEs had a distribution of 7 (1.6%) SEA, 2 (0.46%) SEB, 1 (0.23%) SEC, and 2 (0.46%) SED. SEs were found in 7 (2.3%) cheese and 5 (3.8%) dairy dessert isolates. In conclusion, S.aureus and their SEs were found to be present in sheep cheese and dairy desserts in this study. It is emphasized that the presence of S. aureus and their SEs genes in sheep cheese and dairy desserts may be regarded as a potential risk for human health. Copyright 2010 Elsevier B.V. All rights reserved.

  20. Passive therapy with humanized anti-staphylococcal enterotoxin B antibodies attenuates systemic inflammatory response and protects from lethal pneumonia caused by staphylococcal enterotoxin B-producing Staphylococcus aureus.

    PubMed

    Karau, Melissa J; Tilahun, Mulualem E; Krogman, Ashton; Osborne, Barbara A; Goldsby, Richard A; David, Chella S; Mandrekar, Jayawant N; Patel, Robin; Rajagopalan, Govindarajan

    2017-10-03

    Drugs such as linezolid that inhibit bacterial protein synthesis may be beneficial in treating infections caused by toxigenic Staphylococcus aureus. As protein synthesis inhibitors have no effect on preformed toxins, neutralization of pathogenic exotoxins with anti-toxin antibodies may be beneficial in conjunction with antibacterial therapy. Herein, we evaluated the efficacy of human-mouse chimeric high-affinity neutralizing anti-staphylococcal enterotoxin B (SEB) antibodies in the treatment of experimental pneumonia caused by SEB-producing S. aureus. Since HLA class II transgenic mice mount a stronger systemic immune response following challenge with SEB and are more susceptible to SEB-induced lethal toxic shock than conventional mice strains, HLA-DR3 transgenic mice were used. Lethal pneumonia caused by SEB-producing S. aureus in HLA-DR3 transgenic mice was characterized by robust T cell activation and elevated systemic levels of several pro-inflammatory cytokines and chemokines. Prophylactic administration of a single dose of linezolid 30 min prior to the onset of infection attenuated the systemic inflammatory response and protected from mortality whereas linezolid administered 60 min after the onset of infection failed to confer significant protection. Human-mouse chimeric high-affinity neutralizing anti-SEB antibodies alone, but not polyclonal human IgG, mitigated this response and protected from death when administered immediately after initiation of infection. Further, anti-SEB antibodies as well as intact polyclonal human IgG, but not its Fab or Fc fragments, protected from lethal pneumonia when followed with linezolid therapy 60 min later. In conclusion, neutralization of superantigens with high-affinity antibodies may have beneficial effects in pneumonia.

  1. Toll-Like Receptor 2 Mediates Cellular Activation by the B Subunits of Type II Heat-Labile Enterotoxins

    PubMed Central

    Hajishengallis, George; Tapping, Richard I.; Martin, Michael H.; Nawar, Hesham; Lyle, Elizabeth A.; Russell, Michael W.; Connell, Terry D.

    2005-01-01

    The type II heat-labile enterotoxins (LT-IIa and LT-IIb) of Escherichia coli have an AB5 subunit structure similar to that of cholera toxin (CT) and other type I enterotoxins, despite significant differences in the amino acid sequences of their B subunits and different ganglioside receptor specificities. LT-II holotoxins and their nontoxic B subunits display unique properties as immunological adjuvants distinct from those of CT and its B subunits. In contrast to type II holotoxins, the corresponding pentameric B subunits, LT-IIaB and LT-IIbB, stimulated cytokine release in both human and mouse cells dependent upon Toll-like receptor 2 (TLR2). Induction of interleukin-1β (IL-1β), IL-6, IL-8, or tumor necrosis factor alpha in human THP-1 cells by LT-IIaB or LT-IIbB was inhibited by anti-TLR2 but not by anti-TLR4 antibody. Furthermore, transient expression of TLR1 and TLR2 in human embryonic kidney 293 cells resulted in activation of a nuclear factor-κB-dependent luciferase gene in response to LT-IIaB or LT-IIbB. Moreover, peritoneal macrophages from TLR2-deficient mice failed to respond to LT-IIaB or LT-IIbB, in contrast to wild-type or TLR4-deficient cells. These results demonstrate that besides their established binding to gangliosides, the B subunits of type II enterotoxins also interact with TLR2. Although a ganglioside-nonbinding mutant (T34I) of LT-IIaB effectively induced cytokine release, a phenotypically similar point mutation (T13I) in LT-IIbB abrogated cytokine induction, suggesting a variable requirement for gangliosides as coreceptors in TLR2 agonist activity. TLR2-dependent activation of mononuclear cells by type II enterotoxin B subunits appears to be a novel mechanism whereby these molecules may exert their immunomodulatory and adjuvant activities. PMID:15731031

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

    PubMed

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

    2013-12-01

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

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

    PubMed Central

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

    2013-01-01

    Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection. PMID:24167300

  4. Aeromonas caviae: ecologic adaptation in the intestinal tract of infants coupled to adherence and enterotoxin production as factors in enteropathogenicity.

    PubMed

    Namdari, H; Bottone, E J

    1991-05-15

    Aeromonas caviae isolated from stools of diarrheic formula-fed infants and environmental sources produce acetic acid when grown in glucose broth, which is bactericidal (suicide phenomenon). A. caviae grows anaerobically in a minimal medium or under permissive conditions such as the intestinal tract of formula-fed infants. These isolates adhered to HEp-2 cells and produced a cytotoxic and a cytotonic enterotoxin which underscore their enteropathogenicity.

  5. Characterization of Staphylococcus aureus strains and evidence for the involvement of non-classical enterotoxin genes in food poisoning outbreaks.

    PubMed

    Ciupescu, Laurentiu-Mihai; Auvray, Frederic; Nicorescu, Isabela Madalina; Meheut, Thomas; Ciupescu, Veronica; Lardeux, Anne-Laure; Tanasuica, Rodica; Hennekinne, Jacques-Antoine

    2018-06-05

    To an increasing extent, molecular and genetic characterization is now used to investigate foodborne outbreaks. The aim of this study was to seek molecular links among coagulase-positive staphylococci (CPS) isolated from three recent food poisoning outbreaks in Romania using polymerase chain reaction and pulsed-field gel electrophoresis (PFGE) techniques. Nineteen CPS isolates were identified as Staphylococcus aureus by detection of the 23S rDNA gene. Among them, 15 carried at least one staphylococcal enterotoxin-encoding gene (se). The Calarași outbreak strains grouped in pulsotype 2 and were sed/sej/ser-positive, whereas the Arad outbreak strains clustered in pulsotype 17 and were either sed/seg/sei/sej/ser- or seg/sei-positive. The Pitești outbreak strains clustered in pulsotype 1 and, surprisingly, possessed only one enterotoxin gene, i.e. seh. Similar to other European countries, the seh gene has been identified with increasing frequency in Romanian outbreaks; this highlights the importance of considering the application of methods recommended for staphylococcal enterotoxin regulation in Europe.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    1997-05-01

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

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

    PubMed Central

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

    1997-01-01

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

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

    PubMed

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

    2014-03-01

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

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

    PubMed

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

    2017-01-01

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

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

    PubMed

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

    2014-07-16

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

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

    PubMed

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

    2017-03-01

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

  13. Biosynthesis of staphylococcal enterotoxin A by genetic engineering technology and determination of staphylococcal enterotoxin A in water by HPLC-ESI-TOF.

    PubMed

    Li, Hong-Na; Yuan, Fei; Luo, Yun-Jing; Wang, Jian-Feng; Zhang, Chuan-Bin; Zhou, Wei-E; Ren, Zhi-Qin; Wu, Wen-Jie; Zhang, Feng

    2017-08-01

    Staphylococcal enterotoxin A (SEA) was the major virulence factor of Staphylococcus aureus and a biomarker of S. aureus. To establish a fast, low cost, high accuracy, reliable, and simple method for detecting S. aureus, SEA was analyzed by HPLC-ESI-TOF. SEA was not yet commercially available in universal, so SEA was prepared before it was analyzed by HPLC-ESI-TOF. The result showed that high purified SEA was successfully prepared and SEA has normal distribution in mass spectra. A large amount of recombinant SEA (rSEA) was obtained by engineering technology and was purified by Ni affinity chromatography column, and the expression and purity of rSEA and SEA were analyzed by SDS-PAGE. The factors effected on ionization of SEA were studied, and the qualitative analysis of SEA by HPLC-ESI-TOF. The result showed that large amount of SEs expressed within a short time at 28 °C or thereabouts, and there was no impurity bands in electrophorogram after rSEA was purified by Ni affinity chromatography column. In addition, the SEA which had homologous AA sequence with wild SEA was made by rSEA. The retention of SEA in column and ionization of SEA in ESI-TOF were studied for qualitative analysis of S. aureus. The result showed that the content of formic acid in mobile phase was an important factor for ionization of SEs in ESI-TOF. And the result provided theoretical foundation for qualitative detection of S. aureus. [SEs + nH + + mNH 4 + ] n+m+ was shown on ESI-TOF spectra when SEA was detected by ESI-TOF in positive ion mode, and the numerical value of n+m was less than or equal to the number of basic amino acids in SEs. This method was applied to determine SEA in water samples preliminarily, and the detection limit of SEA in spiked water sample was 3 mg/kg. The limit of detection of 3 mg/kg was low sensitivity for low molecular weight matters, but it was high sensitivity for SEA which had a high molecular weight of 27 kDa. Of SEA, 3 mg/kg was equivalent to 10 -4

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

    PubMed

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

    2016-10-01

    Enterotoxigenic Escherichia coli (ETEC) causes ∼20% of the acute infectious diarrhea (AID) episodes worldwide, often by producing heat-stable enterotoxins (STs), which are peptides structurally homologous to paracrine hormones of the intestinal guanylate cyclase C (GUCY2C) receptor. While molecular mechanisms mediating ST-induced intestinal secretion have been defined, advancements in therapeutics have been hampered for decades by the paucity of disease models that integrate molecular and functional endpoints amenable to high-throughput screening. Here, we reveal that mouse and human intestinal enteroids in three-dimensional ex vivo cultures express the components of the GUCY2C secretory signaling axis. ST and its structural analog, linaclotide, an FDA-approved oral secretagog, induced fluid accumulation quantified simultaneously in scores of enteroid lumens, recapitulating ETEC-induced intestinal secretion. Enteroid secretion depended on canonical molecular signaling events responsible for ETEC-induced diarrhea, including cyclic GMP (cGMP) produced by GUCY2C, activation of cGMP-dependent protein kinase (PKG), and opening of the cystic fibrosis transmembrane conductance regulator (CFTR). Importantly, pharmacological inhibition of CFTR abrogated enteroid fluid secretion, providing proof of concept for the utility of this model to screen antidiarrheal agents. Intestinal enteroids offer a unique model, integrating the GUCY2C signaling axis and luminal fluid secretion, to explore the pathophysiology of, and develop platforms for, high-throughput drug screening to identify novel compounds to prevent and treat ETEC diarrheal disease. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  15. Colorimetric DNAzyme Biosensor for Convenience Detection of Enterotoxin B Harboring Staphylococcus aureus from Food Samples.

    PubMed

    Mondal, Bhairab; N, Bhavanashri; Ramlal, Shylaja; Kingston, Joseph

    2018-02-14

    In the present study, a colorimetric DNAzymes biosensor strategy was devised in combination with immunomagnetic separation for rapid and easy detection of enterotoxin B harboring Staphylococcus aureus from food and clinical samples. The method employs immunocapture of S. aureus and amplification of seb gene by DNAzyme complementary sequence integrated forward primer and with specific reverse primer. The DNAzyme sequence integrated dsDNA PCR products when treated with hemin and TMB (3,3',5,5'-tetramethylbenzidine) in the presence of H 2 O 2 produce colorimetric signal. A linear relationship of optical signal with the initial template of seb was obtained which could be monitored by visually or spectrophotrometrically for qualitative and quantitative detection. The limit of detection for the assay was approximately 10 2 CFU/mL of seb gene harboring target. This method is convenient compared to gel based and ELISA systems. Further, spiking studies and analysis on natural samples emphasized the robustness and applicability of developed method. Altogether, the established assay could be a reliable alternative, low-cost, viable detection tool for the routine investigation of seb from food and clinical sources.

  16. Substitutions of cysteine residues of Escherichia coli heat-stable enterotoxin by oligonucleotide-directed mutagenesis.

    PubMed Central

    Okamoto, K; Okamoto, K; Yukitake, J; Kawamoto, Y; Miyama, A

    1987-01-01

    The Escherichia coli 18-amino-acid, heat-stable enterotoxin STp has six cysteine residues linked intramolecularly by three disulfide bonds. These disulfide bonds are important for toxic activity, but the precise role of each bond is not clear. We substituted cysteine residues of STp in vivo by oligonucleotide-directed site-specific mutagenesis to dissociate each disulfide bond and examined the biological activities of the resulting mutants. The Cys-6----Ala and Cys-17----Ala mutations caused a complete loss of toxic activity. The Cys-5----Ala, Cys-10----Ser, and Gly-16, Cys-17----Cys-16, Gly-17 mutations caused a large decrease in toxic activity. These results mean that all three disulfide bonds formed at fixed positions are required for full expression of the biological activity of STp. However, a weak but significant toxicity still remained after three mutations, Cys-5----Ala, Cys-10----Ser, and Gly-16, Cys-17----Cys-16, Gly-17. This indicates that STp has some flexibilities in its conformation to exert toxic activity and that the role of each disulfide bond exerting toxic activity is not quite the same. Images PMID:3305364

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

    PubMed

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

    2010-05-12

    The foodborne pathogen Staphylococcus aureus produces the virulent staphylococcal enterotoxin A (SEA), a single-chain protein that consists of 233 amino acid residues with a molecular weight of 27 078 Da. SEA is a superantigen that is reported to contribute to animal (mastitis) and human (emesis, diarrhea, atopic dermatitis, arthritis, and toxic shock) syndromes. Changes of the native structural integrity may inactivate the toxin by preventing molecular interaction with cell membrane receptor sites of their host cells. In the present study, we evaluated the ability of one commercial and two freshly prepared apple juices and a commercial apple polyphenol preparation (Apple Poly) to inhibit the biological activity of SEA. Dilutions of freshly prepared apple juices and Apple Poly inhibited the biological activity of SEA without any significant cytotoxic effect on the spleen cells. Additional studies with antibody-coated immunomagnetic beads bearing specific antibodies against the toxin revealed that SEA added to apple juice appears to be largely irreversibly bound to the juice constituents. The results suggest that food-compatible and safe anti-toxin phenolic compounds can be used to inactivate SEA in vitro and possibly also in vivo, even after induction of T-cell proliferation by long-term exposure to SEA. The significance of the results for microbial food safety and human health is discussed.

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

    PubMed

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

    2011-01-01

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

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

    PubMed

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

    2016-04-20

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

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

    PubMed

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

    2016-09-01

    Staphylococcal enterotoxin A (SEA) is an important component of Staphylococcus aureus pathogenesis. SEA induces T lymphocytes activation and proliferation, resulting in the release of a large number of inflammatory cytokines. Blocking the toxic cascade triggered by SEA may be an effective strategy for the treatment of SEA-induced diseases. Through a systematic evolution of ligands by exponential enrichment process, we obtained an aptamer (S3) that could bind SEA with both high affinity and specificity, with a Kd value 36.93 ± 7.29 nM (n = 3). This aptamer antagonist effectively inhibited SEA-mediated human peripheral blood mononuclear cells proliferation and inflammatory cytokines (IFN-γ, TNF-α, IL-2 and IL-6) secretion. Moreover, PEGylated S3 significantly reduced mortality in murine lethal toxic shock models established by lipopolysaccharide-potentiated SEA. Therefore, this novel aptamer antagonist has the potential to become a new strategy for treating S. aureus infections and SEA-induced diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Adipose Tissue-Derived Mesenchymal Stem Cells Attenuate Staphylococcal Enterotoxin A-Induced Toxic Shock

    PubMed Central

    Asano, Krisana; Yoshimura, Sayuri

    2015-01-01

    Adipose tissue-derived stem cells (ASCs), which are mesenchymal stromal cells isolated from adipose tissues, exhibit immunomodulatory effects that are promising for several applications, including the therapeutics of inflammatory diseases. In the present study, the effect of ASCs on bacterial toxin-induced inflammation was investigated. Intraperitoneal administration of ASCs rescued mice from lethal shock induced by staphylococcal enterotoxin A (SEA) potentiated with lipopolysaccharide. In the sera and/or spleens of mice administered ASCs, the production of proinflammatory cytokines, including interferon gamma, tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-2 was reduced. By quantitative real-time PCR, the expression of Foxp3 in the mice administered ASCs was not altered. On the other hand, the expression of IL-12 receptor and STAT4 was decreased with ASC administration. These results imply that the effect of ASCs is not involved in the lineage of regulatory T cells but that these cells may modulate TH1 differentiation. This information provides evidence that ASCs have properties that are effective to attenuate SEA-induced toxic shock and should prompt further exploration on other inflammatory diseases caused by bacterial toxins or bacterial infections. PMID:26099581

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

    PubMed Central

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

    2012-01-01

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

  3. In vivo effect of staphylococcal enterotoxin A on peripheral blood lymphocytes.

    PubMed Central

    Zehavi-Willner, T; Shenberg, E; Barnea, A

    1984-01-01

    Staphylococcal enterotoxin A (SEA) administration to monkeys produced an initial lymphocytic leukopenia lasting approximately 24 h. Lymphocytes isolated from blood circulation (PBL) during this stage had normal or decreased [3H]thymidine incorporating activity. After 48 h, however, a significant increase (five- to sixfold) in [3H]thymidine incorporating activity into PBL was apparent. The peak of incorporating activity (seven- to eightfold) was reached 3 to 4 days after SEA administration, followed by a gradual decline, reaching the baseline after 2 weeks. The increased levels of [3H] thymidine incorporation in PBL were concomitant with the conversion of lymphopenia into lymphocytosis, accompanied by the release of many immature cells into the circulation. Lymphocytes isolated 24 h after SEA administration in vivo did not respond to the mitogenic action of SEA in vitro. Lymphocytes isolated at later stages after SEA challenge were fully activated by toxin. From a series of studies, it was concluded that SEA administered to monkeys caused, during the initial 24 h, the removal of a great proportion of lymphocytes from the circulation, followed by the release of new immature cells with augmented DNA synthesis activity. The lymphocytic leukocytosis state declined gradually and reached normal levels between 3 and 4 weeks after the SEA challenge. The biological implications of the hematological changes occurring after SEA challenge in vivo are discussed. PMID:6715041

  4. In vitro assay of Staphylococcus aureus enterotoxin A activity in food.

    PubMed Central

    Rasooly, L; Rose, N R; Shah, D B; Rasooly, A

    1997-01-01

    Staphylococcus aureus enterotoxin A (SEA) is a leading cause of food poisoning. The current test for functional activity of SEA requires monkeys or kittens. The major drawbacks of animal assays are lack of quantitation, poor reproducibility, low sensitivity, and high cost. In this report we describe and evaluate an alternative assay using T-cell proliferation to measure SEA activity in food. Human and rat lymphocytes proliferate in response to concentrations of SEA as low as 1 pg/ml, well below the pathogenic dose of 100 ng. This proliferation assay is highly sensitive, quantitative, and simple. Nonradioactive assays of T-cell proliferation were also suitable for detecting and measuring SEA, although with a 10-fold lower sensitivity. To evaluate the utility of this assay for food testing, four different food samples were mixed with SEA. In each sample, SEA was detected at a concentration of 1 ng/ml. Heat-inactivated SEA produced no detectable proliferation. These results demonstrate that an in vitro cell proliferation assay is an advantageous alternative to existing animal assays for measuring SEA activity in food. PMID:9172356

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

    PubMed Central

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

    2011-01-01

    Development of a rapid field test is needed capable of determining if field supplies of water are safe to drink by the warfighter during a military operation. The present study sought to assess the effectiveness of handheld assays (HHAs) in detecting ricin and Staphylococcal Enterotoxin B (SEB) in water. Performance of HHAs was evaluated in formulated tap water with and without chlorine, reverse osmosis water (RO) with chlorine, and RO with bromine. Each matrix was prepared, spiked with ricin or SEB at multiple concentrations, and then loaded onto HHAs. HHAs were allowed to develop and then read visually. Limits of detection (LOD) were determined for all HHAs in each water type. Both ricin and SEB were detected by HHAs in formulated tap water at or below the suggested health effect levels of 455 ng/mL and 4.55 ng/mL, respectively. However, in brominated or chlorinated waters, LODs for SEB increased to approximately 2,500 ng/mL. LODs for ricin increased in chlorinated water, but still remained below the suggested health effect level. In brominated water, the LOD for ricin increased to approximately 2,500 ng/mL. In conclusion, the HHAs tested were less effective at detecting ricin and SEB in disinfected water, as currently configured. PMID:21792355

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

    PubMed

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

    2011-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2009-10-01

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

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

    PubMed

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

    1998-01-01

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

  11. Enterotoxin gene profile of methicillin-resistant Staphylococcus pseudintermedius isolates from dogs, humans and the environment.

    PubMed

    Phumthanakorn, Nathita; Fungwithaya, Punpichaya; Chanchaithong, Pattrarat; Prapasarakul, Nuvee

    2018-06-01

    This study aimed to detect and identify staphylococcal enterotoxin (SE) genes in methicillin-resistant Staphylococcus pseudintermedius (MRSP) strains from different sources, and to investigate the relationship between their sequence types (STs) and SE gene patterns. The profiles of 17 SE genes in 93 MRSP strains isolated from dogs (n=43), humans (n=18) and the environment (n=32) were detected by PCR. Multilocus sequence typing (MLST), SCCmec typing and pulsed-field gel electrophoresis (PFGE) were used to analyse the clonal relatedness between the molecular type and SE gene profiles.Results/Key findings. The human MRSP strains harboured the greatest number of SE genes (12/17; sea, sec, seg, sei, sek, sel, sem, sen, seo, sep, seq and tst-1) compared to those from dogs (5/17; sec, sel, sem, seq and tst-1) and environmental sources (3/17; sec, seq and tst-1). Using MLST and PFGE, different SE gene profiles were found within the same clonal type. We show that isolates of MRSP vary in their virulence gene profiles, depending on the source from which they have been isolated. This insight should encourage the development of appropriate monitoring and mitigation strategies to reduce the transmission of MRSP in veterinary hospitals and households.

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

    SciTech Connect

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

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Doll, Viktoria M.

    2013-01-01

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

  15. Crystal structure and electrochemical properties of rare earth non-stoichiometric AB5-type alloy as negative electrode material in Ni-MH battery

    NASA Astrophysics Data System (ADS)

    Zhang, Xinbo; Chai, Yujun; Yin, Wenya; Zhao, Minshou

    2004-07-01

    The La 0.85Mg xNi 4.5Co 0.35Al 0.15 (0.05⩽ x⩽0.35) system compounds have been prepared by arc melting method under Ar atmosphere. X-ray diffraction (XRD) analysis reveals that the as-prepared alloys have different lattice parameters and cell volumes. The electrochemical properties of these alloys have been studied through the charge-discharge recycle testing at different temperatures and discharge currents. It is found that the La 0.85Mg 0.25Ni 4.5Co 0.35Al 0.15 alloy electrode is capable of performing high-rate discharge. Moreover, it has very excellent electrochemical properties as negative electrode materials in Ni-MH battery at low temperature, even at -40°C.

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

    PubMed

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

    2008-06-10

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

  17. Staphylococcal enterotoxin A-activated regulatory T cells promote allergen-specific TH2 response to intratracheal allergen inoculation.

    PubMed

    Zeng, Wei-Ping; McFarland, Margaret M; Zhou, Baohua; Holtfreter, Silva; Flesher, Susan; Cheung, Ambrose; Mallick, Avishek

    2017-02-01

    T H 2 responses are implicated in asthma pathobiology. Epidemiologic studies have found a positive association between asthma and exposure to staphylococcal enterotoxins. We used a mouse model of asthma to determine whether staphylococcal enterotoxins promote T H 2 differentiation of allergen-specific CD4 conventional T (Tcon) cells and asthma by activating allergen-nonspecific regulatory T (Treg) cells to create a T H 2-polarizing cytokine milieu. Ovalbumin (OVA)-specific, staphylococcal enterotoxin A (SEA)-nonreactive naive CD4 Tcon cells were cocultured with SEA-reactive allergen-nonspecific Treg or CD4 Tcon cells in the presence of OVA and SEA. The OVA-specific CD4 T cells were then analyzed for IL-13 and IFN-γ expression. SEA-activated Treg cells were analyzed for the expression of the T H 2-polarizing cytokine IL-4 and the T-cell activation markers CD69 and CD62L. For asthma induction, mice were intratracheally sensitized with OVA or cat dander extract (CDE) alone or together with SEA and then challenged with OVA or CDE. Mice were also subject to transient Treg cell depletion before sensitization with OVA plus SEA. Asthma features and T H 2 differentiation in these mice were analyzed. SEA-activated Treg cells induced IL-13 but suppressed IFN-γ expression in OVA-specific CD4 Tcon cells. SEA-activated Treg cells expressed IL-4, upregulated CD69, and downregulated CD62L. Sensitization with OVA plus SEA but not OVA alone induced asthma, and SEA exacerbated asthma induced by CDE. Depletion of Treg cells abolished these effects of SEA and IL-13 expression in OVA-specific T cells. SEA promoted T H 2 responses of allergen-specific T cells and asthma pathogenesis by activating Treg cells. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

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

    PubMed

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

    2014-12-15

    Due to the fast-acting nature of ricin, staphylococcal enterotoxin B (SEB), and Clostridium perfringens epsilon toxin (ETX), it is necessary that therapeutic interventions following a bioterrorism incident by one of these toxins occur as soon as possible after intoxication. Moreover, because the clinical manifestations of intoxication by these agents are likely to be indistinguishable from each other, especially following aerosol exposure, we have developed a cocktail of chimeric monoclonal antibodies that is capable of neutralizing all three toxins. The efficacy of this cocktail was demonstrated in mouse models of lethal dose toxin challenge. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2015-03-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Yasugi, Mayo; Otsuka, Keisuke; Miyake, Masami

    2016-10-01

    Clostridium perfringens type A is a common source of food-borne illness in humans. Ingested vegetative cells sporulate in the small intestinal tract and in the process produce C. perfringens enterotoxin (CPE). Although sporulation plays a critical role in the pathogenesis of food-borne illness, the molecules triggering/inhibiting sporulation are still largely unknown. It has previously been reported by our group that sporulation is induced in C. perfringens strain NCTC8239 co-cultured with Caco-2 cells in Dulbecco's Modified Eagle Medium (DMEM). In contrast, an equivalent amount of spores was not observed when bacteria were co-cultured in Roswell Park Memorial Institute-1640 medium (RPMI). In the present study it was found that, when these two media are mixed, RPMI inhibits sporulation and CPE production induced in DMEM. When a component of RPMI was added to DMEM, it was found that calcium nitrate (Ca[NO 3 ] 2 ) significantly inhibits sporulation and CPE production. The number of spores increased when Ca(NO 3 ) 2 -deficient RPMI was used. The other nitrate salts significantly suppressed sporulation, whereas the calcium salts used did not. qPCR revealed that nitrate salts increased expression of bacterial nitrate/nitrite reductase. Furthermore, it was found that nitrite and nitric oxide suppress sporulation. In the sporulation stages, Ca(NO 3 ) 2 down-regulated the genes controlled by Spo0A, a master regulator of sporulation, but not spo0A itself. Collectively, these results indicate that nitrate salts suppress sporulation and CPE production by down-regulating Spo0A-regulated genes in C. perfringens strain NCTC8239. Nitrate reduction may be associated with inhibition of sporulation. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

  3. Mechanisms Mediating Enhanced Neutralization Efficacy of Staphylococcal Enterotoxin B by Combinations of Monoclonal Antibodies*

    PubMed Central

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

    2015-01-01

    Staphylococcal enterotoxin B (SEB) is a superantigen that cross-links the major histocompatibility complex class II and specific V-β chains of the T-cell receptor, thus forming a ternary complex. Developing neutralizing mAb to disrupt the ternary complex and abrogate the resulting toxicity is a major therapeutic challenge because SEB is effective at very low concentrations. We show that combining two SEB-specific mAbs enhances their efficacy, even though one of the two mAbs by itself has no effect on neutralization. Crystallography was employed for fine-mapping conformational epitopes in binary and ternary complexes between SEB and Fab fragments. NMR spectroscopy was used to validate and identify subtle allosteric changes induced by mAbs binding to SEB. The mapping of epitopes established that a combination of different mAbs can enhance efficacy of mAb-mediated protection from SEB induced lethal shock by two different mechanisms: one mAb mixture promoted clearance of the toxin both in vitro and in vivo by FcR-mediated cross-linking and clearance, whereas the other mAb mixture induced subtle allosteric conformational changes in SEB that perturbed formation of the SEB·T-cell receptor·major histocompatibility complex class II trimer. Finally structural information accurately predicted mAb binding to other superantigens that share conformational epitopes with SEB. Fine mapping of conformational epitopes is a powerful tool to establish the mechanism and optimize the action of synergistic mAb combinations. PMID:25572397

  4. Anoctamin 6 Contributes to Cl− Secretion in Accessory Cholera Enterotoxin (Ace)-stimulated Diarrhea

    PubMed Central

    Aoun, Joydeep; Hayashi, Mikio; Sheikh, Irshad Ali; Sarkar, Paramita; Saha, Tultul; Ghosh, Priyanka; Bhowmick, Rajsekhar; Ghosh, Dipanjan; Chatterjee, Tanaya; Chakrabarti, Pinak; Chakrabarti, Manoj K.; Hoque, Kazi Mirajul

    2016-01-01

    Accessory cholera enterotoxin (Ace) of Vibrio cholerae has been shown to contribute to diarrhea. However, the signaling mechanism and specific type of Cl− channel activated by Ace are still unknown. We have shown here that the recombinant Ace protein induced ICl of apical plasma membrane, which was inhibited by classical CaCC blockers. Surprisingly, an Ace-elicited rise of current was neither affected by ANO1 (TMEM16A)-specific inhibitor T16A(inh)-AO1(TAO1) nor by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker, CFTR inh-172. Ace stimulated whole-cell current in Caco-2 cells. However, the apical ICl was attenuated by knockdown of ANO6 (TMEM16F). This impaired phenotype was restored by re-expression of ANO6 in Caco-2 cells. Whole-cell patch clamp recordings of ANO currents in HEK293 cells transiently expressing mouse ANO1-mCherry or ANO6-GFP confirmed that Ace induced Cl− secretion. Application of Ace produced ANO6 but not the ANO1 currents. Ace was not able to induce a [Ca2+]i rise in Caco-2 cells, but cellular abundance of phosphatidylinositol 4,5-bisphosphate (PIP2) increased. Identification of the PIP2-binding motif at the N-terminal sequence among human and mouse ANO6 variants along with binding of PIP2 directly to ANO6 in HEK293 cells indicate likely PIP2 regulation of ANO6. The biophysical and pharmacological properties of Ace stimulated Cl− current along with intestinal fluid accumulation, and binding of PIP2 to the proximal KR motif of channel proteins, whose mutagenesis correlates with altered binding of PIP2, is comparable with ANO6 stimulation. We conclude that ANO6 is predominantly expressed in intestinal epithelia, where it contributes secretory diarrhea by Ace stimulation in a calcium-independent mechanism of RhoA-ROCK-PIP2 signaling. PMID:27799301

  5. New insights into the structure-function relationships and therapeutic applications of cholera-like enterotoxins.

    PubMed

    Hirst, Timothy R; Fraser, Sylvia; Soriani, Marco; Aman, A Tholib; de, Haan Lolke; Hearn, Arron; Merritt, Ethan

    2002-02-01

    Cholera toxin and E. coli heat-labile enterotoxin are structurally homologous proteins comprised of an enzymatically active A-subunit and five B-subunits that bind with high affinity to GM1-ganglioside receptors found on the surface of mammalian cells. The B-subunits have long been thought of simply as trafficking vehicles that trigger entry and subsequent delivery of the 'toxic' A-subunit into cells. Indeed, such is the capacity of the B-subunits to enter cells, that they have been developed as generic carriers for attachment and delivery of a variety of peptides into mammalian cells. However, the B-subunits also appear to possess discrete 'signalling functions', that induce both transcription factor and cell activation. These are thought to be directly responsible for the potent immunomodulatory properties of the B-subunits, and have resulted in their use as adjuvants and as agents to suppress inflammatory immune disorders. The relationship between the signalling properties of the B-subunits and their capacity to act as trafficking vehicles has remained unclear. In an effort to understand the structural requirements for these two functions, a set of mutant B-subunits, with amino acid substitutions at position His-57, have been generated and studied. Importantly, such mutant B-subunits retain an ability to bind with high affinity to GM1 and to traffic into cells, but have entirely lost their capacity to activate immune cell populations. Thus, while binding via GM1 appears to be sufficient to trigger cellular uptake it is not sufficient to activate signal transduction. The His-57 region is therefore speculated to be actively engaged in triggering signalling events, possibly via cognate interaction with other cell surface molecules.

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

    PubMed

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

    2014-12-05

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

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

    DOE PAGES

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

    2015-01-08

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

  8. [Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolates with toxic shock syndrome toxin and staphylococcal enterotoxin C genes].

    PubMed

    Kim, Jae Seok; Kim, Han Sung; Song, Wonkeun; Cho, Hyoun Chan; Lee, Kyu Man; Kim, Eui Chong

    2007-04-01

    Many methicillin-resistant Staphylococcus aureus (MRSA) isolates in Korea possess a specific profile of staphylococcal enterotoxins in that the toxic shock syndrome toxin gene (tst) coexists with the staphylococcal enterotoxin C gene (sec). Because the analysis of staphylococcal cassette chromosome mec (SCCmec), a mobile genetic element mecA gene encoding methicillin resistance, showed that majority of these are SCCmec type II, these MRSA isolates with tst and sec may be genetically related with each other. This study was performed to investigate the genetic relatedness of tstand sec-harboring MRSA strains isolated in Korea by using pulsed-field gel electrophoresis (PFGE). A total of 59 strains of MRSA isolates of SCCmec type II possessing tst and sec were selected for PFGE and phylogenetic analyses. These isolates were collected from 13 health care facilities during nationwide surveillance of antimicrobial resistance in 2002. The 59 MRSA isolates were clustered into 11 PFGE types, including one major group of 26 strains (44.1%) isolated from 7 healthcare facilities. Seven PFGE types contained 2 or more isolates each, comprising 55 isolates in total. Most of SCCmec type II MRSA isolates containing tst and sec showed closely related PFGE patterns. Moreover, MRSA isolates collected from different healthcare facilities showed identical PFGE patterns. These findings suggested a clonal spread of MRSA strains possessing tst and sec in Korean hospitals.

  9. Detection with synthetic oligonucleotide probes of nucleotide sequence variations in the genes encoding enterotoxins of Escherichia coli.

    PubMed Central

    Nishibuchi, M; Murakami, A; Arita, M; Jikuya, H; Takano, J; Honda, T; Miwatani, T

    1989-01-01

    We examined variations in the genes encoding heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT) in 88 strains of Escherichia coli isolated from individuals with traveler's diarrhea to find suitable sequences for use as oligonucleotide probes. Four oligonucleotide probes of the gene encoding ST of human origin (STIb or STh), one oligonucleotide probe of the gene encoding ST of porcine origin (STIa or STp), and three oligonucleotide probes of the gene encoding LT of human origin (LTIh) were used in DNA colony hybridization tests. In 15 of 22 strains possessing the STh gene and 28 of 42 strains producing LT, the sequences of all regions tested were identical to the published sequences. One region in the STh gene examined with a 18-mer probe was relatively well conserved and was shown to be closely associated with the enterotoxicity of the E. coli strains in suckling mice. This oligonucleotide, however, hybridized with strains of Vibrio cholerae O1, V. parahaemolyticus, and Yersinia enterocolitica that gave negative results in the suckling mouse assay. PMID:2685027

  10. Characterization of an outbreak of Clostridium perfringens food poisoning by quantitative fecal culture and fecal enterotoxin measurement.

    PubMed Central

    Birkhead, G; Vogt, R L; Heun, E M; Snyder, J T; McClane, B A

    1988-01-01

    Published criteria for implicating Clostridium perfringens as the cause of food-poisoning outbreaks include finding a median fecal C. perfringens spore count of greater than 10(6)/g among specimens from ill persons. We investigated a food-poisoning outbreak with the epidemiologic characteristics of C. perfringens-related disease in a nursing home in which the median fecal spore count for ill patients (2.5 X 10(7)/g) was similar to that for well patients (4.0 X 10(6)/g), making the etiology of the outbreak uncertain. All ill and well patients tested had eaten turkey, the implicated food item. C. perfringens enterotoxin was detected by reverse passive latex agglutination in fecal specimens from six of six ill and none of four well patients who had eaten turkey (P = 0.005), suggesting that this organism had caused the outbreak. This investigation suggests that detection of fecal C. perfringens enterotoxin is a specific way to identify this organism as the causative agent in food-poisoning outbreaks. PMID:2895776

  11. Comparison of Genotypes and Enterotoxin Genes Between Staphylococcus aureus Isolates from Blood and Nasal Colonizers in a Korean Hospital

    PubMed Central

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

    2009-01-01

    In this study, we investigated the genetic background of 70 Staphylococcus aureus isolates (36 methicillin-resistant S. aureus [MRSA] and 34 methicillin-susceptible S. aureus [MSSA]) obtained from blood at a Korean tertiary-care hospital, using spa typing, multilocus sequence typing, and SCCmec typing. In addition, the prevalence of enterotoxin (sea, seb, sec, sed, see, seg, seh, sei, and sek), tst, and pvl genes among the samples was assessed via polymerase chain reaction, and the results were compared with those of 95 isolates of S. aureus obtained from nasal swabs. All MRSA isolates from blood, except one, belonged to three major clones: sequence type (ST)5-MRSA-II, ST72-MRSA-II (or IVA), and ST239-MRSA-III, among which ST5-MRSA-II was the predominant clone. The prevalence of enterotoxin genes in the S. aureus isolates obtained from blood differed significantly from those from the nasal swabs for the sea, seb, sec, and seh gene. In particular, the seb and sec genes were detected exclusively in the MRSA isolates of ST5 or spa-CC002, thereby suggesting the co-adaptation of virulence genes with the genetic background and their contribution to biological fitness. PMID:19654937

  12. Inhibitory effects of food additives derived from polyphenols on staphylococcal enterotoxin A production and biofilm formation by Staphylococcus aureus.

    PubMed

    Shimamura, Yuko; Hirai, Chikako; Sugiyama, Yuka; Shibata, Masaharu; Ozaki, Junya; Murata, Masatsune; Ohashi, Norio; Masuda, Shuichi

    2017-12-01

    In this study, we examined the inhibitory effects of 14 food additives derived from polyphenol samples on staphylococcal enterotoxin A (SEA) production and biofilm formation by Staphylococcus aureus. Tannic acid AL (TA), Purephenon 50 W (PP) and Polyphenon 70A (POP) at 0.25 mg/mL and Gravinol®-N (GN), Blackcurrant polyphenol AC10 (BP), and Resveratrol-P5 (RT) at 1.0 mg/mL significantly decreased SEA production by S. aureus C-29 (p < 0.05). TA, GN, BP, and RT significantly inhibited the expression of the sea gene in S. aureus C-29 (p < 0.05), while suppression attempts by PP and POP proved unsuccessful. After result analysis, it can be derived that TA, GN, BP, and RT inhibit the production of SEA. Of the six samples, each one significantly inhibited biofilm formation (p < 0.05). Food additives derived from polyphenols have viability to be used as a means to inhibit the enterotoxin production and control the biofilm formation of foodborne pathogens.

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

    PubMed

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

    2013-01-01

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

  14. Presence of Classical Enterotoxin Genes, agr Typing, Antimicrobial Resistance, and Genetic Diversity of Staphylococcus aureus from Milk of Cows with Mastitis in Southern Brazil.

    PubMed

    Kroning, Isabela S; Iglesias, Mariana A; Mendonça, Karla S; Lopes, Graciela V; Silva, Wladimir P

    2018-05-01

    Staphylococcus aureus is a common causative agent of bovine mastitis in dairy cows and commonly associated with foodborne disease outbreaks. The aim of this study was to evaluate the presence of enterotoxin genes, agr typing, antimicrobial resistance, and genetic diversity of S. aureus isolated from milk of cows with mastitis in dairy farms from southern Brazil. Results showed that 7 (22.6%) of 31 S. aureus isolates were positive for enterotoxin genes. Specifically, the genes encoding for enterotoxins A ( n = 4), C ( n = 2), and B ( n = 1) were detected. Isolates belonging to the agr group III (10 of 31, 32.2%) and agr group I (7 of 31, 22.5%) were the most common. To our knowledge, this is the first report of both agr I and III in the same S. aureus isolate from milk of cows with bovine mastitis. The antimicrobial resistance test showed that 54% of the isolates were multiresistant to antimicrobial agents. The macrorestriction analysis produced 16 different major SmaI pulsed-field gel electrophoresis patterns, with up to two subpatterns. Moreover, the presence of some S. aureus clones in a distinct area was observed. Although this study characterized a limited number of S. aureus isolates, the presence of classical enterotoxin genes and resistance to multiple antimicrobial agents reinforces the importance of this microorganism to animal and human health. In addition, similar genetic profiles have been identified in distinct geographic areas, suggesting clonal dissemination of S. aureus in dairy herds from southern Brazil.

  15. Growth and enterotoxin production of Staphylococcus aureus during the manufacture and ripening of Camembert-type cheeses from raw goats' milk.

    PubMed

    Meyrand, A; Boutrand-Loei, S; Ray-Gueniot, S; Mazuy, C; Gaspard, C E; Jaubert, G; Perrin, G; Lapeyre, C; Vernozy-Rozand, C

    1998-09-01

    Tests were carried out to determine the effect of manufacturing procedures for a Camembert-type cheese from raw goats' milk on the growth and survival of Staphylococcus aureus organisms added to milk at the start of the process, and to study the possible presence of staphylococcal enterotoxin A in these cheeses. The initial staphylococcal counts were, respectively, 2, 3, 4, 5 and 6 log cfu ml-1. Cheese was prepared following the industrial specifications and ripened for 41 d. Detection of enterotoxins was done by the Vidas SET test and by an indirect double-sandwich ELISA technique using antienterotoxin monoclonal antibodies. Generally, numbers of microbes increased at a similar rate during manufacture in all cheeses until salting. During the ripening period, the aerobic plate count population and Staph. aureus levels remained stable and high. There was an approximately 1 log reduction of Staph. aureus in cheeses made with an initial inoculum of Staph. aureus greater than 10(3) cfu ml-1 at the end of the ripening period (41 d) compared with the count at 22 h. The level of staphylococcal enterotoxin A recovered varied from 1 to 3.2 ng g-1 of cheese made with an initial population of 10(3)-10(6) cfu ml-1. No trace of enterotoxin A was detected in cheeses made with the lowest Staph. aureus inoculum used in this study.

  16. Survey of Genes Encoding Staphylococcal Enterotoxins, Toxic Shock Syndrome Toxin 1, and Exfoliative Toxins in Members of the Staphylococcus sciuri Group

    PubMed Central

    Dakić, Ivana; Vuković, Dragana; Stepanović, Srdjan; Hauschild, Tomasz; Ježek, Petr; Petráš, Petr; Morrison, Donald

    2005-01-01

    Genes encoding staphylococcal enterotoxins (sea to see, seg, and seh), toxic shock syndrome toxin 1 (tst), and exfoliative toxins (eta and etb) were not detected in a large panel of 48 Staphylococcus sciuri group isolates tested. This strongly suggests that production of the staphylococcal exotoxins by these bacteria is highly unlikely. PMID:16145164

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

    PubMed Central

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

    2013-01-01

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

  18. Clostridium perfringens enterotoxin as a potential drug for intravesical treatment of bladder cancer.

    PubMed

    Gabig, Theodore G; Waltzer, Wayne C; Whyard, Terry; Romanov, Victor

    2016-09-16

    The current intravesical treatment of bladder cancer (BC) is limited to a few chemotherapeutics that show imperfect effectiveness and are associated with some serious complications. Thus, there is an urgent need for alternative therapies, especially for patients with high-risk non-muscle invasive (NMIBC). Clostridium perfringens enterotoxin (CPE), cytolytic protein binds to its receptors: claudin 3 and 4 that are expressed in epithelial cells. This binding is followed by rapid cell death. Claudin 4 is present in several epithelial tissue including bladder urothelium and its expression is elevated in some forms of BC. In addition to directly targeting BC cells, binding of CPE to claudins increases urothelium permeability that creates conditions for better accession of the tumor. Therefore, we evaluated CPE as a candidate for intravesical treatment of BC using a cellular model. We examined cytotoxicity of CPE against BC cells lines and 3D cultures of cells derived from surgical samples. To better elucidate cellular mechanisms, activated by CPE and to consider the use of CPE non-toxic fragment (C-CPE) for combination treatment with other drugs we synthesized C-CPE, compared its cytotoxic activity with CPE and examined claudin 4 expression and intracellular localization after C-CPE treatment. CPE induced cell death after 1 h in low aggressive RT4 cells, in moderately aggressive 5637 cells and in the primary 3D cultures of BC cells derived from NMIBC. Conversely, non-transformed urothelial cells and cells derived from highly aggressive tumor (T24) survived this treatment. The reason for this resistance to CPE might be the lower expression of CLDNs or their inaccessibility for CPE in these cells. C-CPE treatment for 48 h did not affect cell viability in tested cells, but declined expression of CLDN4 in RT4 cells. C-CPE increased sensitivity of RT4 cells to Mitommycin C and Dasatinib. To better understand mechanisms of this effect we examined expression and

  19. Suppression of colorectal tumorigenesis by recombinant Bacteroides fragilis enterotoxin-2 in vivo.

    PubMed

    Lv, You; Ye, Tao; Wang, Hui-Peng; Zhao, Jia-Ying; Chen, Wen-Jie; Wang, Xin; Shen, Chen-Xia; Wu, Yi-Bin; Cai, Yuan-Kun

    2017-01-28

    To evaluate the impact of recombinant Bacteroides fragilis enterotoxin-2 (BFT-2, or Fragilysin) on colorectal tumorigenesis in mice induced by azoxymethane/dextran sulfate sodium (AOM/DSS). Recombinant proBFT-2 was expressed in Escherichia coli strain Rosetta (DE3) and BFT-2 was obtained and tested for its biological activity via colorectal adenocarcinoma cell strains SW-480. Seventy C57BL/6J mice were randomly divided into a blank (BC; n = 10), model (AD; n = 20), model + low-dose toxin (ADLT; n = 20, 10 μg), and a model + high-dose toxin (ADHT; n = 20, 20 μg) group. Mice weight, tumor formation and pathology were analyzed. Immunohistochemistry determined Ki-67 and Caspase-3 expression in normal and tumor tissues of colorectal mucosa. Recombinant BFT-2 was successfully obtained, along with its biological activity. The most obvious weight loss occurred in the AD group compared with the ADLT group (21.82 ± 0.68 vs 23.23 ± 0.91, P < 0.05) and the ADHT group (21.82 ± 0.68 vs 23.57 ± 1.06, P < 0.05). More tumors were found in the AD group than in the ADLT and ADHT groups (19.75 ± 3.30 vs 6.50 ± 1.73, P < 0.05; 19.75 ± 3.30 vs 6.00 ± 2.16, P < 0.05). Pathology showed that 12 mice had adenocarcinoma and 6 cases had adenoma in the AD group. Five mice had adenocarcinoma and 15 had adenoma in the ADLT group. Four mice had adenocarcinoma and 16 had adenoma in the ADHT group. The incidence of colorectal adenocarcinoma in both the ADHT group and the ADHT group was reduced compared to that in the AD group ( P < 0.05, P < 0.05). The positive rate of Ki-67 in the ADLT group and the ADHT group was 50% and 40%, respectively, both of which were lower than that found in the AD group (94.44%, P < 0.05, P < 0.05). Caspase-3 expression in the ADLT group and the ADHT group was 45% and 55%, both of which were higher than that found in the BC group (16.67%, P < 0.05, P < 0.05). Oral administration with lower-dose biologically active recombinant BFT-2 inhibited colorectal

  20. Suppression of colorectal tumorigenesis by recombinant Bacteroides fragilis enterotoxin-2 in vivo

    PubMed Central

    Lv, You; Ye, Tao; Wang, Hui-Peng; Zhao, Jia-Ying; Chen, Wen-Jie; Wang, Xin; Shen, Chen-Xia; Wu, Yi-Bin; Cai, Yuan-Kun

    2017-01-01

    AIM To evaluate the impact of recombinant Bacteroides fragilis enterotoxin-2 (BFT-2, or Fragilysin) on colorectal tumorigenesis in mice induced by azoxymethane/dextran sulfate sodium (AOM/DSS). METHODS Recombinant proBFT-2 was expressed in Escherichia coli strain Rosetta (DE3) and BFT-2 was obtained and tested for its biological activity via colorectal adenocarcinoma cell strains SW-480. Seventy C57BL/6J mice were randomly divided into a blank (BC; n = 10), model (AD; n = 20), model + low-dose toxin (ADLT; n = 20, 10 μg), and a model + high-dose toxin (ADHT; n = 20, 20 μg) group. Mice weight, tumor formation and pathology were analyzed. Immunohistochemistry determined Ki-67 and Caspase-3 expression in normal and tumor tissues of colorectal mucosa. RESULTS Recombinant BFT-2 was successfully obtained, along with its biological activity. The most obvious weight loss occurred in the AD group compared with the ADLT group (21.82 ± 0.68 vs 23.23 ± 0.91, P < 0.05) and the ADHT group (21.82 ± 0.68 vs 23.57 ± 1.06, P < 0.05). More tumors were found in the AD group than in the ADLT and ADHT groups (19.75 ± 3.30 vs 6.50 ± 1.73, P < 0.05; 19.75 ± 3.30 vs 6.00 ± 2.16, P < 0.05). Pathology showed that 12 mice had adenocarcinoma and 6 cases had adenoma in the AD group. Five mice had adenocarcinoma and 15 had adenoma in the ADLT group. Four mice had adenocarcinoma and 16 had adenoma in the ADHT group. The incidence of colorectal adenocarcinoma in both the ADHT group and the ADHT group was reduced compared to that in the AD group (P < 0.05, P < 0.05). The positive rate of Ki-67 in the ADLT group and the ADHT group was 50% and 40%, respectively, both of which were lower than that found in the AD group (94.44%, P < 0.05, P < 0.05). Caspase-3 expression in the ADLT group and the ADHT group was 45% and 55%, both of which were higher than that found in the BC group (16.67%, P < 0.05, P < 0.05). CONCLUSION Oral administration with lower-dose biologically active recombinant BFT-2

  1. Staphylococcal enterotoxin-specific IgE antibodies in atopic dermatitis.

    PubMed

    Ide, Fumihito; Matsubara, Tomoyo; Kaneko, Miho; Ichiyama, Takashi; Mukouyama, Tokuko; Furukawa, Susumu

    2004-06-01

    The authors clarified the clinical significance of the measurement of serum concentrations of specific IgE antibodies to staphylococcal enterotoxin (SE) A- and SEB in atopic dermatitis (AD). The serum concentrations of SEA- and SEB-specific IgE antibodies in 140 pediatric patients with AD were measured with an immuno CAP -radioallergosorbent test system (RAST). To check the cross-reaction of specific IgE antibodies to SEA/SEB and other allergens, the CAP RAST fluorescent enzyme immunoassay inhibition test was performed. Forty-seven patients (33.6%) tested positive for either SEA- or SEB-specific IgE antibodies. School children showed higher positive rates of SEA/SEB-specific IgE antibodies than infants or young children. The patients with severe AD and those with exacerbation of symptoms in summer, had higher positive rates of SEA/SEB-specific IgE antibodies than patients with mild AD or those with exacerbation in winter. In addition, the positive rates of specific IgE antibodies to both dog-dander and cat-dander were higher in patients with positive SEA/SEB-specific IgE antibodies than in patients with negative ones. No cross-reactions occurred among specific IgE antibodies to SEA/SEB and dog/cat dander with one patient's serum, which had positive IgE-specific antibodies against cat/dog dander and SEA/SEB. The positive rate of SEA/SEB-specific IgE antibodies in the patients with dogs and/or cats as pets was 48.4%, which was higher than in those with no pets. Atopic dermatitis patients who exhibit high positive rates of SEA/SEB-specific IgE antibodies were found to be school children, severe cases, cases with high serum concentrations of total IgE, cases with exacerbation in summer, and cases with dogs and/or cats as pets. The measurement of serum concentrations of specific IgE antibodies to SEA and SEB, thus has some value for evaluating AD patients.

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

    SciTech Connect

    Takeshita, W.M.; Gushiken, V.O.; Ferreira-Duarte, A.P.

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

  3. Lab-On-a-Chip for carbon nanotubes based immunoassay detection of Staphylococcal Enterotoxin B (SEB).

    PubMed

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

    2010-04-21

    We describe a new eight channel Lab-On-a-Chip (LOC) for a Carbon Nanotube (CNT) based immunoassay with optical detection of Staphylococcal Enterotoxin B (SEB) for food safety applications. In this work, we combined four biosensing elements: (1) CNT technology for primary antibody immobilization, (2) Enhanced Chemiluminescence (ECL) for light signal generation, (3) a cooled charge-coupled device (CCD) for detection and (4) polymer lamination technology for developing a point of care immunological assay for SEB detection. Our concept for developing versatile LOCs, which can be used for many different applications, is to use a modular design with interchangeable recognition elements (e.g. various antibodies) to determine the specificity. Polymer lamination technology was used for the fabrication of a six layer, syringe operated LOC capable of analyzing eight samples simultaneously. An anti-SEB antibody-nanotube mixture was immobilized onto a polycarbonate strip, to serve as an interchangeable ligand surface that was then bonded onto the LOC. SEB samples are loaded into the device and detected by an ELISA assay using Horse Radish Peroxidase (HRP) conjugated anti-SEB IgG as a secondary antibody and ECL, with detection by a previously described portable cooled CCD detector. Eight samples of SEB in buffer or soy milk were assayed simultaneously with a limit of detection of 0.1 ng mL(-1). CNT immobilization of the antibody increased the sensitivity of detection six fold. Use of a simple interchangeable immunological surface allows this LOC to be adapted to any immunoassay by simply replacing the ligand surface. A syringe was used to move fluids for this assay so no power is needed to operate the device. Our versatile portable point-of-care CCD detector combined with the LOC immunoassay method described here can be used to reduce the exposure of users to toxins and other biohazards when working outside the lab, as well as to simplify and increase sensitivity for many other

  4. Metaphase yields from staphylococcal enterotoxin A stimulated peripheral blood lymphocytes of unirradiated and irradiated aged rhesus monkeys

    NASA Technical Reports Server (NTRS)

    Hill, F. S.; Cox, A. B.; Salmon, Y. L.; Cantu, A. O.; Lucas, J. N.

    1994-01-01

    The mitogen phytohemagglutinin (PHA) works well in both human and cynomolgus monkey (Macaca fascicularis) lymphocyte cultures to stimulate T cell proliferation. T cells from rhesus monkeys (Macaca mulatta) are less responsive than human cells, producing few metaphases when thousands are required, e.g. in biological dosimetry studies. We show that staphylococcal enterotoxin A (SEA), one of the most potent mitogens known, at a concentration of 0.5 microgram/ml stimulated peripheral lymphocytes to grow with a mitotic index (MI) averaging 0.13 metaphases/cell in old, irradiated rhesus macaques. This was significantly greater (p < 0.001) than that produced by PHA (MI < 0.01) in lymphocytes from the same animals. Whole blood was cultured for 96, 120 and 144 h for five irradiated individuals and for two controls. All cells cultured with SEA produced a high MI with a peak response at 120 h whereas the same cultures showed low MI for each PHA stimulated culture.

  5. Expression of the B subunit of Escherichia coli heat-labile enterotoxin as a fusion protein in transgenic tomato.

    PubMed

    Walmsley, A M; Alvarez, M L; Jin, Y; Kirk, D D; Lee, S M; Pinkhasov, J; Rigano, M M; Arntzen, C J; Mason, H S

    2003-06-01

    Epitopes often require co-delivery with an adjuvant or targeting protein to enable recognition by the immune system. This paper reports the ability of transgenic tomato plants to express a fusion protein consisting of the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) and an immunocontraceptive epitope. The fusion protein was found to assemble into pentamers, as evidenced by its ability to bind to gangliosides, and had an average expression level of 37.8 microg g(-1) in freeze-dried transgenic tissues. Processing of selected transgenic fruit resulted in a 16-fold increase in concentration of the antigen with minimal loss in detectable antigen. The species-specific nature of this epitope was shown by the inability of antibodies raised against non-target species to detect the LTB fusion protein. The immunocontraceptive ability of this vaccine will be tested in future pilot mice studies.

  6. Clostridium Perfringens Enterotoxin (CPE) and CPE-Binding Domain (c-CPE) for the Detection and Treatment of Gynecologic Cancers

    PubMed Central

    Black, Jonathan D.; Lopez, Salvatore; Cocco, Emiliano; Schwab, Carlton L.; English, Diana P.; Santin, Alessandro D.

    2015-01-01

    Clostridium perfringens enterotoxin (CPE) is a three-domain polypeptide, which binds to Claudin-3 and Claudin-4 with high affinity. Because these receptors are highly differentially expressed in many human tumors, claudin-3 and claudin-4 may provide an efficient molecular tool to specifically identify and target biologically aggressive human cancer cells for CPE-specific binding and cytolysis. In this review we will discuss these surface proteins as targets for the detection and treatment of chemotherapy-resistant gynecologic malignancies overexpressing claudin-3 and -4 using CPE-based theranostic agents. We will also discuss the use of fluorescent c-CPE peptide in the operative setting for real time detection of micro-metastatic tumors during surgery and review the potential role of CPE in other medical applications. PMID:25835384

  7. Lack of evidence in vivo for a remote effect of Escherichia coli heat stable enterotoxin on jejunal fluid absorption.

    PubMed

    Lucas, M L; Duncan, N W; o'reilly, N F; McIlvenny, T J; Nelson, Y B

    2008-05-01

    On contact with the mucosa, heat stable (STa) enterotoxin from Escherichia coli reduces fluid absorption in vivo in the perfused jejunum of the anaesthetized rat. The question of whether it also has a vagally mediated remote action on jejunal absorption, when instilled into the ileum, was re-examined, given contradictory findings in the literature. A standard perfused loop preparation was used to measure luminal uptake of fluid in vivo by means of volume recovery. STa in the ileum was found to have no effect on jejunal absorption, regardless of cervical or sub-diaphragmatic vagotomy and also regardless of the nature of the perfusate anion. The batches of toxin were shown in parallel experiments to reduce fluid absorption directly in the jejunum and also in the ileum. Similarly, vagal nerves prior to section had demonstrable in vivo physiological function. There was therefore no evidence for an indirect, vagally mediated ileal effect of STa on proximal fluid absorption.

  8. Comparative Genomics and Identification of an Enterotoxin-Bearing Pathogenicity Island, SEPI-1/SECI-1, in Staphylococcus epidermidis Pathogenic Strains.

    PubMed

    Argemi, Xavier; Nanoukon, Chimène; Affolabi, Dissou; Keller, Daniel; Hansmann, Yves; Riegel, Philippe; Baba-Moussa, Lamine; Prévost, Gilles

    2018-02-25

    Staphylococcus epidermidis is a leading cause of nosocomial infections, majorly resistant to beta-lactam antibiotics, and may transfer several mobile genetic elements among the members of its own species, as well as to Staphylococcus aureus ; however, a genetic exchange from S. aureus to S. epidermidis remains controversial. We recently identified two pathogenic clinical strains of S. epidermidis that produce a staphylococcal enterotoxin C3-like (SEC) similar to that by S. aureus pathogenicity islands. This study aimed to determine the genetic environment of the SEC-coding sequence and to identify the mobile genetic elements. Whole-genome sequencing and annotation of the S. epidermidis strains were performed using Illumina technology and a bioinformatics pipeline for assembly, which provided evidence that the SEC-coding sequences were located in a composite pathogenicity island that was previously described in the S. epidermidis strain FRI909, called SePI-1/SeCI-1, with 83.8-89.7% nucleotide similarity. Various other plasmids were identified, particularly p_3_95 and p_4_95, which carry antibiotic resistance genes ( hsrA and dfrG , respectively), and share homologies with SAP085A and pUSA04-2-SUR11, two plasmids described in S. aureus . Eventually, one complete prophage was identified, ΦSE90, sharing 30 out of 52 coding sequences with the Acinetobacter phage vB_AbaM_IME200. Thus, the SePI-1/SeCI-1 pathogenicity island was identified in two pathogenic strains of S. epidermidis that produced a SEC enterotoxin causing septic shock. These findings suggest the existence of in vivo genetic exchange from S. aureus to S. epidermidis .

  9. Comparative Genomics and Identification of an Enterotoxin-Bearing Pathogenicity Island, SEPI-1/SECI-1, in Staphylococcus epidermidis Pathogenic Strains

    PubMed Central

    Nanoukon, Chimène; Affolabi, Dissou; Keller, Daniel; Hansmann, Yves; Riegel, Philippe; Baba-Moussa, Lamine; Prévost, Gilles

    2018-01-01

    Staphylococcus epidermidis is a leading cause of nosocomial infections, majorly resistant to beta-lactam antibiotics, and may transfer several mobile genetic elements among the members of its own species, as well as to Staphylococcus aureus; however, a genetic exchange from S. aureus to S. epidermidis remains controversial. We recently identified two pathogenic clinical strains of S. epidermidis that produce a staphylococcal enterotoxin C3-like (SEC) similar to that by S. aureus pathogenicity islands. This study aimed to determine the genetic environment of the SEC-coding sequence and to identify the mobile genetic elements. Whole-genome sequencing and annotation of the S. epidermidis strains were performed using Illumina technology and a bioinformatics pipeline for assembly, which provided evidence that the SEC-coding sequences were located in a composite pathogenicity island that was previously described in the S. epidermidis strain FRI909, called SePI-1/SeCI-1, with 83.8–89.7% nucleotide similarity. Various other plasmids were identified, particularly p_3_95 and p_4_95, which carry antibiotic resistance genes (hsrA and dfrG, respectively), and share homologies with SAP085A and pUSA04-2-SUR11, two plasmids described in S. aureus. Eventually, one complete prophage was identified, ΦSE90, sharing 30 out of 52 coding sequences with the Acinetobacter phage vB_AbaM_IME200. Thus, the SePI-1/SeCI-1 pathogenicity island was identified in two pathogenic strains of S. epidermidis that produced a SEC enterotoxin causing septic shock. These findings suggest the existence of in vivo genetic exchange from S. aureus to S. epidermidis. PMID:29495323

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

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

    1991-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2010-09-01

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

  14. Evaluation of BW942C, a novel antidiarrheal agent, against enterotoxins of Escherichia coli and Vibrio cholerae.

    PubMed Central

    Morgan, D R; Sellin, J; Gutierrez, L; DuPont, H L; Wood, L V

    1985-01-01

    BW942C, an enkephalin-like pentapeptide with anti-diarrheal activity, was tested against crude toxins of Escherichia coli and Vibrio cholerae in the Y-1 adrenal cell assay, rabbit ileal loop assay, and suckling mouse assay. The effects of BW942C on in vitro ion transport were measured in rabbit ileum mounted in Ussing chambers. In vitro, BW942C decreased basal short-circuit current (2.26 and 3.15 mueq cm-2 h-1 in experimental samples and controls, respectively; n = 7, P less than 0.05) and increased basal net Cl absorption (1.59 and 0.50 mueq cm-2 h-1 in experimental samples and controls, respectively; P less than 0.025). Net Na absorption was also increased, but not significantly. BW942C did not block the secretory response to a maximal dose of purified heat-stable toxin. BW942C directly enhanced intestinal fluid absorption. In the Y-1 adrenal cell assay, 5 mg of BW942C per ml inhibited the cytopathic effect caused by cholera toxin or heat-labile enterotoxin of E. coli. In the rabbit ileal loop assay, E. coli heat-stable toxin, E. coli heat-labile enterotoxin, and cholera toxin were inhibited 35 to 70% by administration of BW942C. With the suckling mouse model, the fluid accumulation caused by E. coli heat-stable toxin was ablated by prior treatment with BW942C. The drug is currently being evaluated in patients with acute secretory diarrhea to determine its effect on clinical symptoms. PMID:3838969

  15. Staphylococcus aureus and Staphylococcal Enterotoxin A in Breaded Chicken Products: Detection and Behavior during the Cooking Process

    PubMed Central

    Pepe, Olimpia; Blaiotta, Giuseppe; Bucci, Francesca; Anastasio, Marilena; Aponte, Maria; Villani, Francesco

    2006-01-01

    In this study we examined the presence of Staphylococcus aureus and staphylococcal enterotoxin A (SEA) in 20 industrial breaded chicken products obtained from different retail butchers and supermarket stores in Italy. The levels of contamination in the products analyzed were quite low, although the pH values and water activities (aw) in the samples considered were in ranges favorable for S. aureus growth. As demonstrated by phenotypic and molecular characterization, in spite of the high percentage of coagulase-positive Staphylococcus strains, only three strains could be referred to the species S. aureus. Moreover, all the strains were negative in PCR assays targeting staphylococcal enterotoxin genes (seA to seE, seG to seJ, and seM to seO), as well as the toxic shock syndrome toxin 1 gene, and no SEA was detected in the retail breaded chicken samples analyzed by a reversed passive latex agglutination assay or by Western blotting. Hence, we evaluated the thermal resistance of two strains of SEA-producing S. aureus in a laboratory-scale preparation of precooked breaded chicken cutlets. The heat treatment employed in the manufacture determined the inactivation of S. aureus cells, but the preformed SEA remained active during product storage at 4°C. The presence of the staphylococci and, in particular, of S. aureus in the retail breaded chicken products analyzed is a potential health risk for consumers since the pH and aw values of these kinds of products are favorable for S. aureus growth. The thermal process used during their manufacture can limit staphylococcal contamination but cannot eliminate preformed toxins. PMID:17088378

  16. Staphylococcus aureus and staphylococcal enterotoxin A in breaded chicken products: detection and behavior during the cooking process.

    PubMed

    Pepe, Olimpia; Blaiotta, Giuseppe; Bucci, Francesca; Anastasio, Marilena; Aponte, Maria; Villani, Francesco

    2006-11-01

    In this study we examined the presence of Staphylococcus aureus and staphylococcal enterotoxin A (SEA) in 20 industrial breaded chicken products obtained from different retail butchers and supermarket stores in Italy. The levels of contamination in the products analyzed were quite low, although the pH values and water activities (a(w)) in the samples considered were in ranges favorable for S. aureus growth. As demonstrated by phenotypic and molecular characterization, in spite of the high percentage of coagulase-positive Staphylococcus strains, only three strains could be referred to the species S. aureus. Moreover, all the strains were negative in PCR assays targeting staphylococcal enterotoxin genes (seA to seE, seG to seJ, and seM to seO), as well as the toxic shock syndrome toxin 1 gene, and no SEA was detected in the retail breaded chicken samples analyzed by a reversed passive latex agglutination assay or by Western blotting. Hence, we evaluated the thermal resistance of two strains of SEA-producing S. aureus in a laboratory-scale preparation of precooked breaded chicken cutlets. The heat treatment employed in the manufacture determined the inactivation of S. aureus cells, but the preformed SEA remained active during product storage at 4 degrees C. The presence of the staphylococci and, in particular, of S. aureus in the retail breaded chicken products analyzed is a potential health risk for consumers since the pH and a(w) values of these kinds of products are favorable for S. aureus growth. The thermal process used during their manufacture can limit staphylococcal contamination but cannot eliminate preformed toxins.

  17. 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

  18. Protective Effect of Immunization with Heat-Labile Enterotoxin in Gnotobiotic Rats Monocontaminated with Enterotoxigenic Escherichia coli

    PubMed Central

    Klipstein, Frederick A.; Engert, Richard F.; Short, Helen B.

    1980-01-01

    The protective effect of active immunization with a purified preparation of the polymyxin-release form of Escherichia coli heat-labile enterotoxin (LT), administered using a parenteral prime and peroral boosts given after ablation of gastric secretion by means of cimetidine, was assessed in gnotobiotic rats which were challenged by monocontamination with enterotoxigenic strains of E. coli. Water transport was evaluated by the in vivo marker perfusion technique at weekly intervals over a 3-week period after contamination. Water transport in unimmunized control rats was consistently in absorption in those contaminated by a nontoxigenic strain, in secretion during only week 2 in those contaminated by an LT+/− strain, in secretion during weeks 2 and 3 in those contaminated by an LT+/ST+ (heat-stable enterotoxin) strain, and consistently in absorption in those contaminated by an −/ST+ strain. Rats immunized with a booster dosage of 250 μg had a significant increase (P < 0.001) in net water absorption as compared to unimmunized rats, with values in the borderline range of absorption, when challenged with either the LT+/− or LT+/ST+ strains. Rats immunized with a 10-fold-higher boosting dosage had a significant increase (P < 0.001) in net water absorption as compared to those boosted at the lower dosage; water absorption was within the normal range. There was no difference between the ileal bacterial counts of unimmunized and immunized rats challenged by the various strains. These observations indicate that this immunization program provides complete protection in an animal model against challenge by intestinal contamination with enterotoxigenic strains of E. coli which produce LT, either alone or in combination with ST. PMID:6991436

  19. Clostridium perfringens enterotoxin is a superantigen reactive with human T cell receptors V beta 6.9 and V beta 22

    PubMed Central

    1992-01-01

    Candidate superantigens were screened for their ability to induce lysis of human histocompatibility leukocyte antigen class II-positive targets by human CD8+ influenza-specific cytotoxic T cell (CTL) lines. Clostridium perfringens enterotoxin (CPET) induced major histocompatibility complex unrestricted killing by some but not all CTL lines. Using "anchored" polymerase chain reactions, CPET was shown to selectively stimulate peripheral blood lymphocytes bearing T cell receptor V beta 6.9 and V beta 22 in five healthy donors. V beta 24, V beta 21, V beta 18, V beta 5, and V beta 6.1-5 appeared to be weakly stimulated. Antigen processing was not required for CPET to induce proliferation. Like the staphylococcal enterotoxins, CPET is a major cause of food poisoning. These data suggest that superantigenic and enterotoxigenic properties may be closely linked. PMID:1512551

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

    PubMed

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

    2017-08-04

    Asymptomatic carriers of toxigenic Staphylococcus aureus are potential source of diseases, including food poisoning. Toxigenic potential and genetic traits of colonizing S. aureus were investigated for 563 healthy food handlers in Myanmar. Carriage of S. aureus was found in 110 individuals (19.5%), and a total of 144 S. aureus isolates were recovered from nasal cavities (110 isolates) and hands (34 isolates). Panton-Valentine leucocidin genes ( pvl ) were detected in 18 isolates (12.5%), among which 11 isolates were classified into coa -VIa, agr type III, and ST1930 (CC96) that had been also detected in pvl -positive clinical isolates in Myanmar. A pvl -positive, ST2250 nasal isolate was identified as S. argenteus , a novel coagulase-positive staphylococcus species. Toxic shock syndrome toxin-1 (TSST-1) gene was detected in five pvl -negative isolates. All of the 144 isolates harbored at least one of the 21 enterotoxin(-like) gene(s). The most prevalent enterotoxin(-like) gene was selw (98%), followed by selx (97%), sei (28%), sely (28%), sem (26%), sel (24%), and sea and sec (22% each). Considerable genetic diversity with five groups was detected for selw . The present study revealed the relatively high rate of pvl , as well as the wide distribution of enterotoxin(-like) genes among colonizing S. aureus in Myanmar.

  1. Influence of holding temperature on the growth and survival of Salmonella spp. and Staphylococcus aureus and the production of staphylococcal enterotoxin in egg products.

    PubMed

    Yang, S E; Yu, R C; Chou, C C

    2001-01-22

    In this study, growth and survival of Salmonella spp. and Staphylococcus aureus in steamed egg and scrambled egg held at 5, 18, 22, 37, 55 and 60 degrees C are investigated. The production of staphylococcal enterotoxin in steamed egg is also examined. Results reveal that Salmonella spp. and Staph. aureus in the egg products multiply best at 37 degrees C, followed closely by 22 and 18 degrees C. Neither pathogen showed growth in the egg products held at 5 degrees C. Initial inoculation dose, holding temperature and holding time affected the population of both organisms found in the egg products. Staphylococcal enterotoxin A (SEA) and B (SEB) are detected only in the egg products held at 37 or 22 degrees C. After holding at 37 degrees C for 36 h, scrambled egg inoculated with ca. 5.0 log cfu/g Staph. aureus contains the highest levels of SEA (> 64 ng/g) and SEB (> 64 ng/g). Although Salmonella spp. and Staph. aureus grow better in steamed eggs than in scrambled eggs, production of staphylococcal enterotoxin, in general, was higher in scrambled eggs than in steamed eggs. On the other hand, a repaid destruction of the test organisms in steamed eggs held at 60 degrees C was observed. Holding the steamed eggs at 60 degrees C, Salmonella spp. and Staph. aureus with an initial population of ca. 5.9 and 5.6 log cfu/g, respectively, reduced to a non-detectable level in 1 h.

  2. Development and application of a multiplex PCR assay for detection of the Clostridium perfringens enterotoxin-encoding genes cpe and becAB.

    PubMed

    Yonogi, Shinya; Kanki, Masashi; Ohnishi, Takahiro; Shiono, Masami; Iida, Tetsuya; Kumeda, Yuko

    2016-08-01

    Clostridium perfringens causes food-borne gastroenteritis following the consumption of contaminated food by producing C. perfringens enterotoxin (CPE) in the intestines. Recently, we reported a novel enterotoxin, binary enterotoxin of C. perfringens (BEC) in C. perfringens isolates, which caused two disease outbreaks in Japan. Consequently, in the event of food poisoning outbreaks caused by C. perfringens, it is now necessary to screen for both the cpe and becAB genes by diagnostic PCR. Here, we present a simple multiplex PCR method for simultaneous detection of cpe, becAB and a C. perfringens control locus, phospholipase C (plc). Applying this method, we investigated the prevalence of cpe- or becAB-carrying C. perfringens strains in human stool and bovine rectum swab samples. Using a total of 169 isolates, we found that the percentage of becAB-carrying strains was very small (0.59%), one-tenth that of cpe-carrying strains. The simple method presented in this study with high specificity and sensitivity to C. perfringens will be a useful tool to survey the global prevalence of becAB-carrying C. perfringens strains. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    PubMed

    Lucas, M L

    2013-10-01

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

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

    NASA Astrophysics Data System (ADS)

    Hwang, Joonki; Lee, Sangyeop; Choo, Jaebum

    2016-06-01

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

  5. Prevalence of serum IgE antibodies to the Staphylococcus aureus enterotoxins (SAE, SEB, SEC, SED, TSST-1) in patients with persistent allergic rhinitis.

    PubMed

    Rossi, Renato Enzo; Monasterolo, Giorgio

    2004-03-01

    Enterotoxins produced by Staphylococcus aureus and their specific IgE antibodies were thought to be important in worsening atopic dermatitis. However, few studies have documented an association between S. aureus or its exotoxins and exacerbations of upper airway/nasal disease. In the current study, we determined the prevalence of serum-specific IgE towards staphylococcal enterotoxin A, B, C, D (SEA, SEB, SEC, SED) and toxic shock syndrome toxin 1 (TSST-1) in patients suffering from rhinitis and/or asthma due to allergy. Therefore, we examined whether SEA, SEB, SEC, SED and TSST-1 were important in worsening the clinical status of patients allergic to house dust mites by means of assessing serum eosinophil cationic protein (ECP), which is thought to be a reliable marker of asthma and rhinitis severity. 198 patients with persistent allergic rhinitis and/or asthma due to house dust mites were evaluated. Specific IgE towards SEA, SEB, SEC, SED, TSST-1, timothy grass and birch pollen recombinant allergens, and other aeroallergen extracts from common allergen sources were evaluated by the Pharmacia CAP System. Serum ECP was assessed, too. The percentages of sensitization to staphylococcal enterotoxins of 198 house dust mite-allergic patients were as follows: TSST-1-specific IgE 24.7% (n=49), SEC-specific IgE 22.2% (n=44), SEB-specific IgE 15.1% (n=30), SEA-specific IgE 9.1% (n=18), and SED-specific IgE 5.5% (n=11). Out of 198 individuals allergic to house dust mites 136 patients suffering from persistent rhinitis were subdivided into two subgroups: 53 patients with serum-specific IgE to at least one staphylococcal enterotoxin and 83 patients without specific IgE towards staphylococcal enterotoxins. Patients sensitive to staphylococcal enterotoxins had higher serum ECP levels than patients lacking specific IgE to SEA, SEB, SEC, SED and TSST-1(geometric mean 24.3 vs. 16.6 microg/100 ml; p=0.029), as well as total IgE levels (geometric mean 564 vs. 161 kU/l, p=0.00063) and

  6. Synthesis and assembly of Escherichia coli heat-labile enterotoxin B subunit in transgenic lettuce (Lactuca sativa).

    PubMed

    Kim, Tae-Geum; Kim, Mi-Young; Kim, Bang-Geul; Kang, Tae-Jin; Kim, Young-Sook; Jang, Yong-Suk; Arntzen, Charles J; Yang, Moon-Sik

    2007-01-01

    Escherichia coli heat-labile enterotoxin B subunit (LTB) strongly induces immune responses and can be used as an adjuvant for co-administered antigens. Synthetic LTB (sLTB) based on optimal codon usage by plants was introduced into lettuce cells (Lactuca sativa) by Agrobacterium tumefaciens-mediated transformation methods. The sLTB gene was detected in the genomic DNA of transgenic lettuce leaf cells by PCR DNA amplification. Synthesis and assembly of the sLTB protein into oligomeric structures of pentameric size was observed in transgenic plant extracts using Western blot analysis. The binding of sLTB pentamers to intestinal epithelial cell membrane glycolipid receptors was confirmed by G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA). Based on the results of ELISA, sLTB protein comprised approximately 1.0-2.0% of total soluble protein in transgenic lettuce leaf tissues. The synthesis and assembly of sLTB monomers into biologically active oligomers in transgenic lettuce leaf tissues demonstrates the feasibility of the use of edible plant-based vaccines consumed in the form of raw plant materials to induce mucosal immunity.

  7. Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene.

    PubMed

    Mason, H S; Haq, T A; Clements, J D; Arntzen, C J

    1998-08-01

    The authors have designed and constructed a plant-optimize synthetic gene encoding the Escherichia coli heat-labile enterotoxin B subunit (LT-B), for use in transgenic plants as an edible vaccine against enterotoxigenic E. coli. Expression of the synthetic LT-B gene in potato plants under the control of a constitutive promoter yielded increased accumulation of LT-B in leaves and tubers, as compared to the bacterial LT-B gene. The plant-derived LT-B assembled into native pentameric structures as evidenced by its ability to bind ganglioside. The authors demonstrated immunogenicity by feeding mice the raw tubers and comparing the anti-LT-B serum IgG and faecal IgA to that produced in mice gavaged with bacterial LT-B. Mice were fed three weekly doses of 5 g tuber tissue containing either 20 or 50 micrograms LT-B, or gavaged weekly with 5 micrograms of LT-B from recombinant E. coli. One week after the third dose, mice immunized with potato LT-B had higher levels of serum and mucosal anti-LT-B than those gavaged with bacterial LT-B. Mice were challenged by oral administration of 25 micrograms LT, and protection assessed by comparing the gut/carcass mass ratios. Although none of the mice were completely protected, the higher dose potato vaccine compared favourably with the bacterial vaccine. These findings show that an edible vaccine against E. coli LT-B is feasible.

  8. Expression of the B subunit of E. coli heat-labile enterotoxin in the chloroplasts of plants and its characterization.

    PubMed

    Kang, Tae-Jin; Loc, Nguyen-Hoang; Jang, Mi-Ok; Jang, Yong-Suk; Kim, Young-Sook; Seo, Jo-Eun; Yang, Moon-Sik

    2003-12-01

    Transgenic chloroplasts have become attractive systems for heterologous gene expressions because of unique advantages. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (LTB) via chloroplast transformation of tobacco. Stable site-specific integration of the LTB gene into chloroplast genome was confirmed by PCR and genomic Southern blot analysis in transformed plants. Immunoblot analysis indicated that plant-derived LTB protein was oligomeric, and dissociated after boiling. Pentameric LTB molecules were the dominant molecular species in LTB isolated from transgenic tobacco leaf tissues. The amount of LTB protein detected in transplastomic tobacco leaf was approximately 2.5% of the total soluble plant protein, approximately 250-fold higher than in plants generated via nuclear transformation. The GM1-ELISA binding assay indicated that chloroplast-synthesized LTB protein bound to GM1-ganglioside receptors. LTB protein with biochemical properties identical to native LTB protein in the chloroplast of edible plants opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals.

  9. Ultrasensitive electrochemical immunoassay of staphylococcal enterotoxin B in food using enzyme-nanosilica-doped carbon nanotubes for signal amplification.

    PubMed

    Tang, Dianping; Tang, Juan; Su, Biling; Chen, Guonan

    2010-10-27

    A new sandwich-type electrochemical immunoassay for ultrasensitive detection of staphylococcal enterotoxin B (SEB) in food was developed using horseradish peroxidase-nanosilica-doped multiwalled carbon nanotubes (HRPSiCNTs) for signal amplification. Rabbit polyclonal anti-SEB antibodies immobilized on the screen-printed carbon electrode (SPCE) and covalently bound to the HRPSiCNTs were used as capture antibodies and detection antibodies, respectively. In the presence of SEB analyte, the sandwich-type immunocomplex could be formed between the immobilized anti-SEB on the SPCE and anti-SEB-labeled HRPSiCNTs, and the carried HRP could catalyze the electrochemical reduction of H2O2 with the help of thionine. The high content of HRP in the HRPSiCNTs could greatly amplify the electrochemical signal. Under optimal conditions, the reduction current increased with the increase of SEB in the sample, and exhibited a dynamic range of 0.05-15 ng/mL with a low detection limit (LOD) of 10 pg/mL SEB (at 3σ). Intra- and interassay coefficients of variation were below 10%. In addition, the assay was evaluated with SEB spiked samples including watermelon juice, soymilk, apple juice, and pork food, receiving excellent correlation with results from commercially available enzyme-linked immunosorbent assay (ELISA).

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

    PubMed

    Ram, Siya; Vajpayee, Poornima; Shanker, Rishi

    2007-11-01

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

  11. Highly Sensitive Colorimetric Biosensor for Staphylococcal Enterotoxin B by a Label-Free Aptamer and Gold Nanoparticles

    PubMed Central

    Mondal, Bhairab; Ramlal, Shylaja; Lavu, Padma S.; N, Bhavanashri; Kingston, Joseph

    2018-01-01

    A simple, sensitive and selective colorimetric biosensor for the detection of Staphylococcal enterotoxin B (SEB) was developed using SEB-binding aptamer (SEB2) as recognition element and unmodified gold nanoparticles (AuNPs) as colorimetric probes. The assay is based on color change from red to purple due to conformational change of aptamer in the presence of SEB, and the phenomenon of salt-induced AuNPs aggregation which could be monitored by naked eye or UV–vis spectrometer. Results showed that the AuNPs can effectively differentiate the SEB induced conformational change of the aptamer in the presence of a given high salt concentration. A linear response in the range of 50 μg/mL to 0.5 ng/mL of SEB concentration was obtained. The assay was highly specific to SEB as compared to other related toxins. The limit of detection (LOD) of SEB achieved within few minutes was 50 ng/mL visually and spectrometric method improved it to 0.5 ng/mL. Robustness of the assay was tested in artificially spiked milk samples and cross-checked using in house developed sandwich ELISA (IgY as capturing and SEB specific monoclonal as revealing antibody) and PCR. This colorimetric assay could be a suitable alternative over existing methods during biological emergencies due to its simplicity, sensitive and cost effectiveness. PMID:29487580

  12. Rapid and simultaneous detection of ricin, staphylococcal enterotoxin B and saxitoxin by chemiluminescence-based microarray immunoassay.

    PubMed

    Szkola, A; Linares, E M; Worbs, S; Dorner, B G; Dietrich, R; Märtlbauer, E; Niessner, R; Seidel, M

    2014-11-21

    Simultaneous detection of small and large molecules on microarray immunoassays is a challenge that limits some applications in multiplex analysis. This is the case for biosecurity, where fast, cheap and reliable simultaneous detection of proteotoxins and small toxins is needed. Two highly relevant proteotoxins, ricin (60 kDa) and bacterial toxin staphylococcal enterotoxin B (SEB, 30 kDa) and the small phycotoxin saxitoxin (STX, 0.3 kDa) are potential biological warfare agents and require an analytical tool for simultaneous detection. Proteotoxins are successfully detected by sandwich immunoassays, whereas competitive immunoassays are more suitable for small toxins (<1 kDa). Based on this need, this work provides a novel and efficient solution based on anti-idiotypic antibodies for small molecules to combine both assay principles on one microarray. The biotoxin measurements are performed on a flow-through chemiluminescence microarray platform MCR3 in 18 minutes. The chemiluminescence signal was amplified by using a poly-horseradish peroxidase complex (polyHRP), resulting in low detection limits: 2.9 ± 3.1 μg L(-1) for ricin, 0.1 ± 0.1 μg L(-1) for SEB and 2.3 ± 1.7 μg L(-1) for STX. The developed multiplex system for the three biotoxins is completely novel, relevant in the context of biosecurity and establishes the basis for research on anti-idiotypic antibodies for microarray immunoassays.

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  14. Development of a Protein Standard Absolute Quantification (PSAQ™) assay for the quantification of Staphylococcus aureus enterotoxin A in serum.

    PubMed

    Adrait, Annie; Lebert, Dorothée; Trauchessec, Mathieu; Dupuis, Alain; Louwagie, Mathilde; Masselon, Christophe; Jaquinod, Michel; Chevalier, Benoît; Vandenesch, François; Garin, Jérôme; Bruley, Christophe; Brun, Virginie

    2012-06-06

    Enterotoxin A (SEA) is a staphylococcal virulence factor which is suspected to worsen septic shock prognosis. However, the presence of SEA in the blood of sepsis patients has never been demonstrated. We have developed a mass spectrometry-based assay for the targeted and absolute quantification of SEA in serum. To enhance sensitivity and specificity, we combined an immunoaffinity-based sample preparation with mass spectrometry analysis in the selected reaction monitoring (SRM) mode. Absolute quantification of SEA was performed using the PSAQ™ method (Protein Standard Absolute Quantification), which uses a full-length isotope-labeled SEA as internal standard. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were estimated at 352pg/mL and 1057pg/mL, respectively. SEA recovery after immunocapture was determined to be 7.8±1.4%. Therefore, we assumed that less than 1femtomole of each SEA proteotypic peptide was injected on the liquid chromatography column before SRM analysis. From a 6-point titration experiment, quantification accuracy was determined to be 77% and precision at LLOQ was lower than 5%. With this sensitive PSAQ-SRM assay, we expect to contribute to decipher the pathophysiological role of SEA in severe sepsis. This article is part of a Special Issue entitled: Proteomics: The clinical link. Copyright © 2011 Elsevier B.V. All rights reserved.

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

    PubMed Central

    2012-01-01

    Staphylococcus aureus is an important cause of nosocomial and community-acquired infections in humans and animals, as well as the cause of mastitis in dairy cattle. Vaccines aimed at preventing S. aureus infection in bovine mastitis have been studied for many years, but have so far been unsuccessful due to the complexity of the bacteria, and the lack of suitable vaccine delivery vehicles. The current study developed an Escherichia coli protein expression system that produced a recombinant staphylococcal enterotoxin A (rSEA) encapsulated into biodegradable microparticles generated by polylactic-co-glycolic acid (PLGA) dissolved in methylene chloride and stabilized with polyvinyl acetate. Antigen loading and surface properties of the microparticles were investigated to optimize particle preparation protocols. The prepared PLGA-rSEA microspheres had a diameter of approximately 5 μm with a smooth and regular surface. The immunogenicity of the PLGA-rSEA vaccine was assessed using mice as an animal model and showed that the vaccine induced a strong humoral immune response and increased the percent survival of challenged mice and bacterial clearance. Histological analysis showed moderate impairment caused by the pathogen upon challenge afforded by immunization with PLGA-rSEA microspheres. Antibody titer in the sera of mice immunized with PLGA-rSEA microparticles was higher than in vaccinated mice with rSEA. In conclusion, the PLGA-rSEA microparticle vaccine developed here could potentially be used as a vaccine against enterotoxigenic S. aureus. PMID:22429499

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

    PubMed

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

    2012-01-01

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

  17. Tissue culture and expression of Escherichia coli heat-labile enterotoxin B subunit in transgenic Peperomia pellucida.

    PubMed

    Loc, Nguyen Hoang; Bach, Nguyen Hoang; Kim, Tae-Geum; Yang, Moon-Sik

    2010-07-01

    The B subunit of Escherichia coli heat-labile enterotoxin (LTB), a non-toxic molecule with potent biological properties, is a powerful mucosal and parenteral adjuvant that induces a strong immune response against co-administered or coupled antigens. We synthesized a gene encoding the LTB adapted to the optimized coding sequences in plants and fused to the endoplasmic reticulum retention signal SEKDEL to enhance its expression level and protein assembly in plants. The synthetic LTB gene was located into a plant expression vector under the control of CaMV 35S promoter and was introduced into Peperomia pellucida by biolistic transformation method. The integration of synthetic LTB gene into genomic DNA of transgenic plants was confirmed by genomic DNA PCR amplification method. The assembly of plant-produced LTB was detected by western blot analysis. The amount of LTB protein produced in transgenic P. pellucida leaves was approximately 0.75% of the total soluble plant protein. Enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to GM1-ganglioside, which is receptor for LTB on the cell surface, suggesting that the LTB subunits formed biological active pentamers. Copyright 2010 Elsevier Inc. All rights reserved.

  18. 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.

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

    PubMed Central

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

    2015-01-01

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

  20. Attenuation of massive cytokine response to the staphylococcal enterotoxin B superantigen by the innate immunomodulatory protein lactoferrin

    PubMed Central

    Hayworth, J L; Kasper, K J; Leon-Ponte, M; Herfst, C A; Yue, D; Brintnell, W C; Mazzuca, D M; Heinrichs, D E; Cairns, E; Madrenas, J; Hoskin, D W; McCormick, J K; Haeryfar, S M M

    2009-01-01

    Staphylococcal enterotoxin B (SEB) is a pyrogenic exotoxin and a potent superantigen which causes massive T cell activation and cytokine secretion, leading to profound immunosuppression and morbidity. The inhibition of SEB-induced responses is thus considered a goal in the management of certain types of staphylococcal infections. Lactoferrin (LF) is a multi-functional glycoprotein with both bacteriostatic and bactericidal activities. In addition, LF is known to have potent immunomodulatory properties. Given the anti-microbial and anti-inflammatory properties of this protein, we hypothesized that LF can modulate T cell responses to SEB. Here, we report that bovine LF (bLF) was indeed able to attenuate SEB-induced proliferation, interleukin-2 production and CD25 expression by human leucocyte antigen (HLA)-DR4 transgenic mouse T cells. This inhibition was not due to bLF's iron-binding capacity, and could be mimicked by the bLF-derived peptide lactoferricin. Cytokine secretion by an engineered SEB-responsive human Jurkat T cell line and by peripheral blood mononuclear cells from healthy donors was also inhibited by bLF. These findings reveal a previously unrecognized property of LF in modulation of SEB-triggered immune activation and suggest a therapeutic potential for this naturally occurring protein during toxic shock syndrome. PMID:19659771

  1. The Type II Heat-Labile Enterotoxins LT-IIa and LT-IIb and Their Respective B Pentamers Differentially Induce and Regulate Cytokine Production in Human Monocytic Cells

    PubMed Central

    Hajishengallis, George; Nawar, Hesham; Tapping, Richard I.; Russell, Michael W.; Connell, Terry D.

    2004-01-01

    The type II heat-labile enterotoxins, LT-IIa and LT-IIb, exhibit potent adjuvant properties. However, little is known about their immunomodulatory activities upon interaction with innate immune cells, unlike the widely studied type I enterotoxins that include cholera toxin (CT). We therefore investigated interactions of LT-IIa and LT-IIb with human monocytic THP-1 cells. We found that LT-II enterotoxins were inactive in stimulating cytokine release, whereas CT induced low levels of interleukin-1β (IL-1β) and IL-8. However, all three enterotoxins potently regulated cytokine induction in cells activated by bacterial lipopolysaccharide or fimbriae. Induction of proinflammatory (tumor necrosis factor α [TNF-α]) or chemotactic (IL-8) cytokines was downregulated, whereas induction of cytokines with anti-inflammatory (IL-10) or mucosal adjuvant properties (IL-1β) was upregulated by the enterotoxins. These effects appeared to depend on their A subunits, because isolated B-pentameric subunits lacked regulatory activity. Enterotoxin-mediated inhibition of proinflammatory cytokine induction in activated cells was partially attributable to synergism for endogenous production of IL-10 and to an IL-10-independent inhibition of nuclear factor κB (NF-κB) activation. In sharp contrast to the holotoxins, the B pentamers (LT-IIaB and, to a greater extent, LT-IIbB) stimulated cytokine production, suggesting a link between the absence of the A subunit and increased proinflammatory properties. In this regard, the ability of LT-IIbB to activate NF-κB and induce TNF-α and IL-8 was antagonized by the LT-IIb holotoxin. These findings support distinct immunomodulatory roles for the LT-II holotoxins and their respective B pentamers. Moreover, the anti-inflammatory properties of the holotoxins may serve to suppress innate immunity and promote the survival of the pathogen. PMID:15501764

  2. Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin

    PubMed Central

    Toniti, Waraphan; Yoshida, Toru; Tsurumura, Toshiharu; Irikura, Daisuke; Monma, Chie; Kamata, Yoichi

    2017-01-01

    Unusual outbreaks of food poisoning in Japan were reported in which Clostridium perfringens was strongly suspected to be the cause based on epidemiological information and fingerprinting of isolates. The isolated strains lack the typical C. perfringens enterotoxin (CPE) but secrete a new enterotoxin consisting of two components: C. perfringens iota-like enterotoxin-a (CPILE-a), which acts as an enzymatic ADP-ribosyltransferase, and CPILE-b, a membrane binding component. Here we present the crystal structures of apo-CPILE-a, NAD+-CPILE-a and NADH-CPILE-a. Though CPILE-a structure has high similarity with known iota toxin-a (Ia) with NAD+, it possesses two extra-long protruding loops from G262-S269 and E402-K408 that are distinct from Ia. Based on the Ia–actin complex structure, we focused on actin-binding interface regions (I-V) including two protruding loops (PT) and examined how mutations in these regions affect the ADP-ribosylation activity of CPILE-a. Though some site-directed mutagenesis studies have already been conducted on the actin binding site of Ia, in the present study, mutagenesis studies were conducted against both α- and β/γ-actin in CPILE-a and Ia. Interestingly, CPILE-a ADP-ribosylates both α- and β/γ-actin, but its sensitivity towards β/γ-actin is 36% compared with α-actin. Our results contrast to that only C2-I ADP-ribosylates β/γ-actin. We also showed that PT-I and two convex-concave interactions in CPILE-a are important for actin binding. The current study is the first detailed analysis of site-directed mutagenesis in the actin binding region of Ia and CPILE-a against both α- and β/γ-actin. PMID:28199340

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

    PubMed

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

    2016-03-01

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

  4. Native or Proteolytically Activated NanI Sialidase Enhances the Binding and Cytotoxic Activity of Clostridium perfringens Enterotoxin and Beta Toxin.

    PubMed

    Theoret, James R; Li, Jihong; Navarro, Mauricio A; Garcia, Jorge P; Uzal, Francisco A; McClane, Bruce A

    2018-01-01

    Many Clostridium perfringens strains produce NanI as their major sialidase. Previous studies showed that NanI could potentiate C. perfringens epsilon toxin cytotoxicity by enhancing the binding of this toxin to host cells. The present study first determined that NanI exerts similar cytotoxicity-enhancing effects on C. perfringens enterotoxin and beta toxin, which are also important toxins for C. perfringens diseases (enteritis and enterotoxemia) originating in the gastrointestinal (GI) tract. Building upon previous work demonstrating that purified trypsin can activate NanI activity, this study next determined that purified chymotrypsin or mouse intestinal fluids can also activate NanI activity. Amino acid sequencing then showed that this effect involves the N-terminal processing of the NanI protein. Recombinant NanI (rNanI) species corresponding to major chymotrypsin- or small intestinal fluid-generated NanI fragments possessed more sialidase activity than did full-length rNanI, further supporting the proteolytic activation of NanI activity. rNanI species corresponding to proteolysis products also promoted the cytotoxic activity and binding of enterotoxin and beta toxin more strongly than did full-length rNanI. Since enterotoxin and beta toxin are produced in the intestines during human and animal disease, these findings suggest that intestinal proteases may enhance NanI activity, which in turn could further potentiate the activity of intestinally active toxins during disease. Coupling these new results with previous findings demonstrating that NanI is important for the adherence of C. perfringens to enterocyte-like cells, NanI sialidase is now emerging as a potential auxiliary virulence factor for C. perfringens enteritis and enterotoxemia. Copyright © 2017 American Society for Microbiology.

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

    SciTech Connect

    Laiosa, Michael D.; Eckles, Kevin G.; Langdon, Margaret

    2007-12-15

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

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

    PubMed

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

    2016-04-13

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

  7. Spontaneous water secretion in T84 cells: effects of STa enterotoxin, bumetanide, VIP, forskolin, and A-23187.

    PubMed

    Toriano, R; Kierbel, A; Ramirez, M A; Malnic, G; Parisi, M

    2001-09-01

    The regulated Cl(-) secretory apparatus of T84 cells responds to several pharmacological agents via different second messengers (Ca(2+), cAMP, cGMP). However, information about water movements in T84 cells has not been available. In the absence of osmotic or chemical gradient, we observed a net secretory transepithelial volume flux (J(w) = -0.16 +/- 0.02 microl.min(-1).cm(-2)) in parallel with moderate short-circuit current values (I(sc) = 1.55 +/- 0.23 microA/cm(2)). The secretory J(w) reversibly reverted to an absorptive value when A-23187 was added to the serosal bath. Vasoactive intestinal polypeptide increased I(sc), but, unexpectedly, J(w) was not affected. Bumetanide, an inhibitor of basolateral Na(+)-K(+)-2Cl(-) cotransporter, completely blocked secretory J(w) with no change in I(sc). Conversely, serosal forskolin increased I(sc), but J(w) switched from secretory to absorptive values. Escherichia coli heat-stable enterotoxin increased secretory J(w) and I(sc). No difference between the absorptive and secretory unidirectional Cl(-) fluxes was observed in basal conditions, but after STa stimulation, a significant net secretory Cl(-) flux developed. We conclude that, under these conditions, the presence of secretory or absorptive J(w) values cannot be shown by I(sc) and ion flux studies. Furthermore, RT-PCR experiments indicate that aquaporins were not expressed in T84 cells. The molecular pathway for water secretion appears to be transcellular, moving through the lipid bilayer or, as recently proposed, through water-solute cotransporters.

  8. Bone marrow - mesenchymal stem cells impact on the U937 cells in the presence of staphylococcal enterotoxin B (SEB).

    PubMed

    Ejtehadifar, Mostafa; Halabian, Raheleh; Ghazavi, Ali; Khansarinejad, Behzad; Mosayebi, Ghasem; Imani Fooladi, Abbas Ali

    2018-04-14

    The growing resistance against conventional chemotherapy in acute myeloid leukemia (AML) is a noticeable clinical concern. Therefore, many researchers are looking for novel substances to overcome drug resistance in cancer. Staphylococcal enterotoxin B (SEB) is a superantigen (SAg) and a promising compound which has lethal effects on malignant cells. In this unprecedented study, SEB was used against U937 cells in a co-culture system in the presence of human bone marrow-mesenchymal stem cells (hBM-MSCs). The effects of hBM-MSCs on the proliferation and survival of U937 cell line with SEB was assessed using MTT assay and AnnexinV/PI flowcytometry, respectively. Moreover, the expression of IL-6, IL-10, TGF-β, and inhibitor of nuclear factor kappa-B kinase (IKKb) was evaluated by real-time PCR technique. The same experiments were also carried out using hBM-MSCs-conditioned medium (hBM-MSCs-CM). The results showed that SEB reduced the proliferation and survival of U937 cell line, but hBM-MSCs or hBM-MSCs-CM suppressed the effects of SEB. Furthermore, real-timePCR demonstrated that SEB could decrease the expression of IL-6, IL-10, and TGF-β in hBM-MSCs (P < .05), while the production of IKKb was increased in comparison with the control group. These findings help us to have a broader understanding ofthe usage of SEB in the treatment of haematological malignancies, especially if it is targeted against hBM-MSCs to disrupt their supportive effects on malignant cells. © 2018 John Wiley & Sons Australia, Ltd.

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

  10. Rapid and sensitive sandwich enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxin B in cheese.

    PubMed Central

    Morissette, C; Goulet, J; Lamoureux, G

    1991-01-01

    A rapid and sensitive screening sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of staphylococcal enterotoxin B (SEB) in cheese by using a highly avid anti-SEB antibody (Ab) as the capture Ab (CAb) and as the biotinylated Ab conjugate. The glutaraldehyde fixation method for the immobilization of CAb on polystyrene dipsticks was superior to the adsorption fixation and the adsorption-glutaraldehyde fixation methods. The glutaraldehyde fixation method resulted in a higher surface-saturating CAb concentration as evaluated by the peroxidase saturation technique and by the ability of the CAb-coated dipstick to discriminate between positive and negative controls (index of discrimination). Of nine blocking agents used alone or in pairs, lysine-human serum albumin, bovine serum albumin, human serum albumin, and gelatin effectively saturated available sites on the CAb-coated dipsticks without causing interference with the antigen-Ab reactions. The addition of 1% polyethylene glycol to the diluent of the biotinylated anti-SEB Ab conjugate improved the detection of SEB. A concentration of 4% polyethylene glycol allowed a 5-min reaction time for the streptavidin-biotin-horseradish peroxidase conjugate. Cheddar cheese homogenate reduced the sensitivity of the SEB assay; however, the sensitivity was restored when 1.6% (wt/vol) of either a nonionic detergent (Mega-9) or two zwitterionic detergents (Zwittergent 3-10 and 3-12 detergent) was added to the diluent. By using the rapid sandwich ELISA, a minimum of 0.5 to 1.0 ng of SEB per ml was detected within 45 min. The whole procedure for the analysis of the cheddar cheese samples was completed within 1 h.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2039234

  11. Identification of Escherichia coli enterotoxin inhibitors from traditional medicinal herbs by in silico, in vitro, and in vivo analyses.

    PubMed

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

    2009-01-30

    Glycyrrhiza uralensis has been used for the treatment of gastrointestinal disorders, such as diarrhea, in several ancient cultures. Glycyrrhizin is the principal component of liquorice and lots of pharmacological effects have been demonstrated. Heat-labile enterotoxin (LT), the virulence factor of enterotoxigenic Escherichia coli, induces diarrhea by initially binding to the GM1 on the surfaces of intestinal epithelial cells and consequently leading to the massive loss of fluid and ions from cells. Therefore, we evaluated the inhibitory effects of traditional medicinal herbs (TMH) on the B subunit of LT (LTB) and GM1 interaction. The inhibitory effects of TMH on LTB-GM1 interaction were evaluated by GM1-enzyme-linked immunosorbent assay (ELISA). The likely active phytochemicals of these TMH were then predicted by in silico model (docking) and analyzed by in vitro (GM1-ELISA) and in vivo (patent mouse gut assay) models. We found that various TMH, which have been ethnomedically used for the treatment of diarrhea, inhibited the LTB-GM1 interaction. Docking data showed that triterpenoids were the most active phytochemicals and the oleanane-type triterpenoids presented better LTB-binding abilities than other types of triterpenoids. Moreover, by in vitro and in vivo models, we demonstrated that glycyrrhizin was the most effective oleanane-type triterpenoid that significantly suppressed both the LTB-binding ability (IC50=3.26+/-0.17 mM) and the LT-induced fluid accumulation in mice. We found an LT inhibitor, glycyrrhizin, from TMH by in silico, in vitro, and in vivo analyses.

  12. Specific binding of Clostridium perfringens enterotoxin fragment to Claudin-b and modulation of zebrafish epidermal barrier.

    PubMed

    Zhang, Jingjing; Ni, Chen; Yang, Zhenguo; Piontek, Anna; Chen, Huapu; Wang, Sijie; Fan, Yiming; Qin, Zhihai; Piontek, Joerg

    2015-08-01

    Claudins (Cldn) are the major components of tight junctions (TJs) sealing the paracellular cleft in tissue barriers of various organs. Zebrafish Cldnb, the homolog of mammalian Cldn4, is expressed at epithelial cell-cell contacts and is important for regulating epidermal permeability. The bacterial toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to a subset of mammalian Cldns. In this study, we used the Cldn-binding C-terminal domain of CPE (194-319 amino acids, cCPE 194-319 ) to investigate its functional role in modulating zebrafish larval epidermal barriers. In vitro analyses show that cCPE 194-319 removed Cldn4 from epithelial cells and disrupted the monolayer tightness, which could be rescued by the removal of cCPE 194-319. Incubation of zebrafish larvae with cCPE 194-319 removed Cldnb specifically from the epidermal cell membrane. Dye diffusion analysis with 4-kDa fluorescent dextran indicated that the permeability of the epidermal barrier increased due to cCPE 194-319 incubation. Electron microscopic investigation revealed reversible loss of TJ integrity by Cldnb removal. Collectively, these results suggest that cCPE 194-319 could be used as a Cldnb modulator to transiently open the epidermal barrier in zebrafish. In addition, zebrafish might be used as an in vivo system to investigate the capability of cCPE to enhance drug delivery across tissue barriers. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Escherichia coli heat-labile enterotoxin B subunit prevents autoimmune arthritis through induction of regulatory CD4+ T cells.

    PubMed

    Luross, Jeffrey A; Heaton, Tricia; Hirst, Timothy R; Day, Michael J; Williams, Neil A

    2002-06-01

    The receptor-binding B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a highly stable, nontoxic protein that is capable of modulating immune responses. This study was conducted to determine whether mucosal administration of EtxB can block collagen-induced arthritis (CIA) and to investigate the mechanisms involved. Clinical arthritis in DBA/1 mice was monitored following mucosal administration of EtxB on 4 occasions. The dependence of disease prevention on receptor binding by EtxB and the associated alterations to the immune response to type II collagen (CII) were assessed. Adoptive transfer experiments and lymph node cell cocultures were used to investigate the underlying mechanisms. Both intranasal and intragastric delivery of EtxB were effective in preventing CIA; a 1-microg dose of EtxB was protective after intranasal administration. A non-receptor-binding mutant of EtxB failed to prevent disease. Intranasal EtxB lowered both the incidence and severity of arthritis when given either at the time of disease induction or 25 days later. EtxB markedly reduced levels of anti-CII IgG2a antibodies and interferon-gamma (IFNgamma) production while not affecting levels of IgG1, interleukin-4 (IL-4), or IL-10. Disease protection could be transferred by CD4+ T cells from treated mice, an effect that was abrogated upon depletion of the CD25+ population. In addition, CD4+CD25+ T cells from treated mice were able to suppress anti-CII IFNgamma production by CII-primed lymph node cells. Mucosal administration of EtxB can be used to prevent or treat CIA. Modulation of the anti-CII immune response by EtxB is associated with a reduction in Th1 cell reactivity without a concomitant shift toward Th2. Instead, EtxB mediates its effects through enhancing the activity of a population of CD4+ regulatory T cells.

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

    PubMed

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

    2016-03-10

    Cutaneous T-cell lymphoma (CTCL) is characterized by proliferation of malignant T cells in a chronic inflammatory environment. With disease progression, bacteria colonize the compromised skin barrier and half of CTCL patients die of infection rather than from direct organ involvement by the malignancy. Clinical data indicate that bacteria play a direct role in disease progression, but little is known about the mechanisms involved. Here, we demonstrate that bacterial isolates containing staphylococcal enterotoxin A (SEA) from the affected skin of CTCL patients, as well as recombinant SEA, stimulate activation of signal transducer and activator of transcription 3 (STAT3) and upregulation of interleukin (IL)-17 in immortalized and primary patient-derived malignant and nonmalignant T cells. Importantly, SEA induces STAT3 activation and IL-17 expression in malignant T cells when cocultured with nonmalignant T cells, indicating an indirect mode of action. In accordance, malignant T cells expressing an SEA-nonresponsive T-cell receptor variable region β chain are nonresponsive to SEA in monoculture but display strong STAT3 activation and IL-17 expression in cocultures with SEA-responsive nonmalignant T cells. The response is induced via IL-2 receptor common γ chain cytokines and a Janus kinase 3 (JAK3)-dependent pathway in malignant T cells, and blocked by tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross talk-dependent activation of STAT3 and expression of IL-17 in malignant T cells, suggesting a mechanism whereby SEA-producing bacteria promote activation of an established oncogenic pathway previously implicated in carcinogenesis. © 2016 by The American Society of Hematology.

  15. Protective Effect of Immunization of Rats with Holotoxin or B Subunit of Escherichia coli Heat-Labile Enterotoxin

    PubMed Central

    Klipstein, Frederick A.; Engert, Richard F.

    1981-01-01

    The relative immunogenicities of three forms of the Escherichia coli heatlabile enterotoxin (LT), the holotoxin, its B subunit, and the polymyxin-release form (PM LT) were compared by immunizing rats with various dosages of each given exclusively by the parenteral (IP/IP) or peroral (PO/PO) routes or by a combination of the two (IP/PO). The degree of protection was evaluated by challenge in ligated ileal loops, and the serum antitoxin response was determined by an enzyme-linked immunosorbent assay with homologous antigens. When given by the PO/PO route, each LT antigen provided only weak protection against the toxin and virtually none against viable LT-producing strains; serum antitoxin titers were not significantly increased. When the toxins were given after a parental primary immunization by either the IP/IP or the IP/PO routes, each LT antigen provided a dose-related increase in serum antitoxin titers and in the degree of protection against the toxin as well as against viable strains which produce LT alone (LT+/ST−) or in combination with the heat-stable toxin (LT+/ST+). The degree of protection against viable bacteria, particularly the LT+/ST+ strain, was stronger in animals which received booster immunizations by the PO route. When expressed on the basis of molar equivalents, holotoxin provided significant protection (a protection index of >5 against toxin challenge and >50% reduced secretion with bacterial challenge) with 4 to 15 times fewer moles than PM LT and up to 50 times fewer moles than the B subunit. These observations indicate that, on the basis of molar equivalents, the holotoxin (which contains one A plus five or six B subunits) is a more potent immunogen than either PM LT (which contains one A and probably one B subunit) or the B subunit. PMID:7011990

  16. Transcutaneous immunization with tetanus toxoid and mutants of Escherichia coli heat-labile enterotoxin as adjuvants elicits strong protective antibody responses.

    PubMed

    Tierney, Rob; Beignon, Anne-Sophie; Rappuoli, Rino; Muller, Sylviane; Sesardic, Dorothea; Partidos, Charalambos D

    2003-09-01

    In this study, the adjuvanticity of 2 nontoxic derivatives (LTK63 and LTR72) of heat-labile enterotoxin of Escherichia coli (LT) was evaluated and was compared with that of a cytosine phosphodiester-guanine (CpG) motif, after transcutaneous immunization with tetanus toxoid (TT). TT plus LTR72 elicited the strongest antibody responses, compared with those elicited by the other vaccines (TT, TT plus LTK63, TT plus CpG, and TT plus LTK63 plus CpG); it neutralized the toxin and conferred full protection after passive transfer in mice. Preexisting immunity to LT mutants did not adversely affect their adjuvant potency. Both LTK63 and LTR72 promoted the induction of IgG1 antibodies. In contrast, mice receiving either CpG motif alone or CpG motif plus LTK63 produced strong IgG2a anti-TT antibody responses. Overall, these findings demonstrate that mutants of enterotoxins with reduced toxicity are effective adjuvants for transcutaneous immunization.

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

    PubMed Central

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

    2013-01-01

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

  18. Clostridium perfringens enterotoxin and Clostridium difficile toxin A/B do not play a role in acute haemorrhagic diarrhoea syndrome in dogs.

    PubMed

    Busch, K; Suchodolski, J S; Kühner, K A; Minamoto, Y; Steiner, J M; Mueller, R S; Hartmann, K; Unterer, S

    2015-03-07

    Although an association between clostridial pathogens and canine idiopathic acute haemorrhagic diarrhoea syndrome (AHDS) has been described, the relevance of those bacteria and their toxins remains unclear. The aim of this study was to evaluate the association between severity of clinical signs and presence of Clostridium perfringens enterotoxin (CPE) and Clostridium difficile toxin A/B (CDT A/B) in faeces of dogs with AHDS. Faecal samples of 54 dogs with idiopathic AHDS were tested by qualitative CPE and CDT A/B ELISA, and PCR was performed to detect enterotoxin genes of C. perfringens (cpe) and toxin B genes of C. difficile (cdt b). Prevalence of cdt b and CDT A/B in dogs with AHDS was 10/54 and 2/54 versus 3/23 and 0/23 in control dogs. Prevalence of cpe was 35/54 in affected versus 9/23 in control dogs. Prevalence of CPE in dogs with AHDS (13/54) was higher compared with control dogs (0/23). No significant difference was detected between CPE-positive and -negative and between cpe-positive and -negative dogs in severity of clinical signs, duration of hospitalisation, mortality rate and selected laboratory parameters. This study suggests that CPE and CDT A/B do not play a role in idiopathic AHDS, are not associated with clinical parameters in affected dogs and cannot be used to predict disease outcome. British Veterinary Association.

  19. Safety and efficacy of E coli enterotoxin adjuvant for urease-based rectal immunization against Helicobacter pylori.

    PubMed

    Sougioultzis, Stavros; Lee, Cynthia K; Alsahli, Mazen; Banerjee, Subhas; Cadoz, Michel; Schrader, Robert; Guy, Bruno; Bedford, Philip; Monath, Thomas P; Kelly, Ciaran P; Michetti, Pierre

    2002-12-13

    Low dose E. coli heat-labile enterotoxin (LT), delivered orally or enterically, has been used as an adjuvant for Helicobacter pylori (H. pylori) urease in healthy adults. In this study we aim to test the safety and adjuvant efficacy of LT delivered rectally together with recombinant H. pylori urease. Eighteen healthy adults without present or past H. pylori infection were enrolled in a double blind, randomized, ascending dose study to receive either urease (60 mg), or urease (60 mg) + LT (5 or 25 microg). The immunization preparation was administered per rectum on days 0, 14 and 28. Serum, stool and saliva anti-urease and anti-LT IgG and IgA antibodies (Abs) were measured and urease-specific and LT-specific antigen secreting cells (ASCs) were counted in peripheral blood at baseline and 7 (ASC counts) or 14 days (antibody levels) after each dosing. Peripheral blood lymphoproliferation assays were also performed at baseline and at the end of the study. Rectally delivered urease and LT were well tolerated. Among the 12 subjects assigned to urease+LT, 2 (16.7%) developed anti-urease IgG Abs, 1 (8.3%) developed anti-urease IgA Abs, and 3 (25%) showed urease-specific IgA(+) ASCs. Immune responses to LT were more vigorous, especially in subjects exposed to 5 microg LT. In the urease+ 5 microg LT group, anti-LT IgG and IgA Abs developed in 60 and 80% of the subjects, respectively, while LT-specific IgG(+) and IgA(+) ASCs were detected in all subjects. The magnitude of the anti-LT response was much higher than the response to urease. No IgA anti-urease or anti-LT Abs were detected in stool or saliva and lymphocyte proliferative responses to urease were unsatisfactory. In conclusion, rectal delivery of 5 microg LT is safe and induces vigorous systemic anti-LT immune responses. Further studies are needed to determine if LT can be an effective adjuvant for rectally delivered antigens.

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

    PubMed

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

    1996-03-01

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

  1. Expression of a synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat labile enterotoxin in rice endosperm.

    PubMed

    Oszvald, Maria; Kang, Tae-Jin; Tomoskozi, Sandor; Tamas, Cecilia; Tamas, Laszlo; Kim, Tae-Geum; Yang, Moon-Sik

    2007-03-01

    Epitopes often require co-delivery with adjuvant and targeting proteins to enable recognition by the immune system, and this approach may also increase the efficacy of the antigen. In this study, we assess and describe the ability of transgenic rice plants to express a fusion protein consisting of the B-subunit of the Escherichia coli heat-labile enterotoxin (LTB) and a synthetic core-neutralizing epitope (COE) of porcine epidemic diarrhea virus (PEDV), inducing an enteric disease that is seen most predominantly in piglets. Both components of the fusion proteins were detected with Western blot analysis. The fusion protein was determined to assemble into pentamers, as was evidenced by its ability to bind to GM1 gangliosides, and evidenced an average level of expression in a transgenic rice endosperm. This indicates that the expression system of the plant is capable of generating a sizable amount of antigen, possibly allowing for the successful development of an edible vaccine.

  2. Structure of the superantigen staphylococcal enterotoxin B in complex with TCR and peptide-MHC demonstrates absence of TCR-peptide contacts.

    PubMed

    Rödström, Karin E J; Elbing, Karin; Lindkvist-Petersson, Karin

    2014-08-15

    Superantigens are immune-stimulatory toxins produced by Staphylococcus aureus, which are able to interact with host immune receptors to induce a massive release of cytokines, causing toxic shock syndrome and possibly death. In this article, we present the x-ray structure of staphylococcal enterotoxin B (SEB) in complex with its receptors, the TCR and MHC class II, forming a ternary complex. The structure, in combination with functional analyses, clearly shows how SEB adopts a wedge-like position when binding to the β-chain of TCR, allowing for an interaction between the α-chain of TCR and MHC. Furthermore, the binding mode also circumvents contact between TCR and the peptide presented by MHC, which enables SEB to initiate a peptide-independent activation of T cells. Copyright © 2014 by The American Association of Immunologists, Inc.

  3. [Cloning and sequence analysis of recombinant fusion gene of Escherichia coli heat-liable enterotoxin B subunit and Actinobacillus actinomycetemcomitans fimbria associative protein].

    PubMed

    Li, Yi; Sun, Hong-chen; Guo, Xue-jun; Feng, Shu-zhang

    2005-02-01

    To clone the recombinant fusion gene of Escherichia coli heat-liable enterotoxin B subunit (Ltb) and Actinobacillus actinomycetemcomitans fimbria associative protein (Fap). Two couples of primers were designed for PCR according to the known sequence of ltb and fap. The ltb and fap gene were obtained by amplification PCR technique from plasmid EWD299 of Escherichia coli and Actinobacillus actinomycetemcomitans 310 DNA respectively, and fused them by PCR. The fusion gene ltb-fap were cloning into plasmid pET28a(+). The recombined plasmid pET28a ltb-fap was transformed into Escherichia coli DH5alpha. The recombinant was screened and identified by restriction enzyme and PCR. The cloned gene was sequenced. The ltb-fap about 531bp in size was obtained successfully, and identified by PCR, restrictive enzyme and sequence analysis. The vector of pET28a ltb-fap was obtained.

  4. Sequencing and diversity analyses reveal extensive similarities between some epsilon-toxin-encoding plasmids and the pCPF5603 Clostridium perfringens enterotoxin plasmid.

    PubMed

    Miyamoto, Kazuaki; Li, Jihong; Sayeed, Sameera; Akimoto, Shigeru; McClane, Bruce A

    2008-11-01

    Clostridium perfringens type B and D isolates produce epsilon-toxin, the third most potent clostridial toxin. The epsilon-toxin gene (etx) is plasmid borne in type D isolates, but etx genetics have been poorly studied in type B isolates. This study reports the first sequencing of any etx plasmid, i.e., pCP8533etx, from type B strain NCTC8533. This etx plasmid is 64.7 kb, carries tcp conjugative transfer genes, and encodes additional potential virulence factors including beta2-toxin, sortase, and collagen adhesin but not beta-toxin. Interestingly, nearly 80% of pCP8533etx open reading frames (ORFs) are also present on pCPF5603, an enterotoxin-encoding plasmid from type A isolate F5603. Pulsed-field gel electrophoresis and overlapping PCR indicated that a pCP8533etx-like etx plasmid is also present in most, if not all, other type B isolates and some beta2-toxin-positive, cpe-negative type D isolates, while other type D isolates carry different etx plasmids. Sequences upstream of the etx gene vary between type B isolates and some type D isolates that do not carry a pCP8533etx-like etx plasmid. However, nearly all type B and D isolates have an etx locus with an upstream IS1151, and those etx loci typically reside near a dcm ORF. These results suggest that pCPF5603 and pCP8533etx evolved from insertion of mobile genetic elements carrying enterotoxin or etx genes, respectively, onto a common progenitor plasmid.

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

    PubMed

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

    2015-09-11

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

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

    PubMed Central

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

    2015-01-01

    We previously demonstrated the clonal complex 81 (CC81) subtype 1 lineage is the major staphylococcal food poisoning (SFP)-associated lineage in Japan (Y. Sato'o et al., J Clin Microbiol 52:2637–2640, 2014, http://dx.doi.org/10.1128/JCM.00661-14). Strains of this lineage produce staphylococcal enterotoxin H (SEH) in addition to SEA. However, an evaluation of the risk for the recently reported SEH has not been sufficiently conducted. We first searched for staphylococcal enterotoxin (SE) genes and SE proteins in milk samples that caused a large SFP outbreak in Japan. Only SEA and SEH were detected, while there were several SE genes detected in the samples. We next designed an experimental model using a meat product to assess the productivity of SEs and found that only SEA and SEH were detectably produced in situ. Therefore, we investigated the regulation of SEH production using a CC81 subtype 1 isolate. Through mutant analysis of global regulators, we found the repressor of toxin (Rot) functioned oppositely as a stimulator of SEH production. SEA production was not affected by Rot. seh mRNA expression correlated with rot both in media and on the meat product, and the Rot protein was shown to directly bind to the seh promoter. The seh promoter sequence was predicted to form a loop structure and to hide the RNA polymerase binding sequences. We propose Rot binds to the promoter sequence of seh and unfolds the secondary structure that may lead the RNA polymerase to bind the promoter, and then seh mRNA transcription begins. This alternative Rot regulation for SEH may contribute to sufficient toxin production by the CC81 subtype 1 lineage in foods to induce SFP. PMID:26341202

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

    PubMed

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

    2015-11-01

    We previously demonstrated the clonal complex 81 (CC81) subtype 1 lineage is the major staphylococcal food poisoning (SFP)-associated lineage in Japan (Y. Sato'o et al., J Clin Microbiol 52:2637-2640, 2014, http://dx.doi.org/10.1128/JCM.00661-14). Strains of this lineage produce staphylococcal enterotoxin H (SEH) in addition to SEA. However, an evaluation of the risk for the recently reported SEH has not been sufficiently conducted. We first searched for staphylococcal enterotoxin (SE) genes and SE proteins in milk samples that caused a large SFP outbreak in Japan. Only SEA and SEH were detected, while there were several SE genes detected in the samples. We next designed an experimental model using a meat product to assess the productivity of SEs and found that only SEA and SEH were detectably produced in situ. Therefore, we investigated the regulation of SEH production using a CC81 subtype 1 isolate. Through mutant analysis of global regulators, we found the repressor of toxin (Rot) functioned oppositely as a stimulator of SEH production. SEA production was not affected by Rot. seh mRNA expression correlated with rot both in media and on the meat product, and the Rot protein was shown to directly bind to the seh promoter. The seh promoter sequence was predicted to form a loop structure and to hide the RNA polymerase binding sequences. We propose Rot binds to the promoter sequence of seh and unfolds the secondary structure that may lead the RNA polymerase to bind the promoter, and then seh mRNA transcription begins. This alternative Rot regulation for SEH may contribute to sufficient toxin production by the CC81 subtype 1 lineage in foods to induce SFP. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  8. Development and evaluation of IgY ImmunoCapture PCR ELISA for detection of Staphylococcus aureus enterotoxin A devoid of protein A interference.

    PubMed

    Reddy, Prakash; Ramlal, Shylaja; Sripathy, Murali Harishchandra; Batra, Harsh Vardhan

    2014-06-01

    In the present study, a sensitive and specific IgY mediated ImmunoCapture-PCR-ELISA (IC-PCR-ELISA) was developed for the detection of staphylococcal enterotoxin A (SEA) from culture supernatants and suspected contaminated samples. Due to the virtue of avian immunoglobulins (IgY) to have the least affinity towards staphylococcal protein A (SpA) responsible for false positives, we employed anti-SEA IgY for capture of SEA toxin and revealed with SEA specific rabbit antibodies conjugated to a 524bp DNA marker. Biotin-11-dUTP was incorporated during PCR amplification and post PCR analysis was performed by PCR-ELISA. Unlike IgG immunocapture, IgY mediated immunocapture of SEA was free from false positives due to protein A. The developed assay was specific to SEA except for minor cross reactivity with staphylococcal enterotoxin E (SEE). Several raw milk samples were evaluated for the presence of SEA with and without enrichment. Three samples were found to be positive for SEA after enrichment for 8h. Though IC-PCR-ELISA for SEA showed 100% correlation with PCR analysis for sea gene, the assay was unique in terms of sensitivity of detecting ~10pg/ml of SEA toxin from spiked milk samples. Result of IC-PCR-ELISA was further confirmed by conventional methods of isolation and characterization. The presented method can be very useful for rapid analysis of milk samples for SEA contamination and can be further extended for detection of multiple SE's in different wells of same PCR plate using common DNA substrate. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Immunization with recombinant bivalent chimera r-Cpae confers protection against alpha toxin and enterotoxin of Clostridium perfringens type A in murine model.

    PubMed

    Shreya, Das; Uppalapati, Siva R; Kingston, Joseph J; Sripathy, Murali H; Batra, Harsh V

    2015-05-01

    Clostridium perfringens type A, an anaerobic pathogen is the most potent cause of soft tissue infections like gas gangrene and enteric diseases like food poisoning and enteritis. The disease manifestations are mediated via two important exotoxins, viz. myonecrotic alpha toxin (αC) and enterotoxin (CPE). In the present study, we synthesized a bivalent chimeric protein r-Cpae comprising C-terminal binding regions of αC and CPE using structural vaccinology rationale and assessed its protective efficacy against both alpha toxin (αC) and enterotoxin (CPE) respectively, in murine model. Active immunization of mice with r-Cpae generated high circulating serum IgG (systemic), significantly increased intestinal mucosal s-IgA antibody titres and resulted in substantial protection to the immunized animals (100% and 75% survival) with reduced tissue morbidity when administered with 5×LD(100) doses of αC (intramuscular) and CPE (intra-gastric gavage) respectively. Mouse RBCs and Caco-2 cells incubated with a mixture of anti-r-Cpae antibodies and αC and CPE respectively, illustrated significantly higher protection against the respective toxins. Passive immunization of mice with a similar mixture resulted in 91-100% survival at the end of the 15 days observation period while mice immunized with a concoction of sham sera and respective toxins died within 2-3 days. This work demonstrates the efficacy of the rationally designed r-Cpae chimeric protein as a potential sub unit vaccine candidate against αC and CPE of C. perfringens type A toxemia. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. A two-step non-flowcytometry-based naïve B cell isolation method and its application in Staphylococcal enterotoxin B (SEB) presentation.

    PubMed

    Chokeshai-u-saha, Kaj; Buranapraditkun, Supranee; Jacquet, Alain; Nguyen, Catherine; Ruxrungtham, Kiat

    2012-09-01

    To study the role of human naïve B cells in antigen presentation and stimulation to naïve CD4+ T cell, a suitable method to reproducibly isolate sufficient naïve B cells is required. To improve the purity of isolated naive B cells obtained from a conventional one-step magnetic bead method, we added a rosetting step to enrich total B cell isolates from human whole blood samples prior to negative cell sorting by magnetic beads. The acquired naïve B cells were analyzed for phenotypes and for their role in Staphylococcal enterotoxin B (SEB) presentation to naïve CD4+ T cells. The mean (SD) naïve B cell (CD19+/CD27-) purity obtained from this two-step method compared with the one-step method was 97% (1.0) versus 90% (1.2), respectively. This two-step method can be used with a sample of whole blood as small as 10 ml. The isolated naive B cells were phenotypically at a resting state and were able to prime naïve CD4+ T cell activation by Staphylococcal enterotoxin B (SEB) presentation. This two-step non-flow cytometry-based approach improved the purity of isolated naïve B cells compared with conventional one-step magnetic bead method. It also worked well with a small blood volume. In addition, this study showed that the isolated naïve B cells can present a super-antigen "SEB" to activate naïve CD4 cells. These methods may thus be useful for further in vitro characterization of human naïve B cells and their roles as antigen presenting cells in various diseases.

  11. Maternal immune activation with staphylococcal enterotoxin A produces unique behavioral changes in C57BL/6 mouse offspring.

    PubMed

    Glass, Ruthy; Norton, Sara; Fox, Nicholas; Kusnecov, Alexander W

    2018-05-14

    Stimulation of the immune system during pregnancy, known as maternal immune activation (MIA), can cause long-lasting neurobiological and behavioral changes in the offspring. This phenomenon has been implicated in the etiology of developmental psychiatric disorders, such as autism and schizophrenia. Much of this evidence is predicated on animal models using bacterial agents such as LPS and/or viral mimics such as Poly I:C, both of which act through toll-like receptors. However, fewer studies have examined the role of direct activation of maternal T-cells during pregnancy using microbial agents. Bacterial superantigens, such as Staphylococcal Enterotoxin A and B (SEA; SEB), are microbial proteins that activate CD4 + T-cells and cause prominent T-cell proliferation and cytokine production. We injected pregnant and non-pregnant adult female C57BL/6 mice with 200 μg/Kg of SEA, SEB, or 0.9% saline, and measured splenic T-cell-derived cytokine concentrations (viz., IL-2, IFN-γ, IL-6, and IL-4) 2 h later; animals injected with SEA were also measured for splenic concentrations of TNF-α and IL-17A. Half of the injected pregnant animals were brought to term, and their offspring were tested on a series of behavioral tasks starting at six weeks of age (postnatal day 42 [P42]). These tasks included social interaction, the elevated plus maze (EPM), an open field and object recognition (OR) task, prepulse inhibition (PPI) of sensorimotor gating, and the Morris water maze (MWM). Results showed that SEA and SEB induced significant concentrations of all measured cytokines, and in particular IFN-γ, although cytokine responses were greater following SEA exposure. In addition, pregnancy induced an inhibitory effect on cytokine production. Behavioral results showed distinct phenotypes among offspring from SEA- or SEB-injected mothers, very likely due to differences in the magnitude of cytokines generated in response to each toxin. Offspring from SEA-injected mothers displayed

  12. Clostridium perfringens Type A Strains Carrying a Plasmid-Borne Enterotoxin Gene (Genotype IS1151-cpe or IS1470-like-cpe) as a Common Cause of Food Poisoning▿

    PubMed Central

    Lahti, Päivi; Heikinheimo, Annamari; Johansson, Tuula; Korkeala, Hannu

    2008-01-01

    The prevalences of various genotypes of enterotoxin gene-carrying (cpe-positive) Clostridium perfringens type A in 24 different food poisoning outbreaks were 75% (chromosomal IS1470-cpe), 21% (plasmid-borne IS1470-like-cpe), and 4% (plasmid-borne IS1151-cpe). These results show that C. perfringens type A carrying the plasmid-borne cpe is a common cause of food poisoning. PMID:18003798

  13. Prevalence of Vibrio cholerae with heat-stable enterotoxin (NAG-ST) and cholera toxin genes; restriction fragment length polymorphisms of NAG-ST genes among V. cholerae O serogroups from a major shrimp production area in Thailand.

    PubMed

    Dalsgaard, A; Serichantalergs, O; Shimada, T; Sethabutr, O; Echeverria, P

    1995-09-01

    A total of 148 Vibrio cholerae isolates from a major shrimp production area in Southern Thailand were examined by colony hybridisation for genes encoding heat-stable enterotoxin (NAG-ST) and cholera toxin (CT). Only non-O1 V. cholerae strains were found to harbour NAG-ST (14 of 146) whereas no strains hybridised with the CT probe. NAG-ST-positive V. cholerae non-O1 strains were isolated from shrimp farms situated close to urban areas. Five different O serogroups were found among NAG-ST positive non-O1 strains. Southern blot and restriction endonuclease analysis of NAG-ST-positive strains revealed a high degree of genetic divergence. A total of seven classes of enterotoxin gene patterns were found with HindIII and EcoRI restriction endonucleases. Enterotoxin gene patterns correlated with O-antigen expression in 84% of isolates tested. In combination with other molecular techniques Southern blot analysis with an NAG-ST oligonucleotide probe could be useful for studying the molecular epidemiology of V. cholerae non-O1 strains.

  14. [The staphylococcal enterotoxin burden determines the ultrastructure of ciliated epithelia and inflammatory changes in maxillary sinus mucosa of rabbits].

    PubMed

    Wei, Hongqi; Zhu, Zhengwen; Cao, Zhongsheng; Liu, Zhiyong; Wu, Xiaofan; Yuan, Hui

    2014-12-01

    To investigate the ultrastructure of ciliated epithelia and inflammatory changes upon repeated exposure to staphylococcal enterotoxin A (SEA) of different concentrations in the maxillary sinus mucosa of rabbits. The rabbits were randomly divided into 2 groups (24 rabbits per group): low-dose SEA group and high-dose SEA group. The low-dose SEA group and high-dose SEA group received daily injections of 0.6 ng of SEA (2 ml) and 60 ng of SEA (2 ml) into the left maxillary sinus of rabbits for 28 days, respectively. Concurrent treatment of the right maxillary sinus with normal saline was used as control. Six rabbits chosen randomly in two groups were examined by computed tomography (CT) scans and then sacrificed to obtain the sinus mucosa from the two-side of maxillary sinuses for histological assessment on days 3, 7, 14 and 28. To characterize the inflammatory changes of the sinus mucosa examined using light microscope, hematoxylin and eosin (HE) and toluidine blue staining was performed. Scanning and transmission electron microscopy were performed to observe ultrastructure of ciliated epithelia in the maxillary sinus mucosa. SPSS 13.0 software was used to analyze the data. On days 14 and 28, CT images showed opacification of the left maxillary sinus in the high-dose SEA group. The percentage of epithelial disruption was (22.73 ± 5.72) % and (30.79 ± 4.30)% in the high-dose SEA group respectively, and were significantly greater than those in the low-dose SEA group (5.12% ± 1.98% and 5.38% ± 1.64%, q value was 10.079 and 19.132) and control group (4.08% ± 1.29% and 4.81% ± 1.62%, q value was 11.016 and 19.592, respectively, all P < 0.01). The subepithelial thickness in the high-dose SEA group was (113.34 ± 14.81)µm and (120.86 ± 12.35) µm respectively, and were significantly different from those of the low-dose SEA group [(71.08 ± 10.39)µm and (81.63 ± 9.32)µm, q value was 8.090 and 8.782] and control group [(37.45 ± 7.67)µm and (38.79 ± 7.68)µm, q

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

    PubMed

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

    2015-12-01

    This study evaluated Staphylococcus aureus growth and subsequent staphylococcal enterotoxin A production in tryptone soy broth and on ready-to-eat cooked fish paste at 12 to 37 °C, as well as cross-contamination between stainless steel, polyethylene, and latex glove at room temperature. A model was developed using Barany and Roberts's growth model, which satisfactorily described the suitable growth of S. aureus with R(2)-adj from 0.94 to 0.99. Except at 12 °C, S. aureus cells in TSB presented a lag time lower (14.64 to 1.65 h), grew faster (0.08 to 0.31 log CFU/h) and produced SEA at lower cell density levels (5.65 to 6.44 log CFU/mL) compare to those inoculated on cooked fish paste with data of 16.920 to 1.985 h, 0.02 to 0.23 log CFU/h, and 6.19 to 7.11 log CFU/g, respectively. Staphylococcal enterotoxin type A (SEA) visual immunoassay test showed that primary SEA detection varied considerably among different storage temperature degrees and media. For example, it occurred only during exponential phase at 30 and 37 °C in TSB, but in cooked fish paste it took place at late exponential phase of S. aureus growth at 20 and 25 °C. The SEA detection test was negative on presence of S. aureus on cooked fish paste stored at 12 and 15 °C, although cell density reached level of 6.12 log CFU/g at 15 °C. Cross-contamination expressed as transfer rate of S. aureus from polyethylene surface to cooked fish paste surface was slower than that observed with steel surface to cooked fish paste under same conditions. These results provide helpful information for controlling S. aureus growth, SEA production and cross-contamination during processing of cooked fish paste. © 2015 Institute of Food Technologists®

  16. The fecal presence of enterotoxin and F4 genes as an indicator of efficacy of treatment with colistin sulfate in pigs.

    PubMed

    Rhouma, Mohamed; Fairbrother, John Morris; Thériault, William; Beaudry, Francis; Bergeron, Nadia; Laurent-Lewandowski, Sylvette; Letellier, Ann

    2017-01-05

    Enterotoxigenic Escherichia coli (ETEC) strains producing multiple enterotoxins are important causes of post-weaning diarrhea (PWD) in pigs. The aim of the present study was to investigate the fecal presence of ETEC enterotoxin as well as F4 and F18 genes as an indicator of colistin sulfate (CS) efficacy for treatment of PWD in pigs. Forty-eight piglets were weaned at the age of 21 days, and were divided into four groups: challenged treated, challenged untreated, unchallenged treated, and unchallenged untreated. Challenge was performed using 10 9  CFU of an ETEC: F4 strain, and treatment was conducted using oral CS at the dose of 50,000 IU/kg. The fecal presence of genes encoding for STa, STb, LT, F4 and F18 was detected using PCR. The PCR amplification of ETEC virulence genes showed that nearly 100% of pigs excreted genes encoding for STa and STb toxins in the feces before the challenge. These genes, in the absence of the gene encoding F4, were considered as a marker for F4-negative ETEC. One day after ETEC: F4 oral challenge pigs in the two challenged groups excreted the genes encoding LT and F4 in the feces. These genes were considered as a marker for F4-positive ETEC. However, the gene encoding F18 was not detected in any fecal samples of the 4 groups throughout the experiment. After only 3 days of successive oral treatment with CS, a significant reduction in both the F4-positive and negative ETEC populations was observed in the challenged treated group compared to the challenged untreated group (p < 0.0001). Our study is among the first to report that under controlled farming conditions, oral CS treatment had a significant effect on both fecal F4-positive and F4-negative ETEC in pigs. However, CS clinical efficiency was correlated with non-detection of F4-positive ETEC in the feces. Furthermore the fecal presence of F4-negative ETEC was not associated with clinical symptoms of post-weaning diarrhea in pigs.

  17. Enterotoxin Gene Cluster-Encoded SEI and SElN from Staphylococcus aureus Isolates are Crucial for the Induction of Human Blood Cell Proliferation and Pathogenicity in Rabbits

    PubMed Central

    Roetzer, Andreas; Gruener, Corina S.; Haller, Guenter; Beyerly, John; Model, Nina; Eibl, Martha M.

    2016-01-01

    Among the toxin family of bacterial superantigens, the six members of the enterotoxin gene cluster (egc) seem to have unusual characteristics. They are present in the majority of Staphylococcus aureus strains, but their role in disease remains uncertain. We assessed secretion levels, immunogenicity, and toxicity of native and recombinant egc proteins. After having developed enzyme-linked immunosorbent assays, we found different quantities of egc proteins secreted by bacterial isolates. Supernatants induced proliferation of human peripheral blood mononuclear cells. However, purified recombinant egc proteins were shown to have differing superantigenicity potentials. Immunization with identical amounts of all members of egc, and the prominent toxic agent SEB, resulted in neutralizing antisera. Two egc proteins, SEI and SElN, were found to play a predominant role within the cluster. Both displayed the highest potential to activate blood cells, and were essential to be neutralized in supernatants. The application of a supernatant of a strain bearing only egc was sufficient for a lethal outcome in a rabbit model. Again, neutralization of SEI and SElN led to the survival of all tested animals. Finally, nanogram amounts of purified rSEI and rSElN led to lethality in vivo, pointing out the importance of both as virulence determinants among egc superantigens. PMID:27801832

  18. Enterotoxin Gene Cluster-Encoded SEI and SElN from Staphylococcus aureus Isolates are Crucial for the Induction of Human Blood Cell Proliferation and Pathogenicity in Rabbits.

    PubMed

    Roetzer, Andreas; Gruener, Corina S; Haller, Guenter; Beyerly, John; Model, Nina; Eibl, Martha M

    2016-10-28

    Among the toxin family of bacterial superantigens, the six members of the enterotoxin gene cluster (egc) seem to have unusual characteristics. They are present in the majority of Staphylococcus aureus strains, but their role in disease remains uncertain. We assessed secretion levels, immunogenicity, and toxicity of native and recombinant egc proteins. After having developed enzyme-linked immunosorbent assays, we found different quantities of egc proteins secreted by bacterial isolates. Supernatants induced proliferation of human peripheral blood mononuclear cells. However, purified recombinant egc proteins were shown to have differing superantigenicity potentials. Immunization with identical amounts of all members of egc, and the prominent toxic agent SEB, resulted in neutralizing antisera. Two egc proteins, SEI and SE l N, were found to play a predominant role within the cluster. Both displayed the highest potential to activate blood cells, and were essential to be neutralized in supernatants. The application of a supernatant of a strain bearing only egc was sufficient for a lethal outcome in a rabbit model. Again, neutralization of SEI and SE l N led to the survival of all tested animals. Finally, nanogram amounts of purified rSEI and rSE l N led to lethality in vivo, pointing out the importance of both as virulence determinants among egc superantigens.

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

    PubMed

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

    2015-10-01

    Outbreaks of Staphylococcus aureus are common in neonatal intensive care units (NICUs). Usually they are documented for methicillin-resistant strains, while reports involving methicillin-susceptible S. aureus (MSSA) strains are rare. In this study we report the epidemiological and molecular investigation of an MSSA outbreak in a NICU among preterm neonates. Infection control measures and interventions were commissioned by the Local Public Health Authority and supported by the Robert Koch Institute. To support epidemiological investigations molecular typing was done by spa-typing and Multilocus sequence typing; the relatedness of collected isolates was further elucidated by DNA SmaI-macrorestriction, microarray analysis and bacterial whole genome sequencing. A total of 213 neonates, 123 healthcare workers and 205 neonate parents were analyzed in the period November 2011 to November 2012. The outbreak strain was characterized as a MSSA spa-type t021, able to produce toxic shock syndrome toxin-1 and Enterotoxin A. We identified seventeen neonates (of which two died from toxic shock syndrome), four healthcare workers and three parents putatively involved in the outbreak. Whole-genome sequencing permitted to exclude unrelated cases from the outbreak and to discuss the role of healthcare workers as a reservoir of S. aureus on the NICU. Genome comparisons also indicated the presence of the respective clone on the ward months before the first colonized/infected neonates were detected. Copyright © 2015 Elsevier GmbH. All rights reserved.

  20. Preliminary investigation of human serum albumin-Vβ inhibition on toxic shock syndrome induced by staphylococcus enterotoxin B in vitro and in vivo.

    PubMed

    Yuan, Qifeng; Li, Lin; Pian, Yaya; Hao, Huaijie; Zheng, Yuling; Zang, Yating; Jiang, Hua; Jiang, Yongqiang

    2016-04-01

    Staphylococcus enterotoxin B (SEB) is a superantigen that can induce massive activation of T cells with specific Vβ and inflammatory cytokine cascades, which mediate shock. To date, no SEB vaccine has been developed for preventing toxic shock syndrome (TSS). Here, we evaluated the therapeutic effect of a fusion protein human serum albumin-Vβ (HSA-Vβ) on TSS induced by SEB. Compared with Vβ, the preparation of HSA-Vβ was much easier to handle owing to its solubility. Affinity testing showed that HSA-Vβ had high affinity for SEB. In vitro results showed that HSA-Vβ could effectively inhibit interferon (IFN)-γ and tumor necrosis factor (TNF)-α secretion by human peripheral blood mononuclear cells. Moreover, in vivo, HSA-Vβ reduced IFN-γ and TNF-α levels in the serum and protected mice from SEB lethal challenge when administered simultaneously with SEB or 30 min after SEB. In summary, we simplified the preparation of Vβ by fusion with HSA, creating the HSA-Vβ protein, which effectively inhibited cytokine production and protected mice from lethal challenge with SEB. These data indicated that HSA-Vβ may represent a novel therapeutic strategy for the treatment of SEB-induced TSS. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Frequency of enterotoxins, toxic shock syndrome toxin-1, and biofilm formation genes in Staphylococcus aureus isolates from cows with mastitis in the Northeast of Brazil.

    PubMed

    Costa, F N; Belo, N O; Costa, E A; Andrade, G I; Pereira, L S; Carvalho, I A; Santos, R L

    2018-06-01

    Staphylococcus aureus is among the microorganisms more frequently associated with subclinical bovine mastitis. S. aureus may produce several virulence factors. This study aimed at determining the frequency of virulence factors such as enterotoxins, toxic shock syndrome toxin 1, and ica adhesion genes. In addition, we assessed antimicrobial drug resistance in S. aureus isolated from clinical and subclinical cases of mastitis. A total of 88 cows with clinical or subclinical mastitis were sampled, resulting in 38 S. aureus isolates, from which 25 (65.78%) carried toxin genes, including seb, sec, sed, tst, and icaD adhesion gene. These S. aureus isolates belong to 21 ribotypes and three S. aureus strains belonged to the same ribotype producing ica adhesion gene. Approximately 90% of S. aureus strains obtained in our study demonstrated multiple resistance to different antimicrobial agents. The most efficacious antimicrobial agents against the isolates were gentamicin, amoxicillin, and norfloxacin. Gentamicin was the most efficacious agent inhibiting 78.95% of the S. aureus isolates. The least efficacious were penicillin, streptomycin, and ampicillin. Our results can help in understanding the relationship between virulence factors and subclinical mastitis caused by S. aureus. Further research about diversity of S. aureus isolates and genes responsible for the pathogenicity of subclinical mastitis is essential.

  2. Reduction of Non-Specific Protein Adsorption Using Poly(ethylene) Glycol (PEG) Modified Polyacrylate Hydrogels In Immunoassays for Staphylococcal Enterotoxin B Detection

    PubMed Central

    Charles, Paul T.; Stubbs, Veronte R.; Soto, Carissa M.; Martin, Brett D.; White, Brandy J.; Taitt, Chris R.

    2009-01-01

    Three PEG molecules (PEG-methacrylate, -diacrylate and -dimethacrylate) were incorporated into galactose-based polyacrylate hydrogels and their relative abilities to reduce non-specific protein adsorption in immunoassays were determined. Highly crosslinked hydrogels containing amine-terminated functionalities were formed and used to covalently attach antibodies specific for staphylococcal enterotoxin B (SEB). Patterned arrays of immobilized antibodies in the PEG-modified hydrogels were created with a PDMS template containing micro-channels for use in sandwich immunoassays to detect SEB. Different concentrations of the toxin were applied to the hydrogel arrays, followed with a Cy3-labeled tracer antibody specific for the two toxins. Fluorescence laser scanning confocal microscopy of the tracer molecules provided both qualitative and quantitative measurements on the detection sensitivity and the reduction in non-specific binding as a result of PEG incorporation. Results showed the PEG-modified hydrogel significantly reduced non-specific protein binding with a detection limit for SEB of 1 ng/mL. Fluorescence signals showed a 10-fold decrease in the non-specific binding and a 6-fold increase in specific binding of SEB. PMID:22389622

  3. [Experimental study on the activity of Staphyloccocal enterotoxin A liposome for inducing cytotoxicity of TIL from human hepatocellular carcinoma against tumor cells].

    PubMed

    Li, Mao-de; Li, Zhi-yu; He, Sheng; Xue, Hua

    2004-01-01

    To investigate the activity of Staphyloccocal enterotoxin A liposome (L-SEA) for inducing cytotoxicity of tumor infiltrating lymphocytes (TIL) against tumor cells. TIL were isolated from the tumor tissues of five hepatocellular carcinoma patients. L-SEA, SEA and IL-2 were tested in vitro for their activity levels in stimulating TIL proliferation. The TNF-alpha and IFN-gamma secretion and cytotoxicity of TIL against HepG-2 liver cancer cells were estimated by ELISA and MTT, respectively. Both L-SEA and SEA significantly stimulated the proliferation of TIL. The cytokine secretion of L-SEA group was significantly higher than that of IL-2 group (P < 0.05). There was no significantly statistical difference in cytokine secretion between L-SEA group and SEA group (P > 0.05) except that IFN-gamma secretion of L-SEA group was lower than that of SEA group at day 4 (P < 0.05). Both L-SEA and SEA had potent ability to induce TIL cytotoxicity against HepG-2 cells. And no significant difference was observed between these two groups (P > 0.05). These results suggest that L-SEA is as efficient as SEA in activating TIL.

  4. In vitro cytotoxicity induced by Clostridium perfringens isolate carrying a chromosomal cpe gene is exclusively dependent on sporulation and enterotoxin production.

    PubMed

    Yasugi, Mayo; Sugahara, Yuki; Hoshi, Hidenobu; Kondo, Kaori; Talukdar, Prabhat K; Sarker, Mahfuzur R; Yamamoto, Shigeki; Kamata, Yoichi; Miyake, Masami

    2015-08-01

    Clostridium perfringens type A is a common source of food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases in humans. In the intestinal tract, the vegetative cells sporulate and produce a major pathogenic factor, C. perfringens enterotoxin (CPE). Most type A FP isolates carry a chromosomal cpe gene, whereas NFB type A isolates typically carry a plasmid-encoded cpe. In vitro, the purified CPE protein binds to a receptor and forms pores, exerting a cytotoxic activity in epithelial cells. However, it remains unclear if CPE is indispensable for C. perfringens cytotoxicity. In this study, we examined the cytotoxicity of cpe-harboring C. perfringens isolates co-cultured with human intestinal epithelial Caco-2 cells. The FP strains showed severe cytotoxicity during sporulation and CPE production, but not during vegetative cell growth. While Caco-2 cells were intact during co-culturing with cpe-null mutant derivative of strain SM101 (a FP strain carrying a chromosomal cpe gene), the wild-type level cytotoxicity was observed with cpe-complemented strain. In contrast, both wild-type and cpe-null mutant derivative of the NFB strain F4969 induced Caco-2 cell death during both vegetative and sporulation growth. Collectively, the Caco-2 cell cytotoxicity caused by C. perfringens strain SM101 is considered to be exclusively dependent on CPE production, whereas some additional toxins should be involved in F4969-mediated in vitro cytotoxicity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Methicillin-susceptible Staphylococcus aureus endocarditis isolates are associated with clonal complex 30 genotype and a distinct repertoire of enterotoxins and adhesins.

    PubMed

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

    2011-09-01

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

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

    PubMed Central

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

    2011-01-01

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

  7. 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.

  8. Lanthanum inhibition of Vibrio cholerae and Escherichia coli enterotoxin-induced enterosorption and its effects on intestinal mucosa cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate levels.

    PubMed Central

    Leitch, G J; Amer, M S

    1975-01-01

    Several trivalent cations, including lanthanum (La3+), inhibited the secretion (enterosorption) induced by the enterotoxins of Vibrio cholerae and Escherichia coli in the rabbit ileum in vivo. High concentrations (greater than 10 mM) of La3+ were required to inhibit cholera enterotoxin (CE)-induced enterosorption, probably because of the adsorption of the La3+ often potentiated the CE-induced enterosorption. If luminal La3+ exposure followed CE exposure, some recovery of the enterosorptive response was observed. The longer the lag between the CE exposure and the La3+ exposure, the greater was the recovery of the enterosorptive response. Lanthanum inhibited HCO3- secretion more than Cl- secretion. By altering the luminal fluid pH at the time of La3+ exposure, it was found that La3+ was adsorbed to negatively charged luminal sites, having an apparent pK between 2.5 and 3.0. Although La3+ antagonized the enterosorptive response to CE, it mimicked rather than antagonized the cyclic adenosine 3',5'-monophosphate elevation and cyclic guanosine 3',5'-monophosphate depression induced by the toxin. It is therefore concluded that the La3+ inhibition of the CE-induced enterosorption must have occurred at a site following the generation of the cyclic nucleotides. Cholera enterotoxin caused complex time-dependent changes in the mucosal cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate levels, as revealed by studying tissue cyclic adenosine 3',5'-monophosphate/cyclic guanosine 3',5'-monophosphate ratios. The possible roles these two cyclic nucleotides may play in the pathogenesis of the cholera diarrhea are discussed. PMID:164410

  9. Effects of Small Molecule Calcium-Activated Chloride Channel Inhibitors on Structure and Function of Accessory Cholera Enterotoxin (Ace) of Vibrio cholerae

    PubMed Central

    Chatterjee, Tanaya; Sheikh, Irshad Ali; Chakravarty, Devlina; Chakrabarti, Pinak; Sarkar, Paramita; Saha, Tultul; Chakrabarti, Manoj K.; Hoque, Kazi Mirajul

    2015-01-01

    Cholera pathogenesis occurs due to synergistic pro-secretory effects of several toxins, such as cholera toxin (CTX) and Accessory cholera enterotoxin (Ace) secreted by Vibrio cholerae strains. Ace activates chloride channels stimulating chloride/bicarbonate transport that augments fluid secretion resulting in diarrhea. These channels have been targeted for drug development. However, lesser attention has been paid to the interaction of chloride channel modulators with bacterial toxins. Here we report the modulation of the structure/function of recombinant Ace by small molecule calcium-activated chloride channel (CaCC) inhibitors, namely CaCCinh-A01, digallic acid (DGA) and tannic acid. Biophysical studies indicate that the unfolding (induced by urea) free energy increases upon binding CaCCinh-A01 and DGA, compared to native Ace, whereas binding of tannic acid destabilizes the protein. Far-UV CD experiments revealed that the α-helical content of Ace-CaCCinh-A01 and Ace-DGA complexes increased relative to Ace. In contrast, binding to tannic acid had the opposite effect, indicating the loss of protein secondary structure. The modulation of Ace structure induced by CaCC inhibitors was also analyzed using docking and molecular dynamics (MD) simulation. Functional studies, performed using mouse ileal loops and Ussing chamber experiments, corroborate biophysical data, all pointing to the fact that tannic acid destabilizes Ace, inhibiting its function, whereas DGA stabilizes the toxin with enhanced fluid accumulation in mouse ileal loop. The efficacy of tannic acid in mouse model suggests that the targeted modulation of Ace structure may be of therapeutic benefit for gastrointestinal disorders. PMID:26540279

  10. Detection of target staphylococcal enterotoxin B antigen in orange juice and popular carbonated beverages using antibody-dependent antigen-capture assays.

    PubMed

    Principato, MaryAnn; Njoroge, Joyce M; Perlloni, Andrei; O' Donnell, Michael; Boyle, Thomas; Jones, Robert L

    2010-10-01

    There is a critical need for qualitative and quantitative methodologies that provide the rapid and accurate detection of food contaminants in complex food matrices. However, the sensitivity of the assay can be affected when antigen-capture is applied to certain foods or beverages that are extremely acidic. This study was undertaken to assess the effects of orange juice and popular carbonated soft drink upon the fidelity of antibody-based antigen-capture assays and to develop simple approaches that could rescue assay performance without the introduction of additional or extensive extraction procedures. We examined the effects of orange juice and a variety of popular carbonated soft drink beverages upon a quantitative Interleukin-2 (IL-2) enzyme-linked immunosorbent assay (ELISA) assay system and a lateral flow device (LFD) adapted for the detection of staphylococcal enterotoxin B (SEB) in foods. Alterations in the performance and sensitivity of the assay were directly attributable to the food matrix, and alterations in pH were especially critical. The results demonstrate that approaches such as an alteration of pH and the use of milk as a blocking agent, either singly or in combination, will partially rescue ELISA performance. The same approaches permit lateral flow to efficiently detect antigen. Practical Application: The authors present ways to rescue an ELISA assay compromised by acidity in beverages and show that either the alteration of pH, or the use of milk as a blocking agent are not always capable of restoring the assay to its intended efficiency. However, the same methods, when employed with lateral flow technology, are rapid and extremely successful.

  11. A functional antigen in a practical crop: LT-B producing maize protects mice against Escherichia coli heat labile enterotoxin (LT) and cholera toxin (CT).

    PubMed

    Chikwamba, Rachel; Cunnick, Joan; Hathaway, Diane; McMurray, Jennifer; Mason, Hugh; Wang, Kan

    2002-10-01

    We have produced a functional heat labile enterotoxin (LT-) B subunit of Escherichia coli in maize. LT-B is a multimeric protein that presents an ideal model for an edible vaccine, displaying stability in the gut and inducing mucosal and systemic immune responses. Transgenic maize was engineered to synthesize the LT-B polypeptides, which assembled into oligomeric structures with affinity for G(M1) gangliosides. We orally immunized BALB/c mice by feeding transgenic maize meal expressing LT-B or non-transgenic maize meal spiked with bacterial LT-B. Both treatments stimulated elevated IgA and IgG antibodies against LT-B and the closely related cholera toxin B subunit (CT-B) in serum, and elevated IgA in fecal pellets. The transgenic maize induced a higher anti-LT-B and anti-CT-B mucosal and serum IgA response compared to the equivalent amount of bacterial LT-B spiked into maize. Following challenge by oral administration of the diarrhea inducing toxins LT and CT, transgenic maize-fed mice displayed reduced fluid accumulation in the gut compared to non-immunized mice. Moreover, the gut to carcass ratio of immunized mice was not significantly different from the PBS (non-toxin) challenged control group. We concluded that maize-synthesized LT-B had features of the native bacterial LT-B such as molecular weight, G(M1) binding ability, and induction of serum and mucosal immunity. We have demonstrated that maize, a major food and feed ingredient, can be efficiently transformed to produce, accumulate, and store a fully assembled and functional candidate vaccine antigen.

  12. Bicarbonate and amino acids are co-germinants for spores of Clostridium perfringens type A isolates carrying plasmid-borne enterotoxin gene.

    PubMed

    Alnoman, Maryam; Udompijitkul, Pathima; Banawas, Saeed; Sarker, Mahfuzur R

    2018-02-01

    Clostridium perfringens type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe) are generally linked to food poisoning, while isolates carrying cpe on a plasmid (P-cpe) are associated with non-food-borne gastrointestinal diseases. Both C-cpe and P-cpe isolates can form metabolically dormant spores, which through germination process return to actively growing cells to cause diseases. In our previous study, we showed that only 3 out of 20 amino acids (aa) in phosphate buffer (pH 7.0) triggered germination of spores of P-cpe isolates (P-cpe spores). We now found that 14 out of 20 individual aa tested induced germination of P-cpe spores in the presence of bicarbonate buffer (pH 7.0). However, no significant spore germination was observed with bicarbonate (pH 7.0) alone, indicating that aa and bicarbonate are co-germinants for P-cpe spores. P-cpe strain F4969 gerKC spores did not germinate, and gerAA spores germinated extremely poorly as compared to wild-type and gerKA spores with aa-bicarbonate (pH 7.0) co-germinants. The germination defects in gerKC and gerAA spores were partially restored by complementing gerKC or gerAA spores with wild-type gerKC or gerAA, respectively. Collectively, this study identified aa-bicarbonate as a novel nutrient germinant for P-cpe spores and provided evidence that GerKC and GerAA play major roles in aa-bicarbonate induced germination. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2016-09-01

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

  14. Staphylococcal enterotoxin-like X (SElX) is a unique superantigen with functional features of two major families of staphylococcal virulence factors.

    PubMed

    Langley, Ries J; Ting, Yi Tian; Clow, Fiona; Young, Paul G; Radcliff, Fiona J; Choi, Jeong Min; Sequeira, Richard P; Holtfreter, Silva; Baker, Heather; Fraser, John D

    2017-09-01

    Staphylococcus aureus is an opportunistic pathogen that produces many virulence factors. Two major families of which are the staphylococcal superantigens (SAgs) and the Staphylococcal Superantigen-Like (SSL) exoproteins. The former are immunomodulatory toxins that induce a Vβ-specific activation of T cells, while the latter are immune evasion molecules that interfere with a wide range of innate immune defences. The superantigenic properties of Staphylococcal enterotoxin-like X (SElX) have recently been established. We now reveal that SElX also possesses functional characteristics of the SSLs. A region of SElX displays high homology to the sialyl-lactosamine (sLacNac)-specific binding site present in a sub-family of SSLs. By analysing the interaction of SElX with sLacNac-containing glycans we show that SElX has an equivalent specificity and host cell binding range to the SSLs. Mutation of key amino acids in this conserved region affects the ability of SElX to bind to cells of myeloid origin and significantly reduces its ability to protect S. aureus from destruction in a whole blood killing (WBK) assay. Like the SSLs, SElX is up-regulated early during infection and is under the control of the S. aureus exotoxin expression (Sae) two component gene regulatory system. Additionally, the structure of SElX in complex with the sLacNac-containing tetrasaccharide sialyl Lewis X (sLeX) reveals that SElX is a unique single-domain SAg. In summary, SElX is an 'SSL-like' SAg.

  15. Human Papillomavirus Virus-Like Particles Are Efficient Oral Immunogens when Coadministered with Escherichia coli Heat-Labile Enterotoxin Mutant R192G or CpG DNA

    PubMed Central

    Gerber, S.; Lane, C.; Brown, D. M.; Lord, E.; DiLorenzo, M.; Clements, J. D.; Rybicki, E.; Williamson, A.-L.; Rose, R. C.

    2001-01-01

    Certain human papillomaviruses (HPVs) cause most cervical cancer, which remains a significant source of morbidity and mortality among women worldwide. HPV recombinant virus-like particles (VLPs) are promising vaccine candidates for controlling anogenital HPV disease and are now being evaluated as a parenteral vaccine modality in human subjects. Vaccines formulated for injection generally are more costly, more difficult to administer, and less acceptable to recipients than are mucosally administered vaccines. Since oral delivery represents an attractive alternative to parenteral injection for large-scale human vaccination, the oral immunogenicity of HPV type 11 (HPV-11) VLPs in mice was previously investigated; it was found that a modest systemic neutralizing antibody response was induced (R. C. Rose, C. Lane, S. Wilson, J. A. Suzich, E. Rybicki, and A. L. Williamson, Vaccine 17:2129–2135, 1999). Here we examine whether VLPs of other genotypes may also be immunogenic when administered orally and whether mucosal adjuvants can be used to enhance VLP oral immunogenicity. We show that HPV-16 and HPV-18 VLPs are immunogenic when administered orally and that oral coadministration of these antigens with Escherichia coli heat-labile enterotoxin (LT) mutant R192G (LT R192G) or CpG DNA can significantly improve anti-VLP humoral responses in peripheral blood and in genital mucosal secretions. Our results also suggest that LT R192G may be superior to CpG DNA in this ability. These findings support the concept of oral immunization against anogenital HPV disease and suggest that clinical studies involving this approach may be warranted. PMID:11312347

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

    PubMed Central

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

    2015-01-01

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

  17. Application of IgY to sandwich enzyme-linked immunosorbent assays, lateral flow devices, and immunopillar chips for detecting staphylococcal enterotoxins in milk and dairy products.

    PubMed

    Jin, Wanchun; Yamada, Keiko; Ikami, Mai; Kaji, Noritada; Tokeshi, Manabu; Atsumi, Yusuke; Mizutani, Makoto; Murai, Atsushi; Okamoto, Akira; Namikawa, Takao; Baba, Yoshinobu; Ohta, Michio

    2013-03-01

    Staphylococcal enterotoxins (SEs), produced by Staphylococcus aureus, are a major cause of staphylococcal food poisoning. Traditionally, sandwich enzyme-linked immunosorbent assay (ELISA) and reverse passive latex agglutination with rabbit antibody IgG have been used to detect SEs. However, most of these kits require a long processing time and there is a risk of false-positive results since IgG reacts nonspecifically with protein A produced by S. aureus. In this study, we prepared antienterotoxin chicken IgY antibodies specific for each SE (SEA to SEE) without reaction to protein A, which enabled a drastic reduction in nonspecific reactions. ELISAs, lateral flow device (LFDs), and IgY-based immunopillar chips were developed for SE detection. All the ELISAs developed were as sensitive as commercially available kits. The SEs in milk were successfully detected by the ELISAs, LFDs, and immunopillar chips without any sample pretreatment. The LFD could detect SEA even at the low concentration of 0.2 ng/ml within 15 min in milk. The detection limit of the immunopillar chips for the SEs ranged from 0.01 to 0.1 ng/ml in milk; the SEs were detected within 12 min and specialized skills were not required. The ELISA and LFD detected SEA in dairy products artificially contaminated with S. aureus, including ice cream, yogurt, and café au lait, in a dose-dependent manner. In conclusion, IgY allows highly specific detection of SEs, and ELISAs, LFDs, and immunopillar chips should be useful tools for screening SEs in milk and dairy products. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Biodegradable and biocompatible poly(DL-lactide-co-glycolide) microspheres as an adjuvant for staphylococcal enterotoxin B toxoid which enhances the level of toxin-neutralizing antibodies.

    PubMed Central

    Eldridge, J H; Staas, J K; Meulbroek, J A; Tice, T R; Gilley, R M

    1991-01-01

    Microspheres composed of biocompatible, biodegradable poly(DL-lactide-co-glycolide) (DL-PLG) and staphylococcal enterotoxin B (SEB) toxoid were evaluated as a vaccine delivery system when subcutaneously injected into mice. As measured by circulating immunoglobulin G (IgG) antitoxin titers, the delivery of SEB toxoid via DL-PLG microspheres, 1 to 10 microns in diameter, induced an immune response which was approximately 500 times that seen with nonencapsulated toxoid. The kinetics, magnitude, and duration of the antitoxin response induced with microencapsulated toxoid were similar to those obtained when an equal toxoid dose was administered as an emulsion with complete Freund adjuvant. However, the microspheres did not induce the inflammation and granulomata formation seen with complete Freund adjuvant. The adjuvant activity of the microspheres was not dependent on the superantigenicity of SEB toxin and was equally effective at potentiating circulating IgG antitrinitrophenyl levels in response to microencapsulated trinitrophenyl-keyhole limpet hemocyanin. Empty DL-PLG microspheres were not mitogenic, and SEB toxoid injected as a mixture with empty DL-PLG microspheres was no more effective as an immunogen than toxoid alone. Antigen-containing microspheres 1 to 10 microns in diameter exhibited stronger adjuvant activity than those greater than 10 microns, which correlated with the delivery of the 1- to 10-microns, but not the greater than 10-microns, microspheres into the draining lymph nodes within macrophages. The antibody response induced through immunization with microencapsulated SEB toxoid was protective against the weight loss and splenic V beta 8+ T-cell expansion induced by intravenous toxin administration. These results show that DL-PLG microsphere vaccine delivery systems, which are composed of pharmaceutically acceptable components, possess a strong adjuvant activity for their encapsulated antigens. PMID:1879922

  19. Intestinal receptor for heat-stable enterotoxin of Escherichia coli is tightly coupled to a novel form of particulate guanylate cyclase.

    PubMed Central

    Waldman, S A; Kuno, T; Kamisaki, Y; Chang, L Y; Gariepy, J; O'Hanley, P; Schoolnik, G; Murad, F

    1986-01-01

    A novel form of particulate guanylate cyclase tightly coupled by cytoskeletal components to receptors for heat-stable enterotoxin (ST) produced by Escherichia coli can be found in membranes from rat intestinal mucosa. Intestinal particulate guanylate cyclase was resistant to solubilization with detergent alone, with only 30% of the total enzyme activity being extracted with Lubrol-PX. Under similar conditions, 70% of this enzyme was solubilized from rat lung membranes. The addition of high concentrations of sodium chloride to the extraction buffer resulted in greater solubilization of particulate guanylate cyclase from intestinal membranes. Although extraction of intestinal membranes with detergent and salt resulted in greater solubilization of guanylate cyclase, a small fraction of the enzyme activity remained associated with the particulate fraction. This activity was completely resistant to solubilization with a variety of detergents and chaotropes. Particulate guanylate cyclase and the ST receptor solubilized by detergent retained their abilities to produce cyclic GMP and bind ST, respectively. However, ST failed to activate particulate guanylate cyclase in detergent extracts. In contrast, guanylate cyclase resistant to solubilization remained functional and coupled to the ST receptor since enzyme activation by ST was unaffected by various extraction procedures. The possibility that the ST receptor and particulate guanylate cyclase were the same molecule was explored. ST binding and cyclic GMP production were separated by affinity chromatography on GTP-agarose. Similarly, guanylate cyclase migrated as a 300,000-dalton protein, while the ST receptor migrated as a 240,000-dalton protein on gel filtration chromatography. Also, thiol-reactive agents such as cystamine and N-ethylmaleimide inhibited guanylate cyclase activation by ST, with no effect on receptor binding of ST. These data suggest that guanylate cyclase and the ST receptor are independent proteins

  20. Salmonella enterica Serovar Enteritidis Ghosts Carrying the Escherichia coli Heat-Labile Enterotoxin B Subunit Are Capable of Inducing Enhanced Protective Immune Responses

    PubMed Central

    Jawale, Chetan V.

    2014-01-01

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

  1. MHC Class II Activation and Interferon-γ Mediate the Inhibition of Neutrophils and Eosinophils by Staphylococcal Enterotoxin Type A (SEA).

    PubMed

    Ferreira-Duarte, Ana P; Pinheiro-Torres, Anelize S; Anhê, Gabriel F; Condino-Neto, Antônio; Antunes, Edson; DeSouza, Ivani A

    2017-01-01

    Staphylococcal enterotoxins are classified as superantigens that act by linking T-cell receptor with MHC class II molecules, which are expressed on classical antigen-presenting cells (APC). Evidence shows that MHC class II is also expressed in neutrophils and eosinophils. This study aimed to investigate the role of MHC class II and IFN-γ on chemotactic and adhesion properties of neutrophils and eosinophils after incubation with SEA. Bone marrow (BM) cells obtained from BALB/c mice were resuspended in culture medium, and incubated with SEA (3-30 ng/ml; 1-4 h), after which chemotaxis and adhesion were evaluated. Incubation with SEA significantly reduced the chemotactic and adhesive responses in BM neutrophils activated with IL-8 (200 ng/ml). Likewise, SEA significantly reduced the chemotactic and adhesive responses of BM eosinophils activated with eotaxin (300 ng/ml). The inhibitory effects of SEA on cell chemotaxis and adhesion were fully prevented by prior incubation with an anti-MHC class II blocking antibody (2 μg/ml). SEA also significantly reduced the intracellular Ca 2+ levels in IL-8- and eotaxin-activated BM cells. No alterations of MAC-1, VLA4, and LFA-1α expressions were observed after SEA incubation. In addition, SEA elevated by 3.5-fold ( P < 0.05) the INF-γ levels in BM cells. Incubation of BM leukocytes with IFN-γ (10 ng/ml, 2 h) reduced both neutrophil and eosinophil chemotaxis and adhesion, which were prevented by prior incubation with anti-MHC class II antibody (2 μg/ml). In conclusion, SEA inhibits neutrophil and eosinophil by MHC class II-dependent mechanism, which may be modulated by concomitant release of IFN-γ.

  2. Molecular Typing and Virulence Gene Profiles of Enterotoxin Gene Cluster (egc)-Positive Staphylococcus aureus Isolates Obtained from Various Food and Clinical Specimens.

    PubMed

    Song, Minghui; Shi, Chunlei; Xu, Xuebing; Shi, Xianming

    2016-11-01

    The enterotoxin gene cluster (egc) has been proposed to contribute to the Staphylococcus aureus colonization, which highlights the need to evaluate genetic diversity and virulence gene profiles of the egc-positive population. Here, a total of 43 egc-positive isolates (16.2%) were identified from 266 S. aureus isolates that were obtained from various food and clinical specimens in Shanghai. Seven different egc profiles were found based on the polymerase chain reaction (PCR) result for egc genes. Then, these 43 egc-positive isolates were further typed by multilocus sequence typing, pulsed-field gel electrophoresis (PFGE), multiple-locus variable-number tandem-repeat analysis (MLVA), and accessory gene regulatory (agr) typing. It showed that the 43 egc-positive isolates displayed 17 sequence types, 28 PFGE patterns, 29 MLVA types, and 4 agr types, respectively. Among them, the dominant clonal lineage was CC5-agr II (48.84%). Thirty toxin and 20 adhesion-associated genes were detected by PCR in egc-positive isolates. Notably, invasive toxin genes showed a high prevalence, such as 76.7% for Panton-Valentine leukocidin encoding genes, 27.9% for sec, and 23.3% for tsst-1. Most of the examined adhesion-associated genes were found to be conserved (76.7-100%), whereas the fnbB gene was only found in 8 (18.6%) isolates. In addition, 33 toxin gene profiles and 13 adhesion gene profiles were identified, respectively. Our results imply that isolates belonging to the same clonal lineage harbored similar adhesion gene profiles but diverse toxin gene profiles. Overall, the high prevalence of invasive virulence genes increases the potential risk of egc-positive isolates in S. aureus infection.

  3. Immunogenicity of recombinant Lactobacillus casei-expressing F4 (K88) fimbrial adhesin FaeG in conjunction with a heat-labile enterotoxin A (LTAK63) and heat-labile enterotoxin B (LTB) of enterotoxigenic Escherichia coli as an oral adjuvant in mice.

    PubMed

    Yu, M; Qi, R; Chen, C; Yin, J; Ma, S; Shi, W; Wu, Y; Ge, J; Jiang, Y; Tang, L; Xu, Y; Li, Y

    2017-02-01

    The aims of this study were to develop an effective oral vaccine against enterotoxigenic Escherichia coli (ETEC) infection and to design new and more versatile mucosal adjuvants. Genetically engineered Lactobacillus casei strains expressing F4 (K88) fimbrial adhesin FaeG (rLpPG-2-FaeG) and either co-expressing heat-labile enterotoxin A (LTA) subunit with an amino acid mutation associated with reduced virulence (LTAK63) and a heat-labile enterotoxin B (LTB) subunit of E. coli (rLpPG-2-LTAK63-co-LTB) or fused-expressing LTAK63 and LTB (rLpPG-2-LTAK63-fu-LTB) were constructed. The immunogenicity of rLpPG-2-FaeG in conjunction with rLpPG-2-LTAK63-co-LTB or rLpPG-2-LTAK63-fu-LTB as an orally administered mucosal adjuvant in mice was evaluated. Results showed that the levels of FaeG-specific serum IgG and mucosal sIgA, as well as the proliferation of lymphocytes, were significantly higher in mice orally co-administered rLpPG-2-FaeG and rLpPG-2-LTAK63-fu-LTB compared with those administered rLpPG-2-FaeG alone, and were lower than those co-administered rLpPG-2-FaeG and rLpPG-2-LTAK63-co-LTB. Moreover, effective protection was observed after challenge with F4+ ETEC strain CVCC 230 in mice co-administered rLpPG-2-FaeG and rLpPG-2-LTAK63-co-LTB or rLpPG-2-FaeG and rLpPG-2-LTAK63-fu-LTB group compared with those that received rLpPG-2-FaeG alone. rLpPG-2-FaeG showed greater immunogenicity in combination with LTAK63 and LTB as molecular adjuvants. Recombinant Lactobacillus provides a promising platform for the development of vaccines against F4+ ETEC infection. © 2016 The Society for Applied Microbiology.

  4. Anoctamin 6 Contributes to Cl- Secretion in Accessory Cholera Enterotoxin (Ace)-stimulated Diarrhea: AN ESSENTIAL ROLE FOR PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE (PIP2) SIGNALING IN CHOLERA.

    PubMed

    Aoun, Joydeep; Hayashi, Mikio; Sheikh, Irshad Ali; Sarkar, Paramita; Saha, Tultul; Ghosh, Priyanka; Bhowmick, Rajsekhar; Ghosh, Dipanjan; Chatterjee, Tanaya; Chakrabarti, Pinak; Chakrabarti, Manoj K; Hoque, Kazi Mirajul

    2016-12-23

    Accessory cholera enterotoxin (Ace) of Vibrio cholerae has been shown to contribute to diarrhea. However, the signaling mechanism and specific type of Cl - channel activated by Ace are still unknown. We have shown here that the recombinant Ace protein induced I Cl of apical plasma membrane, which was inhibited by classical CaCC blockers. Surprisingly, an Ace-elicited rise of current was neither affected by ANO1 (TMEM16A)-specific inhibitor T16A (inh) -AO1(TAO1) nor by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker, CFTR inh-172. Ace stimulated whole-cell current in Caco-2 cells. However, the apical I Cl was attenuated by knockdown of ANO6 (TMEM16F). This impaired phenotype was restored by re-expression of ANO6 in Caco-2 cells. Whole-cell patch clamp recordings of ANO currents in HEK293 cells transiently expressing mouse ANO1-mCherry or ANO6-GFP confirmed that Ace induced Cl - secretion. Application of Ace produced ANO6 but not the ANO1 currents. Ace was not able to induce a [Ca 2+ ] i rise in Caco-2 cells, but cellular abundance of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) increased. Identification of the PIP 2 -binding motif at the N-terminal sequence among human and mouse ANO6 variants along with binding of PIP 2 directly to ANO6 in HEK293 cells indicate likely PIP 2 regulation of ANO6. The biophysical and pharmacological properties of Ace stimulated Cl - current along with intestinal fluid accumulation, and binding of PIP 2 to the proximal KR motif of channel proteins, whose mutagenesis correlates with altered binding of PIP 2 , is comparable with ANO6 stimulation. We conclude that ANO6 is predominantly expressed in intestinal epithelia, where it contributes secretory diarrhea by Ace stimulation in a calcium-independent mechanism of RhoA-ROCK-PIP 2 signaling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. 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

  6. 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 (J H +) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and J H + (~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 J H + 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 J H + 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

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

    SciTech Connect

    Busbee, Philip B.; Nagarkatti, Mitzi; Nagarkatti, Prakash S., E-mail: prakash@mailbox.sc.edu

    2014-01-01

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

  8. Sensitive quantification of Clostridium perfringens in human feces by quantitative real-time PCR targeting alpha-toxin and enterotoxin genes.

    PubMed

    Nagpal, Ravinder; Ogata, Kiyohito; Tsuji, Hirokazu; Matsuda, Kazunori; Takahashi, Takuya; Nomoto, Koji; Suzuki, Yoshio; Kawashima, Kazunari; Nagata, Satoru; Yamashiro, Yuichiro

    2015-10-19

    Clostridium perfringens is a widespread pathogen, but the precise quantification of this subdominant gut microbe remains difficult due to its low fecal count (particularly in asymptomatic subjects) and also due to the presence of abundant polymerase-inhibitory substances in human feces. Also, information on the intestinal carriage of toxigenic C. perfringens strains in healthy subjects is sparse. Therefore, we developed a sensitive quantitative real-time PCR assays for quantification of C. perfringens in human feces by targeting its α-toxin and enterotoxin genes. To validate the assays, we finally observed the occurrence of α-toxigenic and enterotoxigenic C. perfringens in the fecal microbiota of healthy Japanese infants and young adults. The plc-specific qPCR assay was newly validated, while primers for 16S rRNA and cpe genes were retrieved from literature. The assays were validated for specificity and sensitivity in pre-inoculated fecal samples, and were finally applied to quantify C. perfringens in stool samples from apparently healthy infants (n 124) and young adults (n 221). The qPCR assays were highly specific and sensitive, with a minimum detection limit of 10(3) bacterial cells/g feces. Alpha-toxigenic C. perfringens was detected in 36% infants and 33% adults, with counts ranging widely (10(3)-10(7) bacterial cells/g). Intriguingly, the mean count of α-toxigenic C. perfringens was significantly higher in infants (6.0±1.5 log10 bacterial cells/g), as compared to that in adults (4.8±1.2). Moreover, the prevalence of enterotoxigenic C. perfringens was also found to be significantly higher in infants, as compared to that in adults. The mean enterotoxigenic C. perfringens count was 5.9±1.9 and 4.8±0.8 log10 bacterial cells/g in infants and adults, respectively. These data indicate that some healthy infants and young adults carry α-toxigenic and enterotoxigenic C. perfringens at significant levels, and may be predisposed to related diseases. Thus, high

  9. 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

  10. [Escherichia coli heat-labile enterotoxin B subunit enhances the immune response against canine parvovirus VP2 in mice immunized by VP2 DNA vaccine].

    PubMed

    Han, Dongmei; Zhong, Fei; Li, Xiujin; Wang, Wei; Wang, Xingxing; Pan, Sumin

    2011-01-01

    To investigate the effect of Escherichia coli heat-labile enterotoxin (LT) B subunit (LTB) gene on canine parvovirus (CPV) VP2 gene vaccine. The LTB gene was amplified by PCR from genomic DNA of E. coli 44815 strain. The VP2-70 fragment (210 bp) encoding major epitope of VP2 (70 amino acids) was amplified by PCR from a plasmid encoding VP2 gene. VP2-70 and LTB genes were inserted into the eukaryotic vector to construct VP2-70 gene,LTB gene and VP2-70-LTB fused gene vectors. The mice were immunized with VP2-70 vector, VP2-70-LTB fused vector, or VP2-70 vector plus LTB vector, respectively. The antibody titers at the different time were measured by using ELISA method. The spleen lymphocyte proliferation activity was analyzed by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The sequence of VP2-70 and LTB genes was identified. The recombinant VP2-70 and LTB proteins could be expressed in HEK293T cells in a secretory manner. The mice immunized with VP2-70 vector, VP2-70-LTB vector or VP2-70 vector plus LTB vector could generate the specific antibody against VP2 protein. The antibody titer immunized with VP2-70-LTB vector reached 1:5120 at 35 d post immunization, significantly higher than that of other two groups (P < 0.01). For antibody isotype analysis, the IgG1 isotype antibody titers in all test groups were significantly higher than of IgG2a (P < 0.01). The high-level spleen lymphocyte stimulation index was observed in the three test groups under the stimulation with Con A, higher than that in control groups (P < 0.01). LTB gene could enhance the humoral immune response of CPV VP2 gene vaccine in mice.

  11. 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, E-mail: cody@hwi.buffalo.edu; University at Buffalo, 700 Ellicott Street, Buffalo, NY 14203; Pace, Jim

    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}, butmore » 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

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

    PubMed

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

    2011-04-01

    The major objective of this study was to determine the influence of the initial headspace and dissolved O(2) level and vacuum packaging on growth and diarrhoeal enterotoxin production by Bacillus weihenstephanensis on potato based ready-to-eat food products. In general, the lower the initial headspace or dissolved O(2) level the slower the maximum growth rate (μ(max), log(10) CFU g(-1) d(-1)), the longer the lag phase duration (λ, d) and the smaller the maximum population density (N(max), log(10) CFU g(-1)) became. The slowest μ(max), the longest λ and the smallest N(max) were generally found for growth under vacuum packaging. This implies shorter shelf-lives will occur at higher initial headspace or dissolved O(2) levels as the growth of B. weihenstephanensis to the infective dose of 10(5) CFU g(-1) in such atmospheres takes a shorter time. Significant consumption of dissolved O(2) only occurred when growth shifted from the lag to the exponential phase and growth generally transitioned from the exponential to the stationary phase when the dissolved O(2) levels fell below ca. 75 ppb. Diarrhoeal enterotoxin production (determined via detection of the L2 component of haemolytic BL) was similar for growth under initial headspace O(2) levels of 1-20.9%, and was only reduced when growth took place under vacuum packaging. The reduction in L2 production when growth took place under vacuum was most probably related to the low final cell densities observed under this condition. Both growth and L2 production were inhibited over a 32-day incubation period at 7 °C by 40% CO(2) irrespective of the headspace or dissolved O(2) levels. The results illustrate the importance of residual O(2) and CO(2) on the shelf-stability and safety of modified atmosphere packaged potato based ready-to-eat food products with regards to B. weihenstephanensis. Copyright © 2010 Elsevier Ltd. All rights reserved.

  13. 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.

  14. Bacteroides fragilis Enterotoxin Induces Formation of Autophagosomes in Endothelial Cells but Interferes with Fusion with Lysosomes for Complete Autophagic Flux through a Mitogen-Activated Protein Kinase-, AP-1-, and C/EBP Homologous Protein-Dependent Pathway.

    PubMed

    Ko, Su Hyuk; Jeon, Jong Ik; Myung, Hyun Soo; Kim, Young-Jeon; Kim, Jung Mogg

    2017-10-01

    Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), plays an essential role in mucosal inflammation. Although autophagy contributes to the pathogenesis of diverse infectious diseases, little is known about autophagy in ETBF infection. This study was conducted to investigate the role of BFT in the autophagic process in endothelial cells (ECs). Stimulation of human umbilical vein ECs (HUVECs) with BFT increased light chain 3 protein II (LC3-II) conversion from LC3-I and protein expression of p62, Atg5, and Atg12. In addition, BFT-exposed ECs showed increased indices of autophagosomal fusion with lysosomes such as LC3-lysosome-associated protein 2 (LAMP2) colocalization and the percentage of red vesicles monitored by the expression of dual-tagged LC3B. BFT also upregulated expression of C/EBP homologous protein (CHOP), and inhibition of CHOP significantly increased indices of autophagosomal fusion with lysosomes. BFT activated an AP-1 transcription factor, in which suppression of AP-1 activity significantly downregulated CHOP and augmented autophagosomal fusion with lysosomes. Furthermore, suppression of Jun N-terminal protein kinase (JNK) mitogen-activated protein kinase (MAPK) significantly inhibited the AP-1 and CHOP signals, leading to an increase in autophagosomal fusion with lysosomes in BFT-stimulated ECs. These results suggest that BFT induced accumulation of autophagosomes in ECs, but activation of a signaling pathway involving JNK, AP-1, and CHOP may interfere with complete autophagy. Copyright © 2017 American Society for Microbiology.

  15. 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 Central

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

    2017-01-01

    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. PMID:27994101

  16. 3,3′-Diindolylmethane Ameliorates Staphylococcal Enterotoxin B–Induced Acute Lung Injury through Alterations in the Expression of MicroRNA that Target Apoptosis and Cell-Cycle Arrest in Activated T Cells

    PubMed Central

    Elliott, David M.; Nagarkatti, Mitzi

    2016-01-01

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

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

    PubMed Central

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

    2007-01-01

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

  18. 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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Byrd, Wyatt; Boedeker, Edgar C

    2013-03-15

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

  1. Influence of Host Interleukin-10 Polymorphisms on Development of Traveler's Diarrhea Due to Heat-Labile Enterotoxin-Producing Escherichia coli in Travelers from the United States Who Are Visiting Mexico▿

    PubMed Central

    Flores, Jose; DuPont, Herbert L.; Lee, Stephanie A.; Belkind-Gerson, Jaime; Paredes, Mercedes; Mohamed, Jamal A.; Armitige, Lisa Y.; Guo, Dong-Chuan; Okhuysen, Pablo C.

    2008-01-01

    Up to 60% of U.S. visitors to Mexico develop traveler's diarrhea (TD), mostly due to enterotoxigenic Escherichia coli (ETEC) strains that produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. Distinct single-nucleotide polymorphisms (SNPs) within the interleukin-10 (IL-10) promoter have been associated with high, intermediate, or low production of IL-10. We conducted a prospective study to investigate the association of SNPs in the IL-10 promoter and the occurrence of TD in ETEC LT-exposed travelers. Sera from U.S. travelers to Mexico collected on arrival and departure were studied for ETEC LT seroconversion by using cholera toxin as the antigen. Pyrosequencing was performed to genotype IL-10 SNPs. Stools from subjects who developed diarrhea were also studied for other enteropathogens. One hundred twenty-one of 569 (21.3%) travelers seroconverted to ETEC LT, and among them 75 (62%) developed diarrhea. Symptomatic seroconversion was more commonly seen in subjects who carried a genotype producing high levels of IL-10; it was seen in 83% of subjects with the GG genotype versus 54% of subjects with the AA genotype at IL-10 gene position −1082 (P, 0.02), in 71% of those with the CC genotype versus 33% of those with the TT genotype at position −819 (P, 0.005), and in 71% of those with the CC genotype versus 38% of those with the AA genotype at position −592 (P, 0.02). Travelers with the GCC haplotype were more likely to have symptomatic seroconversion than those with the ATA haplotype (71% versus 38%; P, 0.002). Travelers genetically predisposed to produce high levels of IL-10 were more likely to experience symptomatic ETEC TD. PMID:18579697

  2. Influence of host interleukin-10 polymorphisms on development of traveler's diarrhea due to heat-labile enterotoxin-producing Escherichia coli in travelers from the United States who are visiting Mexico.

    PubMed

    Flores, Jose; DuPont, Herbert L; Lee, Stephanie A; Belkind-Gerson, Jaime; Paredes, Mercedes; Mohamed, Jamal A; Armitige, Lisa Y; Guo, Dong-Chuan; Okhuysen, Pablo C

    2008-08-01

    Up to 60% of U.S. visitors to Mexico develop traveler's diarrhea (TD), mostly due to enterotoxigenic Escherichia coli (ETEC) strains that produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. Distinct single-nucleotide polymorphisms (SNPs) within the interleukin-10 (IL-10) promoter have been associated with high, intermediate, or low production of IL-10. We conducted a prospective study to investigate the association of SNPs in the IL-10 promoter and the occurrence of TD in ETEC LT-exposed travelers. Sera from U.S. travelers to Mexico collected on arrival and departure were studied for ETEC LT seroconversion by using cholera toxin as the antigen. Pyrosequencing was performed to genotype IL-10 SNPs. Stools from subjects who developed diarrhea were also studied for other enteropathogens. One hundred twenty-one of 569 (21.3%) travelers seroconverted to ETEC LT, and among them 75 (62%) developed diarrhea. Symptomatic seroconversion was more commonly seen in subjects who carried a genotype producing high levels of IL-10; it was seen in 83% of subjects with the GG genotype versus 54% of subjects with the AA genotype at IL-10 gene position -1082 (P, 0.02), in 71% of those with the CC genotype versus 33% of those with the TT genotype at position -819 (P, 0.005), and in 71% of those with the CC genotype versus 38% of those with the AA genotype at position -592 (P, 0.02). Travelers with the GCC haplotype were more likely to have symptomatic seroconversion than those with the ATA haplotype (71% versus 38%; P, 0.002). Travelers genetically predisposed to produce high levels of IL-10 were more likely to experience symptomatic ETEC TD.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2005-10-25

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

  5. DNA adenine methyltransferase (Dam) controls the expression of the cytotoxic enterotoxin (act) gene of Aeromonas hydrophila via tRNA modifying enzyme-glucose-inhibited division protein (GidA).

    PubMed

    Erova, Tatiana E; Kosykh, Valeri G; Sha, Jian; Chopra, Ashok K

    2012-05-01

    Aeromonas hydrophila is both a human and animal pathogen, and the cytotoxic enterotoxin (Act) is a crucial virulence factor of this bacterium because of its associated hemolytic, cytotoxic, and enterotoxic activities. Previously, to define the role of some regulatory genes in modulating Act production, we showed that deletion of a glucose-inhibited division gene (gidA) encoding tRNA methylase reduced Act levels, while overproduction of DNA adenine methyltransferase (Dam) led to a concomitant increase in Act-associated biological activities of a diarrheal isolate SSU of A. hydrophila. Importantly, there are multiple GATC binding sites for Dam within an upstream sequence of the gidA gene and one such target site in the act gene upstream region. We showed the dam gene to be essential for the viability of A. hydrophila SSU, and, therefore, to better understand the interaction of the encoding genes, Dam and GidA, in act gene regulation, we constructed a gidA in-frame deletion mutant of Escherichia coli GM28 (dam(+)) and GM33 (∆dam) strains. We then tested the expressional activity of the act and gidA genes by using a promoterless pGlow-TOPO vector containing a reporter green fluorescent protein (GFP). Our data indicated that in GidA(+) strains of E. coli, constitutive methylation of the GATC site(s) by Dam negatively regulated act and gidA gene expression as measured by GFP production. However, in the ∆gidA strains, irrespective of the presence or absence of constitutively active Dam, we did not observe any alteration in the expression of the act gene signifying the role of GidA in positively regulating Act production. To determine the exact mechanism of how Dam and GidA influence Act, a real-time quantitative PCR (RT-qPCR) assay was performed. The analysis indicated an increase in gidA and act gene expression in the A. hydrophila Dam-overproducing strain, and these data matched with Act production in the E. coli GM28 strain. Thus, the extent of DNA methylation

  6. Investigation of ’Escherichia coli’ Enterotoxins

    DTIC Science & Technology

    1978-05-01

    gain in accordance with the specifications of mouse toxicity tests for Bordetella pertussis . Since the diseases in question are confined to the...usage of Gram-negative bac- teria in several parenteral vaccines (Vibrio cholerae, Bordetella per- tussis, and Salmonella typhosa) provides a precedent

  7. Enterotoxin Vaccine Delivery System With Bioadherence. Phase 1.

    DTIC Science & Technology

    1995-12-05

    Microencapsulation 33 Bioadhesive Biodegradable 16. PRICE CODE Perorally Controlled Delivery 17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19. SECURITY...this magnitude requires a delivery system configured with a bioadhesive polymer that integrates the surface of the microcapsules and the mucosa. SBIR...integrates the surface of the microcapsules and the mucosa. SBIR Phase I Program efforts focused on the development of the most feasible method(s) for

  8. Therapeutic Human Hyperimmune Polyclonal Antibodies Against Staphylococcal Enterotoxin B

    DTIC Science & Technology

    2008-05-01

    antibodies and to develop the required SOPs. Both goals have been achieved. Body Phase I Objective 1: Assemble a cohort of monkey sera from IBT’s...30 serum samples (2 ml each) from monkeys vaccinated either with adjuvant, or two different doses of STEBVax were collected. The monkey sera...originated from a concurrent GLP-safety study for STEBVax in which monkeys received four injections of STEBVax at 50 and 200 μg. However upon assay, the

  9. Immunological Relationship of Different Preparations of Coliform Enterotoxins

    PubMed Central

    Klipstein, Frederick A.; Engert, Richard F.

    1978-01-01

    Antisera raised in rabbits to ultrafiltrate toxin preparations containing either the heat-labile (LT) toxin form obtained from whole cell lysates or broth filtrates or the heat-stable (ST) toxin form prepared from broth filtrates from nontoxigenic and toxigenic strains of Escherichia coli and Klebsiella were examined for their ability to neutralize the secretory effect on water transport of these toxins in the rat jejunum as determined by the in vivo marker perfusion technique. Antisera to the heat-labile toxin derived from whole cell lysate preparations from nontoxigenic strains had no neutralizing effect. Antisera to both types of LT preparation from both toxigenic strains neutralized, with several exceptions, all of the homologous and heterologous LT toxins as well as a heat-labile toxin preparation derived from sequential ultrafiltration of cell-free whole cell lysates which had a defined molecular weight of between 30,000 and 100,000. These antisera also neutralized homologous and heterologous ST preparations obtained from broth filtrates, but they had no neutraliziṅg effect on low-molecular-weight, ST toxin material obtained during the sequential ultrafiltration of cell lysates. Antisera to ST prepared from broth filtrates had no neutralizing capacity against either LT or ST toxin preparations. These observations (i) indicate that the immunological relationship of E. coli and Klebsiella LT and ST toxins extends to antisera raised against LT prepared by several different methods, (ii) raise the possibility that, based on the response to antisera to LT, there may be several immunologically heterogeneous forms of low-molecular-weight ST toxin, and (c) confirm the lack of immunogenicity of ST. PMID:361578

  10. Magnetic and electronic properties of the Cu-substituted Weyl semimetal candidate ZrCo2Sn.

    PubMed

    Kushwaha, S K; Wang, Zhijun; Kong, Tai; Cava, Robert

    2018-01-04

    We report that the partial substitution of Cu for Co has a significant impact on the magnetic properties of the Heusler-phase Weyl fermion candidate ZrCo<sub>2</sub>Sn. Polycrystalline samples of ZrCo<sub>2-&lt;i>x&lt;/i></sub>Cu<sub>&lt;i>x</i></sub>Sn (&lt;i>x</i> = 0.0 to 1.0) exhibited a linearly decreasing ferromagnetic transition temperature and similarly decreasing saturated magnetic moment on increasing Cu substitution x. Materials with Cu contents near &lt;i>x</i> = 1 and several other quaternary materials synthesized at the same &lt;i>x</i> (ZrCo&lt;i>T</i>'Sn (&lt;i>T</i>' = Rh, Pd, Ni)) display what appears to be non-ferromagnetic magnetization behavior with spin glass characteristics. Electronic structure calculations suggest that the half-metallic nature of unsubstituted ZrCo<sub>2</sub>Sn is disrupted significantly by the Cu substitutions, leading to the breakdown of the magnetization vs. electron count guidelines usually followed by Heusler phases, and a more typical metallic non-spin-polarized electronic structure at high &lt;i>x</i>. © 2018 IOP Publishing Ltd.

  11. Thermal annealing and pressure effects on BaFe2-xCoxAs2 single crystals.

    PubMed

    Shin, Dongwon; Jung, Soon-Gil; Prathiba, G; Seo, Soonbeom; Choi, Ki-Young; Kim, Kee Hoon; Park, Tuson

    2017-11-26

    We investigate the pressure and thermal annealing effects on BaFe<sub>2-&lt;i>x&lt;/i></sub>Co<sub>&lt;i>x&lt;/i></sub>As<sub>2</sub> (Co-Ba122) single crystals with &lt;i>x</i> = 0.1 and 0.17 via electrical transport measurements. The thermal annealing treatment not only enhances the superconducting transition temperature (&lt;i>T&lt;/i><sub>c</sub>) from 9.6 to 12.7 K for &lt;i>x</i> = 0.1 and from 18.1 to 21.0 K for &lt;i>x</i> = 0.17, but also increases the antiferromagnetic transition temperature (&lt;i>T&lt;/i><sub>N</sub>). Simultaneous enhancement of &lt;i>T&lt;/i><sub>c</sub> and &lt;i>T&lt;/i><sub>N</sub> by the thermal annealing treatment indicates that thermal annealing could substantially improve the quality of the Co-doped Ba122 samples. Interestingly, &lt;i>T&lt;/i><sub>c</sub> of the Co-Ba122 compounds shows a scaling behavior with a linear dependence on the resistivity value at 290 K, irrespective of tuning parameters, such as chemical doping, pressure, and thermal annealing. These results not only provide an effective way to access the intrinsic properties of the BaFe<sub>2</sub>As<sub>2</sub> system, but also may shed a light on designing new materials with higher superconducting transition temperature. © 2017 IOP Publishing Ltd.

  12. Investigating energy deposition within cell populations using Monte Carlo simulations.

    PubMed

    Oliver, Patricia A K; Thomson, Rowan M

    2018-06-27

    In this work, we develop multicellular models of healthy and cancerous human soft tissues, which are used to investigate energy deposition in subcellular targets, quantify the microdosimetric spread in a population of cells, and determine how these results depend on model details. Monte Carlo (MC) tissue models combining varying levels of detail on different length scales are developed: microscopically-detailed regions of interest (>1500 explicitly-modelled cells) are embedded in bulk tissue phantoms irradiated by photons (20 keV to 1.25 MeV). Specific energy (&lt;i>z</i>; energy imparted per unit mass) is scored in nuclei and cytoplasm compartments using the EGSnrc user-code egs_chamber; specific energy mean, <&lt;i>z</i>>, standard deviation, &lt;i>σ&lt;/i><sub>&lt;i>z</i></sub>, and distribution, &lt;i>f&lt;/i>(&lt;i>z&lt;/i>,<i>D</i>), are calculated for a variety of macroscopic doses, &lt;i>D</i>. MC-calculated &lt;i>f&lt;/i>(&lt;i>z&lt;/i>,<i>D</i>) are compared with normal distributions having the same mean and standard deviation. For mGy doses, there is considerable variation in energy deposition (microdosimetric spread) throughout a cell population: &lt;i>e&lt;/i>.<i>g</i>., for 30 keV photons irradiating melanoma with 7.5 μm cell radius and 3 μm nuclear radius, &lt;i>σ&lt;/i><sub>&lt;i>z&lt;/i></sub>/<&lt;i>z</i>> for nuclear targets is 170%, and the fraction of nuclei receiving no energy deposition, &lt;i>f&lt;/i><sub>&lt;i>z</i>=0</sub>, is 0.31 for a dose of 10 mGy. If cobalt-60 photons are considered instead, then &lt;i>σ&lt;/i><sub>&lt;i>z&lt;/i></sub>/<&lt;i>z</i>> decreases to 84%, and &lt;i>f&lt;/i><sub>&lt;i>z</i>=0</sub> decreases to 0.036. These results correspond to randomly

  13. Expression of non-toxic mutant of Escherichia coli heat-labile enterotoxin in tobacco chloroplasts.

    PubMed

    Kang, Tae-Jin; Han, So-Chon; Kim, Mi-Young; Kim, Young-Sook; Yang, Moon-Sik

    2004-11-01

    Chloroplast transformation systems offer unique advantages in biotechnology, including high level of foreign gene expression, maternal inheritance, and polycistronic expression. We studied chloroplast expression of LTK63 (change Ser-->Lys at position 63 in the A subunit) which is the mutant of Escherichia coli heat-labile toxin. LTK63 is devoid of any toxic activity, but still retains its mucosal adjuvanticity. The LTK63 was cloned into chloroplast targeting vector and transformed to tobacco chloroplasts by particle bombardment. PCR and Southern blot analyses confirmed stable homologous recombination of the LTK63 gene into the chloroplast genome. The amount of LTK63 protein detected in tobacco chloroplasts was approximately 3.7% of the total soluble protein. The GM1-ganglioside binding assay confirmed that chloroplast-synthesized LTB of LTK63 binds to the intestinal membrane GM1-ganglioside receptor. Thus, the expression of LTK63 in chloroplasts provides a potential route toward the development of a plant-based edible vaccine for high expression system and environmentally friendly approach.

  14. Inhibition of Biological Activity of Staphylococcal Enterotoxin A (SEA) by Apple Juice and Apple Polyphenols

    USDA-ARS?s Scientific Manuscript database

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

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

    DTIC Science & Technology

    2015-05-28

    Chemokine (C-X-C motif ) ligand 10 − 77.7 − 53.6 − 14.4 AU138239 INDO Indoleamine- pyrrole 2,3 dioxygenase − 29.3 − 31.5 − 14.0 AV734258 CCL8 Chemokine...immunomodulatory enzyme indoleamine- pyrrole 2,3 dioxygenase (INDO), the growth factors CSF1 and CSF2 and other T-cell response genes. Using NSC...2.8 − 2.2 AK026053 BCL2 B-cell CLL/lymphoma 2 − 2.2 − 2.0 − 2.3 − 2.2 AB015331 INDO Indoleamine- pyrrole 2,3 dioxygenase − 2.1 − 29.3 − 2.2 − 31.5

  16. Intranasal Rapamycin Rescues Mice from Staphylococcal Enterotoxin B-Induced Shock

    DTIC Science & Technology

    2012-09-18

    PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) U.S. Army Medical Research Institute of...Infectious Diseases,Fort Detrick,MD,21702 8. PERFORMING ORGANIZATION REPORT NUMBER 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR...Student’s t-test. Statistical comparisons of survival data were performed by Fisher’s exact test with Stata software (Stata Corp., College Station, TX

  17. Bacillus anthracis Edema Toxin Inhibits Staphylococcus aureus Enterotoxin B Effects in Vitro: A Potential Protein Therapeutic?

    DTIC Science & Technology

    2005-10-01

    5). Inherent characteristics of edema toxin and other procaryotic adenylate cyclases from Bordetella pertussis, Pseudomonas aeruginosa, and Yersinia...by mouse peritoneal macrophages: the role of cellular cyclic AMP. Immunology 64:719–724. 12. Krakauer, T. 1999. Induction of CC chemokines in human

  18. Activation of MyD88 Signaling upon Staphylococcal Enterotoxin Binding to MHC Class II Molecules

    DTIC Science & Technology

    2011-01-20

    TCCTGTGGCATCCACGA- AACT-39; Reverse 59-GAAGCATTTGCGGTGGACGAT-39), TNF-a (Forward 59- CGG GAC GTG GAG CTG GCC GAG G- AG-39; Reverse 59-CAC CAG CTG GTT...Biol Chem 273: 12203–12209. 38. Gray P, Dunne A, Brikos C, Jefferies CA, Doyle SL, et al. (2006) MyD88 adapter-like (Mal) is phosphorylated by Bruton’s...stimulate nuclear translocation of PKC in B lymphocytes. Nature 327: 629–632. 42. Barr TA, Brown S, Mastroeni P, Gray D (2009) B cell intrinsic MyD88

  19. Protective Effect of Active Immunization with Purified Escherichia coli Heat-Labile Enterotoxin in Rats

    PubMed Central

    Klipstein, Frederick A.; Engert, Richard F.

    1979-01-01

    The protective effect of active immunization by different routes with a purified preparation of the polymyxin-release form of Escherichia coli heat-labile toxin was evaluated in rats. Immunized animals were challenged by placing toxin into ligated ileal loops at dosages which produced either 50% or the maximum secretory response in unimmunized rats. Immunization exclusively by the parenteral route yielded significant protection. Rats were also protected when parenteral priming was followed by boosting given either directly into the duodenum or perorally 2 h after intragastric cimetidine, but not when the peroral boosts were given with bicarbonate. Immunization administered entirely by the peroral route with cimetidine yielded protection but only when the immunizing dosage was fivefold greater than that found effective in the parenteral-peroral approach. Rats immunized exclusively by the parenteral route and those boosted perorally with cimetidine were also tested, and found to be protected, against challenge with viable organisms of strains that produce either heat-labile toxin alone or both heat-labile and heat-stable toxin, but they were not protected against a strain which produces just heat-stable toxin. Geometric mean serum antibody titers were increased by 16-fold or more over control values in those groups of rats in which protection was achieved, with the exception of those immunized exclusively by the peroral route. These observations demonstrate that (i) active immunization with purified E. coli heat-labile toxin results in significant protection against both this toxin as well as viable organisms which produce it, but not against viable strains which produce heat-stable toxin only, and (ii) concomitant ablation of gastric secretion by the use of cimetidine renders the peroral route of immunization effective. They suggest that prophylactic immunization against diarrheal disease caused by heat-labile toxin-producing strains of E. coli may be feasible in humans. PMID:378831

  20. Development of New Immunogens and a Controlled Release Delivery System for Oral Immunization Against Staphylococcal Enterotoxin B

    DTIC Science & Technology

    1991-12-06

    DSN 343-7322. N fjGARX\\R. GILBERT COL, MS Deputy Chief of Staff for Information Management REPRODUCTION QUALITY NOTICE This document is the best quality...1976. 35. Ogre, P.L.: Karzon, D.T. Distribution of poliovirus antibody in serum, nasopharymrt and alimentary tract folloving segmental limunization...sysemst, In.- Hisholl, DAI. ; Advances in human fertility and reproductive endocrinology. NWe York, Raven Press Books. Ltd; Vol. 2. 1963: 175-199. 52. Tic

  1. Mass production of somatic embryos expressing Escherichia coli heat-labile enterotoxin B subunit in Siberian ginseng.

    PubMed

    Kang, Tae-Jin; Lee, Won-Seok; Choi, Eun-Gyung; Kim, Jae-Whune; Kim, Bang-Geul; Yang, Moon-Sik

    2006-01-24

    The B subunit of Escherichia coli heat-labile toxin (LTB) is a potent mucosal immunogen and immunoadjuvant for co-administered antigens. In order to produce large scale of LTB for the development of edible vaccine, we used transgenic somatic embryos of Siberian ginseng, which is known as medicinal plant. When transgenic somatic embryos were cultured in 130L air-lift type bioreactor, they were developed to mature somatic embryos through somatic embryogenesis and contained approximately 0.36% LTB of the total soluble protein. Enzyme-linked immunosorbent assay indicated that the somatic embryo-synthesized LTB protein bound specifically to GM1-ganglioside, suggesting the LTB subunits formed active pentamers. Therefore, the use of the bioreactor system for expression of LTB proteins in somatic embryos allows for continuous mass production in a short-term period.

  2. Ingestion of transgenic carrots expressing the Escherichia coli heat-labile enterotoxin B subunit protects mice against cholera toxin challenge.

    PubMed

    Rosales-Mendoza, Sergio; Soria-Guerra, Ruth Elena; López-Revilla, Rubén; Moreno-Fierros, Leticia; Alpuche-Solís, Angel Gabriel

    2008-01-01

    Diarrheal diseases caused by Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) are worldwide health problems that might be prevented with vaccines based on edible plants expressing the B subunit from either the cholera toxin (CTB) or the E. coli heat labile toxin (LTB). In this work we analyzed the immunity induced in Balb/c mice by ingestion of three weekly doses of 10 mug of LTB derived from transgenic carrot material. Although the anti-LTB serum immunoglobulin G (IgG) and intestinal IgA antibody responses were higher with 10 mug-doses of pure bacterial recombinant LTB (rLTB), the transgenic carrot material also elicited significant serum and intestinal antibody responses. Serum anti-LTB IgG1 antibodies predominated over IgG2a antibodies, suggesting that mainly Th2 responses were induced. A decrease of intestinal fluid accumulation after cholera toxin challenge was observed in mice immunized with either rLTB or LTB-containing carrot material. These results demonstrate that ingestion of carrot-derived LTB induces antitoxin systemic and intestinal immunity in mice and suggest that transgenic carrots expressing LTB may be used as an effective edible vaccine against cholera and ETEC diarrhea in humans.

  3. Detection of Staphylococcus Aureus Enterotoxin A and B Genes with PCR-EIA and a Hand-Held Electrochemical Sensor

    DTIC Science & Technology

    2004-06-11

    Streptococcus pneumoniae 33400 Enterobacter cloaceae 49141 S. pyogenes 19615 E. aerogenes m10822 Vibrio cholerae N16961 Enterococcus durans 6056 Yersinia...identified. Thus the sensitivity for both assays was 100%. Of the 56 samples that lacked sea or seb genes, two false positives ( Enterobacter aerogenes ...Comanonas, Enterobacter , Enterococcus, Escherichia, Francisella, Haemophilus, Klebsiella, Listeria, Moraxella, Neisseria, Proteus, Pseudomonas, Salmonella

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

    DTIC Science & Technology

    2004-11-30

    Que, and A. S. Bayer. 2002. Pathogenesis of streptococcal and staphylococcal endocarditis . Infect . Dis. Clin. N. Am. 16:297–318. 26. Nabozny, G. H., J... INFECTION AND IMMUNITY, Apr. 2005, p. 2452–2460 Vol. 73, No. 4 0019-9567/05/$08.000 doi:10.1128/IAI.73.4.2452–2460.2005 Copyright © 2005, American... infections , including scarlet fever, pharyngitis, dermatitis, infectious ar- thritis, and toxic shock syndrome (2, 13, 18, 25, 31). These pathogenic bacteria

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

    SciTech Connect

    Gill, R.

    Epidemiological studies have linked consumption of n-3 PUFAs with a variety of beneficial health benefits, particularly with respect to putative anti-inflammatory effects. Unfortunately, many of these results remain somewhat controversial because in most instances there has not been a linkage to specific molecular mechanisms. For instance, dietary exposure to low levels of mercury has been shown to be damaging to neural development, but concomitant ingestion of n-3 PUFAs as occurs during consumption of fish, has been shown to counteract the detrimental effects. As the mechanisms mediating the neurotoxicity of environmental mercury are not fully delineated, it is difficult to conceptualizemore » a testable molecular mechanism explaining how n-3 PUFAs negate its neurotoxic effects. However, environmental exposure to mercury also has been linked to increased autoimmunity. By way of a molecular understanding of this immuno-toxic association, disruption of CD95 signaling is well established as a triggering factor for autoimmunity, and we have previously shown that environmentally relevant in vitro and dietary exposures to mercury interfere with CD95 signaling. In particular we have shown that activation of caspase 8, as well as downstream activation of caspase 3, in response to CD95 agonist stimulation is depressed by mercury. More recently we have shown in vitro that the n-3 PUFA docosahexaenoic acid counteracts the negative effect of mercury on CD95 signaling by restoring caspase activity. We hypothesized that concomitant ingestion of n-3 PUFAs with mercury might be protective from the immuno-toxic effects of mercury, as it is with mercury's neuro-toxic effects, and in the case of immuno-toxicity this would be related to restoration of CD95 signal strength. We now show that dietary ingestion of n-3 PUFAs generally promotes CD95 signaling by upregulating caspase 8 activation. Apart from accounting for the ability of n-3 PUFAs to specifically counteract autoimmune sequelae of mercury exposure, this novel finding for the first time suggests a testable molecular mechanism explaining the overall anti-inflammatory properties of n-3 PUFAs. - Highlights: • Dietary n-3 PUFAs counter Hg{sup 2+} immunotoxicity • Hg{sup 2+} interference with SEB-mediated signal transduction is ameliorated by n-3 PUFA rich diets. • Dietary n-3 PUFAs augment SEB-mediated activation of caspase 8 in vivo.« less

  6. Food compounds inhibit Staphylococcus aureus bacteria and the toxicity of Staphylococcus Enterotoxin A (SEA) associated with atopic dermatitis

    USDA-ARS?s Scientific Manuscript database

    Atopic dermatitis or eczema is characterized by skin rashes and itching is an inflammatory disease that affects 10-20% of children and 1-3% of adults. Staphylococcus aureus bacteria are present on the skin of nearly all patients with atopic dermatitis. Antibiotics that suppress colonization of S. au...

  7. Immunological Interrelationships Between Cholera Toxin and the Heat-Labile and Heat-Stable Enterotoxins of Coliform Bacteria

    PubMed Central

    Klipstein, Frederick A.; Engert, Richard F.

    1977-01-01

    Cholera toxin (CT) and the heat-labile (LT) toxin of Escherichia coli are known to share antigenic properties. The present study examined the immunological relationship of CT and the LT and heat-stable (ST) toxins of E. coli, Klebsiella pneumoniae, and Enterobacter cloacae. The neutralizing capacity of equine CT antiserum and of antiserum raised in rabbits to the LT toxin of the three species of coliform bacteria was evaluated by determining their capacity to inhibit the action of purified CT and semipurified ultrafiltration preparations of the coliform LT and ST toxins in inducing water secretion as assayed by the in vivo marker perfusion technique in the rat jejunum. One milliliter of antiserum to CT and to E. coli and Klebsiella LT completely neutralized the secretory action of each of these three toxins; effective serial dilutions of CT antiserum extended to 1 to 4, whereas those of the antisera to LT were limited to 1 to 2 in most instances. One milliliter of antiserum to E. cloacae LT partially neutralized each of the three coliform LT toxins; serial dilutions were inactive. Antiserum to E. cloacae LT did not neutralize CT. Antiserum to CT and to each of the three coliform LT toxins also had a weak neutralizing effect on the ST toxins of E. coli and Klebsiella, but they did not affect E. cloacae ST. Adsorption of the antiserum to CT and to each of the three LT toxins by incubation with a heat-inactivated preparation of either the homologous or a heterologous LT toxin completely abolished the neutralizing capacity of the antisera towards both LT and ST. These observations indicate that the immunological interrelationship of CT and E. coli LT extends to the LT toxins of Klebsiella and E. cloacae and, further, that these immunological properties are shared to a lesser extent by the ST toxins of E. coli and Klebsiella. PMID:332637

  8. Virulence factors associated with cytotoxic necrotizing factor type two in bovine diarrheic and septicemic strains of Escherichia coli.

    PubMed Central

    Oswald, E; de Rycke, J; Lintermans, P; van Muylem, K; Mainil, J; Daube, G; Pohl, P

    1991-01-01

    Forty-three bovine isolates of Escherichia coli producing a second type of cytotoxic necrotizing factor (CNF2) and three K-12 strains carrying different Vir plasmids coding for CNF2 were tested for the presence of several virulence factors. Most of the strains were serum resistant (79%), produced an aerobactin (70%), and adhered to calf villi (53%); some of them produced a colicin (32%) and a hemolysin (9%). These strains were also tested by a colony hybridization assay with gene probes for six toxins (classical heat-stable [STaP and STb] and heat-labile [LT-I and LT-IIa] enterotoxins and Shiga-like toxins [SLT-I and SLT-II]) and five adhesion factors (K99, K88, 987P, F17, and F41). Only two gene probes, LT-IIa (9%) and F17A (53%), hybridized with the CNF2 strains. However, antibodies raised against F17 fimbriae did not agglutinate the strains hybridizing with the F17A probe. In contrast, all except one of these strains adhered to calf villi. Interestingly, these two properties, F17A positivity and adherence to calf villi, were the only ones expressed by the K-12 strains carrying different Vir plasmids. In conclusion, this study confirmed that CNF2-producing strains are unrelated to previously described toxigenic E. coli strains and also demonstrated that in half of the strains the production of CNF2 was associated with an adhesion factor genetically related to, but different from, F17, which is more than likely encoded by Vir plasmids. PMID:1774259

  9. Rhombohedral R3c to orthorhombic Pnma phase transition induced by Y-doping in BiFeO3.

    PubMed

    Graf, Monica Elisabet; Di Napoli, Solange; Barral, Maria Andrea Andrea; Saleh Medina, Leila; Negri, R Martín; Sepliarsky, Marcelo; Llois, Ana María

    2018-05-23

    In this work we study, by means of &lt;i>ab initio&lt;/i> calculations, the structural, electronic and magnetic properties of Y-doped BiFeO<sub>3</sub> compounds. We determine that there is a morphotropic phase boundary at an yttrium concentration of (18 ± 2)%, where the structure changes from &lt;i>R3c&lt;/i> to &lt;i>Pnma</i>. This structural transition is driven by the chemical pressure induced by the dopant. By analyzing the evolution of the oxygen octahedral tilts we find an enhanced antiferrodistortive distortion when increasing the Y-doping, together with a reduction of the ferroelectric distorsion, that gives rise to a smaller value of the electric polarization. These cooperative effects should lead to a larger canting of the Fe magnetic moments and to a larger ferromagnetic response in the &lt;i>R3c&lt;/i> phase, as it is observed in the experiments. . © 2018 IOP Publishing Ltd.

  10. The effects of optical sensor-tissue separation in endocavitary photoplethysmography.

    PubMed

    Patel, Zaibaa; Thaha, Mohamed A; Kyriacou, Panayiotis A

    2018-06-12

    &lt;i>Objective:</i> Intestinal anastomotic failure that occurs mainly due to ischaemia is a serious risk in colorectal cancer patients undergoing surgery. Surgeons continue to rely on subjective methods such as visual inspection to assess intestinal viability during surgery and there are no clinical tools to directly monitor viability postoperatively. A dual wavelength, reflectance optical sensor has been developed for continuous and dynamic monitoring of intestinal viability via the intestinal lumen. Maintaining direct contact between the sensor and the inner intestinal wall can be difficult in an intraluminal design, therefore impacting on signal acquisition and quality. This paper investigates the effect of direct contact versus variable distances between the sensor and the tissue surface of the buccal mucosa as a surrogate. &lt;i>Approach:</i> The &lt;i>in-vivo</i> study involved 20 healthy volunteers to measure the effect of optical sensor-tissue distances on the ability to acquire photoplethysmography signals and their quality. Signals were acquired from the buccal mucosa at five optical sensor-tissue distances. &lt;i>Main results:&lt;/i> Distances between 0 mm (contact) to 5 mm were the most optimal, producing signals of high quality and signal-to-noise ratio, resulting in reliable estimations of the blood oxygen saturation. Distances exceeding 5 mm compromised the acquired signals, and were of poor quality, thereby unreliably estimating the blood oxygen saturation. &lt;i>Significance:</i> The developed optical sensor proved to be reliable for acquiring photoplethysmography signals for cases where distances between the optical sensor-tissue may arise during the assessment of intraluminal intestinal viability. © 2018 Institute of Physics and Engineering in Medicine.

  11. Analysis of immune response in young and aged mice vaccinated with corn-derived antigen against Escherichia coli heat-labile enterotoxin.

    PubMed

    Karaman, Sule; Cunnick, Joan; Wang, Kan

    2006-01-01

    Enterotoxigenic strains of Escherichia coli produce a heat-labile holotoxin (LT), which causes diarrhea. We engineered corn seeds to produce LT-B, the nontoxic subunit of LT, to serve as a plant-derived vaccine to traveler's diarrhea and as an adjuvant for co-administered proteins. We previously demonstrated that a strong mucosal and systemic antibody response is elicited in young mice with oral administration of corn-derived LT-B. The present study examined systemic and mucosal antibody responses to LT-B in young and aged mice, and recall responses to oral administration and injection of LT-B in aged mice. Specific IgA and IgG antibodies were detectable during an 11-mo period, although the concentration of antigen-specific antibodies declined gradually. Booster by feeding or injection dramatically increased the concentration of specific IgA from that seen in young mice. Specific IgG levels were boosted to concentrations similar to those in young mice. This effect may be age-dependent and related to prior immunization exposure. Analysis of the antibody response of naïve aged mice against corn-derived LT-B demonstrated an age-related suppression in specific IgG production, but not specific IgA. These results may provide important information for edible vaccine strategies for young and aged individuals.

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

    DTIC Science & Technology

    2012-07-27

    induce MyD88- mediated signaling [15,16]. Seeking to improve the efficacy of Compound 1 as an inhibitor of MyD88 signaling, we synthesized a dimeric...molecule in which two Compound 1 moieties were covalently linked together, reasoning that the dimeric compound would be a more potent inhibitor of protein...pellets in 50 ml of lysis buffer (Active Motif) in the presence of DTT, protease inhibitors and phosphatase inhibitors and incubated on ice for 30–60

  13. X-Ray Crystal Structure of the passenger domain of Plasmid encoded toxin(Pet), an Autotransporter Enterotoxin from enteroaggregative Escherichia coli (EAEC)

    PubMed Central

    Meza-Aguilar, J. Domingo; Fromme, Petra; Torres-Larios, Alfredo; Mendoza-Hernández, Guillermo; Hernandez-Chiñas, Ulises; Monteros, Roberto A. Arreguin-Espinosa de los; Campos, Carlos A. Eslava; Fromme, Raimund

    2014-01-01

    Autotransporters (ATs) represent a superfamily of proteins produced by a variety of pathogenic bacteria, which include the pathogenic groups of Escherichia coli (E. coli) associated with gastrointestinal and urinary tract infections. We present the first X-ray structure of the passenger domain from the Plasmid-encoded toxin (Pet) a 100 kDa protein at 2.3 Å resolution which is a cause of acute diarrhea in both developing and industrialized countries. Pet is a cytoskeleton-altering toxin that induces loss of actin stress fibers. While Pet (pdb code: 4OM9) shows only a sequence identity of 50 % compared to the closest related protein sequence, extracellular serine protease plasmid (EspP) the structural features of both proteins are conserved. A closer structural look reveals that Pet contains a β-pleaded sheet at the sequence region of residues 181-190, the corresponding structural domain in EspP consists of a coiled loop. Secondary, the Pet passenger domain features a more pronounced beta sheet between residues 135-143 compared to the structure of EspP. PMID:24530907

  14. Spreading of genes encoding enterotoxins, haemolysins, adhesin and biofilm among methicillin resistant Staphylococcus aureus strains with staphylococcal cassette chromosome mec type IIIA isolated from burn patients.

    PubMed

    Motallebi, Mitra; Jabalameli, Fereshteh; Asadollahi, Kheirollah; Taherikalani, Morovat; Emaneini, Mohammad

    2016-08-01

    The emergence of antibiotic-resistant Staphylococcus aureus in particular methicillin-resistant S. aureus (MRSA) is an important concern in burn medical centers either in Iran or worldwide. A total of 128 S. aureus isolates were collected from wound infection of burn patients during June 2013 to June 2014. Multiplex-polymerase chain reaction (MPCR) assay was performed for the characterization of the staphylococcal cassette chromosome mec (SCCmec). Genes encoding virulence factors and biofilm were targeted by PCR. Of 128 S. aureus isolates, 77 (60.1%) isolates were MRSA. Fifty four (70.1%) isolates were identified as SCCmec type IIIA. The most frequently detected toxin genes among MRSA isolates with SCCmec type IIIA were sea (64.1%) and hla (51.8%). The rate of coexistence of sea with hla and sea with hla and hlb was 37% and12.9%, respectively. The sec, eta, tst, pvl, hla and hlb genes were not detected in any of the MRSA isolates. The most prevalent genes encoding biofilm was eno, found in 61.1% of isolates, followed by fib and icaA found in 48.1% and 38.8% of the isolates, respectively. The rate of coexistence of fib + eno + icaA + icaD and fib + eno was 20.3% and 9.2%, respectively. The ebps gene was not detected in any of the isolates. In conclusion, our study indicated that the sea, hla, fib and icaA were most frequent genes encoding virulence factors among MRSA with SCCmec type IIIA isolated from burn wound infection. Moreover, the results of this study shows that the rate of coexistence of genes encoding different virulence factor were high. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Acoustic Microsensors III. Direct Detection of Staphylococcal Enterotoxin B Employing a Piezoelectric Crystal Immunosensor with a Flexible Carboxylated Dextran Matrix as the Biochemical Interface.

    DTIC Science & Technology

    1998-03-01

    8217real time’ monitoring van biologische strijdmiddelen en aanverwante verbindingen. TNO- PML onderzoekt de mogelijkheden van een immunoreactie op een...toxine, dat voedselvergiftiging veroorzaakt, behoort tot de biologische strijdmid- delen. SEB werd door PG33 gekozen als teststof om de diverse...onderzoek zieh richten op de detectie van grote antigenen die tot de biologische strijdmiddelen behoren, zoals sommige virussen en bacteri- en. De

  16. X-ray crystal structure of the passenger domain of plasmid encoded toxin(Pet), an autotransporter enterotoxin from enteroaggregative Escherichia coli (EAEC)

    SciTech Connect

    Domingo Meza-Aguilar, J.; Laboratorio de Patogenicidad Bacteriana, Unidad de Hemato Oncología e Investigación, Hospital Infantil de México Federico Gómez 06720, D.F.; Fromme, Petra

    Highlights: • X-ray crystal structure of the passenger domain of Plasmid encoded toxin at 2.3 Å. • Structural differences between Pet passenger domain and EspP protein are described. • High flexibility of the C-terminal beta helix is structurally assigned. - Abstract: Autotransporters (ATs) represent a superfamily of proteins produced by a variety of pathogenic bacteria, which include the pathogenic groups of Escherichia coli (E. coli) associated with gastrointestinal and urinary tract infections. We present the first X-ray structure of the passenger domain from the Plasmid-encoded toxin (Pet) a 100 kDa protein at 2.3 Å resolution which is a cause ofmore » acute diarrhea in both developing and industrialized countries. Pet is a cytoskeleton-altering toxin that induces loss of actin stress fibers. While Pet (pdb code: 4OM9) shows only a sequence identity of 50% compared to the closest related protein sequence, extracellular serine protease plasmid (EspP) the structural features of both proteins are conserved. A closer structural look reveals that Pet contains a β-pleaded sheet at the sequence region of residues 181–190, the corresponding structural domain in EspP consists of a coiled loop. Secondary, the Pet passenger domain features a more pronounced beta sheet between residues 135 and 143 compared to the structure of EspP.« less

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

    PubMed

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

    2012-06-01

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

  18. Magnetization, resistivity, specific heat and ab initio calculations of Gd5Sb3.

    PubMed

    Samatham, S Shanmukharao; Patel, Akhilesh Kumar; Lukoyanov, Alexey V; Suresh, K G

    2018-06-07

    We report on the combined results of structural, magnetic, transport and calorimetric properties of Mn<sub>5</sub>Si<sub>3</sub>-type hexagonal Gd<sub>5</sub>Sb<sub>3</sub>, together with &lt;i>ab-initio</i> calculations. It exhibits a ferromagnetic (FM)-like transition at 265 K, antiferromagnetic (AFM) Néel transition at 95.5 K followed by a spin-orientation transition at 62 K. The system is found to be in AFM state down to 2 K in a field of 70 kOe. The FM-AFM phase coexistence is not noticeable despite large positive Curie-Weiss temperature (θ<sub>CW</sub> = 223.5 ± 0.2 K). Instead, low-temperature AFM and high-temperature FM-like phases are separated in large temperatures. Temperature-magnetic field (&lt;i>H&lt;/i>-<i>T</i>) phase diagram reveals field-driven complex magnetic phases. Within the AFM phase, the system is observed to undergo field-driven spin-orientation transitions. Field-induced tricritical and quantum critical points appear to be absent due to strong AFM nature and by the intervention of FM-like state between paramagnetic and AFM states, respectively. The metallic behavior of the compound is inferred from resistivity along with large Sommerfeld parameter. However, no sign of strong electron-correlations is reasoned from the Kadowaki-Wood's ratio &lt;i>A</i>/γ<sup>2</sup> ∼ 1.9×10<sup>-6</sup> μΩ.cm.(mol.K)<sup>2</sup>(mJ)<sup>-2</sup>, despite heavy γ. Essentially, &lt;i>ab initio&lt;/i> calculations accounting for electronic correlations confirm AFM nature of low-temperature magnetic state in Gd<sub>5</sub>Sb<sub>3</sub> and attainable FM ordering in agreement with experimental data. © 2018 IOP Publishing Ltd.

  19. A Small Molecule that Mimics the BB-Loop in the Toll/IL-1 Receptor Domain of MyD88 Attenuates Staphylococcal Enterotoxin B Induced Pro-Inflammatory Cytokine Production and Toxicity in Mice

    DTIC Science & Technology

    2011-06-01

    121.8, 114.6, 67.0, 55.1, 46.6, 45.7, 31.3, 25.9, 24.0, 19.4, 17.6; HRMS: (ESI-TOF) C17H24N2O3H+ expected: 305.1860. found: 305.1860. (S)-1- benzyl -3...chemiluminescent substrate in the presence of hydrogen peroxide using Immun-Star WesternC Chemiluminescent Kit (BioRad). An imaging system VersaDoc Model

  20. Agents of Bioterrorism: Argument For and Against a List That Needs Cropping

    DTIC Science & Technology

    2003-06-10

    Burkholderia pseudomallei • Coxiella burnetti (Q fever) • Brucella species (brucellosis) • Burkholderia mallei ( glanders ) • Ricin toxin (from...brucellosis) • Burkholderia mallei ( glanders ) • Ricin toxin (from Ricinus communis) • Epsilon toxin of Clostridium perfringens • Staphylococcus enterotoxin B... mallei ( glanders ) • Ricin toxin (from Ricinus communis) • Epsilon toxin of Clostridium perfringens • Staphylococcus enterotoxin B • Typhus fever

  1. Development of Bacterial Display Peptides for use in Biosensing Applications

    DTIC Science & Technology

    2012-09-01

    performance. Specific results on peptides binders to Protective Antigen (PA) protein of Bacillus anthracis and Staphylococcal Enterotoxin B (SEB...reagent, affinity reagent, bacterial display, multi-scale modeling, docking, protective antigen , SEB, biosensing 16. SECURITY CLASSIFICATION OF: 17...performance. Specific results on peptides binders to Protective Antigen (PA) protein of Bacillus anthracis and Staphylococcal Enterotoxin B (SEB) will be

  2. Electrical and thermoelectric transport properties of two-dimensional fermionic systems with k-cubic spin-orbit coupling.

    PubMed

    Mawrie, Alestin; Verma, Sonu; Ghosh, Tarun Kanti

    2017-09-01

    We investigate effect of &lt;i>k</i>-cubic spin-orbit interaction on electrical and thermoelectric transport properties of two-dimensional fermionic systems. We obtain exact analytical expressions of the inverse relaxation time (IRT) and the Drude conductivity for long-range Coulomb and short-range delta scattering potentials. The IRT reveals that the scattering is completely suppressed along the three directions θ = (2n+1)π/3 with n=1,2,3. We also obtain analytical results of the thermopower and thermal conductivity at low temperature. The thermoelectric transport coefficients obey the Wiedemann-Franz law, even in the presence of &lt;i>k</i>-cubic Rashba spin-orbit interaction (RSOI) at low temperature. In the presence of quantizing magnetic field, the signature of the RSOI is revealed through the appearance of the beating pattern in the Shubnikov-de Haas (SdH) oscillations of thermopower and thermal conductivity in low magnetic field regime. The empirical formulae for the SdH oscillation frequencies accurately describe the locations of the beating nodes. The beating pattern in magnetothermoelectric measurement can be used to extract the spin-orbit coupling constant. © 2017 IOP Publishing Ltd.

  3. U.S. EPA, Pesticide Product Label, , 03/20/1975

    EPA Pesticide Factsheets

    2011-04-21

    ... i4 S ~"aula~ Lt;i ... Po ~ct 4rci~ ~~£, tat~~~ ccnd~tic~~ ~av~~ d~{~t !\\r~ 4~~a) :,cat~d. 0: ~ct eO'lt~~i~ar~ ~al ... chemicil descrlntl~" on the la~el dnd '. ...

  4. U.S. EPA, Pesticide Product Label, INSECTICIDE CONCENTRATE, 02/11/1985

    EPA Pesticide Factsheets

    2011-04-21

    ... ". ... w ••• 11 dft ... ln- .... el ..... wi" .... 4 h., • .. 0 .... . cil eft ..... If I"'tet'''' ,11'1. If ••• 111."- • "I' •• Alee -.ltI"._ C.II. ",11.:1 ... 1_ ... ,tI,. ...

  5. Food-Borne Outbreak Investigation and Molecular Typing: High Diversity of Staphylococcus aureus Strains and Importance of Toxin Detection

    PubMed Central

    Denayer, Sarah; Nia, Yacine; Botteldoorn, Nadine

    2017-01-01

    Staphylococcus aureus is an important aetiological agent of food intoxications in the European Union as it can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Reported enterotoxin dose levels causing food-borne illness are scarce and varying. Three food poisoning outbreaks due to enterotoxin-producing S. aureus strains which occurred in 2013 in Belgium are described. The outbreaks occurred in an elderly home, at a barbecue event and in a kindergarten and involved 28, 18, and six cases, respectively. Various food leftovers contained coagulase positive staphylococci (CPS). Low levels of staphylococcal enterotoxins ranging between 0.015 ng/g and 0.019 ng/g for enterotoxin A (SEA), and corresponding to 0.132 ng/g for SEC were quantified in the food leftovers for two of the reported outbreaks. Molecular typing of human and food isolates using pulsed-field gel electrophoresis (PFGE) and enterotoxin gene typing, confirmed the link between patients and the suspected foodstuffs. This also demonstrated the high diversity of CPS isolates both in the cases and in healthy persons carrying enterotoxin genes encoding emetic SEs for which no detection methods currently exist. For one outbreak, the investigation pointed out to the food handler who transmitted the outbreak strain to the food. Tools to improve staphylococcal food poisoning (SFP) investigations are presented. PMID:29261162

  6. Aeromonas Caviae Strain Induces Th1 Cytokine Response in Mouse Intestinal Tract

    EPA Science Inventory

    Aeromonas caviae has been associated with human gastrointestinal disease. Strains of this species typically lack virulence factors (VFs) such as enterotoxins and hemolysins that are produced by other human pathogens of the Aeromonas genus. Microarray profiling of murine small i...

  7. NATO Planning Guide for the Estimation of Chemical, Biological, Radiological, and Nuclear (CBRN) Casualties (AMedP-8(C)) - Parameters for Estimation of Casualties from Exposure to Specified Biological Agents. Addenda to Allied Medical Publication 8

    DTIC Science & Technology

    2011-01-01

    glanders, Q fever, and tularemia , as well as the biotoxin staphylococcal enterotoxin B. Incorporating these five agents into the published NATO guide will...Specified Biological Agents: Brucellosis, Glanders, Q Fever, SEB, and Tularemia . This document describes the research methods used by the study authors...enterotoxin B (SEB), and tularemia . Several editorial changes, such as renumbering figures and tables, updating the corresponding references in the text, and

  8. Resistance of bovine colostral anti-cholera toxin antibody to in vitro and in vivo proteolysis.

    PubMed Central

    McClead, R E; Gregory, S A

    1984-01-01

    Pregnant cows immunized with cholera enterotoxin produce an immunoglobulin G class 1 antibody that enters the colostrum in high titer. After exposure to intestinal enzymes, this antibody remains immunologically reactive and inhibits intestinal fluid secretion in infant and adult rabbits exposed to cholera enterotoxin. Specific bovine colostral antibodies may be a source of passive immune protection for human infants and adults at risk for cholera and other enteric diseases. PMID:6425223

  9. Task Validation for the AN/TPQ-36 Radar System

    DTIC Science & Technology

    1978-09-01

    report presents the method and results of a study to validate personnel task descriptions for the new AN/TyP-Jb radar...TP.J-Sb KAPAK SVSTKM CONTENTS i ■ l.t |i- INTRODUCTION t METHOD 2 RESULTS, CONCLUSIONS, AND RECOMMENDATIONS b Task Validation 5 26B MOS... method , results, conclusions, and recommendations of the validation study. The appendixes contain the following: 1. Appendix A contains

  10. The Effect of High Pressure on Phase Relationships and Some Properties of Ti and Its alloys.

    DTIC Science & Technology

    1981-05-01

    8217. forma--t ion. That- lna\\ p), G id-2 On, n t --: y of 1 tI \\ it t’ -I n( the ? e al fovs after var tcu-s heat treoo’ Ii ’s jn 4 new, : ieLt I u-1J * ri...H. Bernstein, Computer Calculations of Phase Diagrams, Academic Press, N.Y. and.London, 1970. 23, L. Kaufman, Acta Met. 7, 575 (1959). 24, A. Gysler

  11. Pyrometallurgical Extraction of Valuable Elements in Ni-Metal Hydride Battery Electrode Materials

    NASA Astrophysics Data System (ADS)

    Jiang, Yin-ju; Deng, Yong-chun; Bu, Wen-gang

    2015-10-01

    Gas selective reduction-oxidation (redox) and melting separation were consecutively applied to electrode materials of AB5-type Ni-metal hydride batteries leading to the production of a Ni-Co alloy and slag enriched with rare earth oxides (REO). In the selective redox process, electrode materials were treated with H2/H2O at 1073 K and 1173 K (800 °C and 900 °C). Active elements such as REs, Al, and Mn were oxidized whereas relatively inert elements such as Ni and Co were transformed into their elemental states in the treated materials. SiO2 and Al2O3 powders were added into the treated materials as fluxes which were then melted at 1823 K (1550 °C) to yield a Ni-Co alloy and a REO-SiO2-Al2O3-MnO slag. The high-purity Ni-Co alloy produced can be used as a raw material for AB5-type hydrogen-storage alloy. The REO content in slag was very high, i.e., 48.51 pct, therefore it can be used to recycle rare earth oxides.

  12. Staphylococcal food poisoning in the United Kingdom, 1969-90.

    PubMed Central

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

    1993-01-01

    Between 1969 and 1990 strains of Staphylococcus aureus from 359 outbreaks and sporadic cases of staphylococcal food poisoning in the United Kingdom were examined in the PHLS Food Hygiene Laboratory for the production of enterotoxin. In a number of instances the incriminated foods were also examined for the presence of enterotoxin. Strains from 79% of incidents produced enterotoxin A alone or together with another enterotoxin. The level of S. aureus present in the foods ranged from no viable S. aureus detected to 1.5 x 10(10) c.f.u./g with a median of 3.0 x 10(7) c.f.u./g. Enterotoxin was detected in foods in the absence of viable S. aureus in only two outbreaks and in both cheese was the implicated food. Meat, poultry or their products were the vehicle in 75% of incidents with ham and chicken most frequently implicated. Other foods included fish and shellfish (7%) and milk and milk products (8%). Most contamination took place in the home followed by restaurants and shops. Seventy-one percent of the incident strains were lysed by phages of group III or I/III. PMID:8519317

  13. Thermal stability and structural changes in bacterial toxins responsible for food poisoning

    PubMed Central

    Regenthal, Paulina; Hansen, Jesper S.; André, Ingemar

    2017-01-01

    The staphylococcal enterotoxins (SEs) are secreted by the bacteria Staphylococcus aureus and are the most common causative agent in staphylococcal food poisoning. The staphylococcal enterotoxin A (SEA) has been associated with large staphylococcal food poisoning outbreaks, but newer identified SEs, like staphylococcal enterotoxin H (SEH) has recently been shown to be present at similar levels as SEA in food poisoning outbreaks. Thus, we set out to investigate the thermo-stability of the three-dimensional structures of SEA, SEH and staphylococcal enterotoxin E (SEE), since heat inactivation is a common method to inactivate toxins during food processing. Interestingly, the investigated toxins behaved distinctly different upon heating. SEA and SEE were more stable at slightly acidic pH values, while SEH adopted an extremely stable structure at neutral pH, with almost no effects on secondary structural elements upon heating to 95°C, and with reversible formation of tertiary structure upon subsequent cooling to room temperature. Taken together, the data suggests that the family of staphylococcal enterotoxins have different ability to withstand heat, and thus the exact profile of heat inactivation for all SEs causing food poisoning needs to be considered to improve food safety. PMID:28207867

  14. Gram-positive, catalase-positive cocci from dry cured Iberian ham and their enterotoxigenic potential.

    PubMed Central

    Rodríguez, M; Núñez, F; Córdoba, J J; Bermúdez, E; Asensio, M A

    1996-01-01

    Iberian ham is an uncooked, cured meat product ripened under natural uncontrolled conditions for 18 to 24 months. Gram-positive, catalase-positive cocci are the main microbial population in Iberian ham for most of the ripening time. Since some of these organisms are able to produce enterotoxins, adequate characterization and toxicological study are needed. For this, 1,327 gram-positive, catalase-positive cocci, isolated from Iberian hams at different stages and locations, were characterized by physiological and biochemical tests. Selected isolates were further characterized by guanine-cytosine (G+C) content and restriction enzyme analysis of genes coding for 16S rRNA. The toxigenic potential of these organisms was tested with specific DNA gene probes for staphylococcal enterotoxins A, B, C, and D and confirmed by semiquantitative sandwich enzyme immunoassay. The majority of the isolates were identified as Staphylococcus spp. and Micrococcus spp. Non-identified gram-positive, catalase-positive cocci which were moderately halophilic and showed a 42 to 52% G+C content were detected. A great variety of staphylococcal strains were found within the different species at any sampling time. Two strains of Staphylococcus xylosus, one Staphylococcus cohnii strain, and four of the non-identified organisms with 42 to 52% G+C contents hybridized with some of the DNA probes for C and D staphylococcal enterotoxin genes. S. xylosus hybridizing with C-enterotoxin probe reacted with both C and D enterotoxins in the immunological test. In addition, enterotoxin D was confirmed in the nonidentified strains. Some toxigenic organisms were isolated from the final product, posing a health hazard for the consumer. PMID:8787389

  15. Oral immunization with a recombinant bacterial antigen produced in transgenic plants.

    PubMed

    Haq, T A; Mason, H S; Clements, J D; Arntzen, C J

    1995-05-05

    The binding subunit of Escherichia coli heat-labile enterotoxin (LT-B) is a highly active oral immunogen. Transgenic tobacco and potato plants were made with the use of genes encoding LT-B or an LT-B fusion protein with a microsomal retention sequence. The plants expressed the foreign peptides, both of which formed oligomers that bound the natural ligand. Mice immunized by gavage produced serum and gut mucosal anti-LT-B immunoglobulins that neutralized the enterotoxin in cell protection assays. Feeding mice fresh transgenic potato tubers also caused oral immunization.

  16. The three-dimensional crystal structure of cholera toxin

    SciTech Connect

    Zhang, Rong-Guang; Westbrook, M.L.; Nance, S.

    1996-02-01

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

  17. Isolation of Bacillus cereus Group from the Fecal Material of Endangered Wood Turtles.

    PubMed

    Nfor, Nancy Ngvumbo; Lapin, Carly N; McLaughlin, Richard William

    2015-10-01

    Members of the Bacillus cereus group are opportunistic human pathogens. They can be found in a broad range of foods. Diarrheal food poisoning and/or emetic type syndromes can result from eating contaminated food. In this study, seven B. cereus group members were isolated from the fecal material of Wood Turtles (Glyptemys insculpta). The isolates were then assessed for the presence of enterotoxin genes (nheA, entFM, hblC, and cytK) using PCR. The most prevalent is the nonhemolytic enterotoxin gene which was found in all seven isolates.

  18. Inhibition of Clostridium difficile toxin A and B by 1,2-cyclohexanedione modification of an arginine residue.

    PubMed

    Balfanz, J; Rautenberg, P

    1989-12-29

    Toxin A (enterotoxin) and toxin B (cytotoxin) of Clostridium difficile were both inactivated by the arginine specific reagent 1,2-cyclohexanedione. Molecular stability during the inactivation process was demonstrated by SDS-PAGE analysis showing the same migration rates for modified and unmodified forms of the 230 kDa toxin A and of the 250 kDa toxin B. Cytotoxicity of both toxins as well as mouse lethality of the enterotoxin were drastically decreased as a result of the arginine modification. The reaction followed pseudo-first-order kinetics. Analysis of the data suggested that modification of a single arginine residue was sufficient to abolish the activity of both toxins.

  19. Effect of the Cerium Oxide (CeO2) on the Structural and Electrochemical Properties of the LaNi5Ce Metal Hydride Anode

    NASA Astrophysics Data System (ADS)

    Utami Hapsari, Ade; Zulfia, Anne; Raharjo, Jarot; Agustanhakri

    2017-07-01

    One of negative electrode, AB5-type alloy electrodes, have been extensively studied and applied in rechargeable Ni-MH batteries due to their excellent electrochemical characteristics. Some researchers have found that addition of rare earth oxides (La, Ce, Pr, Er, Tm, Yb) to AB5-type alloy (MH) electrode improves battery performance significantly. Cerium Oxide (CeO2) is a light rare earth oxide is widely obtained from the processing of tailings in mining activities. During this time, there is still little data for research applications of cerium oxide for electrode materials. In this paper, the effects of adding CeO2 on the performance metal hydride electrode were investigated. In order to study the effects of CeO2 on the performance of anode material, 1%, 2%, and 3% of weight ratio CeO2 was mixed to LaNi5 as an negative electrode. The powder mixtures were mechanically milled at a speed of rpm 240 for 2 hours using ball mill. The powder mixtures were characterized by X-Ray Diffraction (XRD) and Scanning Electron Microscope (SEM). Electrochemical characteristics were measured using electrochemical impedance spectroscopy (EIS). The powder mixing showed the presence of Ce atom substitution into LaNi5 structures that affect the electrochemical properties of the material. The addition of cerium oxide at LaNi5 increase of the value of impedance. However, the addition of the value of impedance at 1% CeO2 is not significant when compared with the addition of 2% and 3% CeO2 that actually make the electrochemical properties of LaNi5 worst. Although the addition of 1% CeO2 also slightly increases the impedance value of LaNi5, but the addition of 1% CeO2 showed increase the corrosion resistance than without the addition of CeO2 and the addition of 2% and 3% CeO2.

  20. Laboratory Investigation of Expansion and Venting and Plug Response in the MX Trench.

    DTIC Science & Technology

    1979-02-01

    twnch is it hotoi. [’i’-t lb) W~ls t’olld(itd I-r il no0111a I 1rLt I I ,C tLit prl( SIrt OL’f 2 1 1 )S pi. SOil Irf1oil thi IL\\V 1 11051 F5S1 to Ua W...hloc ks bn,;ian to opc -n 111) . SCVCla Il add i tL/Io;il I .r [1ks Ircl :11 OSt immc!Ld 1;ltc lV. (The", (’[ll 1wscl cilsil :Vit t - i IhLc roof I

  1. International Symposium on Mechanism of Cyanide Toxicity and Antagonism (1st), Proceedings Held on 15-19 August 1982.

    DTIC Science & Technology

    1983-10-01

    intraventricular contents, and toxicologically significant concentrations of cyanide could still be measured at 3 weeks STORAGE TIME DAYS) in blood samples ...thi- rhod~,iit m lt~ tilt, h v - lt i~ tni ii t iituMtil tmn 1 hu’I-huct~ irt, ig thtu t\\ttlottiimmtii.tt rtighl% equ~.il %i/emiid thc toinnt tig...loopr’ tnbth’id’ wt~hlu ~ Ih l vmhttiblliii thtu unity o~th t * ’ it thu- tjrt~ikt’ tit thc m lt t lt , l I lit it ttvt’ ’itc’ Io,,-~ it, tlt, thgUrt’ 11

  2. Multi-Technique Study of Ionospheric Structures Causing Degradation in Trans-Ionospheric Communications Systems.

    DTIC Science & Technology

    1987-04-20

    NASA . Dr. Basi was a member of the Guest Investigator Team on the NASA Atmosphere Explorer Program and is currently a member of the Dynamics Explorer...Electric Com- pany in Lynn. MA. At this point he joined the Geomagnctism Branch at the Air Force Cam- Ilerbert E. Whitney. photograph and biography not...crgs ENERGYtlb*V) (cm 2 s)- ’ arc observed. The gaps in between the precipitation I-if; 3 Lt-. .i .etrum obtained ai 14O1.45 UT shoting nil acl- regions

  3. Soviet Frontal Aviation.

    DTIC Science & Technology

    1980-06-01

    Oxplail thLtI th\\ v ,:crL’ v , turiou-, only because of tile " r’ .e.r e.i,:: t, tan. un , ol e - in, :, t.Lictics, as well as the u.-, e of -, V "I 01I o, L...Pilot". Soviet Military Review. No. 2, 1979. Mikryukov , L. "Upravleniye istrebitelyami v vozdushnom boyu" (Controlling Fighter Planes in Aerial Combat...4. TITLE (and Subtitle) S. TYPE OF REPORT & PERIOD CeVERED SOVIET FRONTAL AVIATION,./ - r E -RrSWUN r.oR. *ep**T NumaE.R 7. AUTHOR(a) S. CONTRACT OR

  4. Success Factors for Adoption of Real-Time Java

    DTIC Science & Technology

    2010-04-01

    use Java in an effective way in order to achieve objectives If d ’t l t ti f bj ti d d ’t lt i t l i 9© 2010 Atego. All rights reserved. you on p an o...order to effectively use object-oriented programming practices. Te ps d’execution des algos 400 450 500 550 600 650 700 n m s TacticalPicture...find all garbage, nor to defragment the available free pool Common operations may have surprising effects (e.g. entering a mutual exclusion region

  5. Waterborne Commerce of the United States, Calendar Year 1985. Part 3. Waterways and Harbors. Great Lakes.

    DTIC Science & Technology

    1987-05-01

    Pa -625 11. MIRO C P R E O I O T E S C H ARin l i lT i 1 i l u l I I l m I I n n l l l n I I 0) . . . 0LC.2 ,"- - " IWATERBORNE COMMERCE OF THE... 322 COPPER ALLOTS, U𔃾RDAED. . . . . . . . . . 712’ 750. . . . . . . 3323 LEAD AND ZINC, USMO42ED. . . . . . . . . . . . . .6204 6,204. . . . . 3324... 322 CCPET, ALLOTS, N ... KED-’’ - sor, b9, 2 bq , . . .JN....E.D..-.. 2 2 .-- - - - - - - - -o 3323 EA0 AN D ZIN , UNNOAKE---- --- Ia 900 a3 50

  6. Communication Effectiveness of the Army Civilian Personnel Program

    DTIC Science & Technology

    1966-06-01

    Fort Devens Eastern CONARC/Troops 1880 Fort Lee Southern CONARC/School 2500 Fort MacArthur Western CONARC/Troops 1060 Fort Sam Houston Southern...A- t- I ta1 it ciu.1 rac t v’I I at I vi ii o I tih cim t c lt i oisit Itil Ih cm we𔃻:.’ tin t i f It i ,:i1 v .1 Nil I ii’j’or e’i I ACK NOWI I. M...Southern AMC/Supply 3000 Fort Benjamin Harrison Midwestern Audit/Finance 6000 Fort Benning Southern CONARC/Troops 3300 Fort Carson Western

  7. Molecular characterization of a functional type VI secretion system from a clinical isolate of Aeromonas hydrophila

    EPA Science Inventory

    Our laboratory recently molecularly characterized the type II secretion system (T2SS)-associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrop...

  8. Molecular Characterization of a Functional Type VI Secretion System from a Clinical Isolate of Aeromonas hydrophilia

    EPA Science Inventory

    Our laboratory recently molecularly characterized the type II secretion system (T2SS)-associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrop...

  9. BACTERIOCIN E1073 PRODUCED BY ENTEROCOCCUS FAECIUM LWP1073 IS EFFECTIVE FOR TREATING COMMENSAL CLOSTRIDIUM PERFRINGENS INFECTION IN BROILERS

    USDA-ARS?s Scientific Manuscript database

    Enterotoxin-producing Clostridium perfringens type A bacteria occupy a significant place in the etiological structure of food-borne infections in humans. One potential approach to minimize infections associated with food-borne pathogens is to control the carriage of C. perfringens in broilers. For ...

  10. Molecular Typing of Staphylococcus Aureus Isolate Responsible for Staphylococcal Poisoning Incident in Homemade Food

    PubMed Central

    Bellio, Alberto; Bianchi, Daniela Manila; Gallina, Silvia; Adriano, Daniela; Zuccon, Fabio; Chiesa, Francesco; Acutis, Pier Luigi; Casalinuovo, Francesco; Decastelli, Lucia

    2016-01-01

    In October 2012, two persons fell ill with symptoms consistent with staphylococcal food poisoning after eating home-canned tuna fish and tomatoes. Laboratory investigation detected the enterotoxins in the home-canned tuna and molecular analysis of the isolated Staphylococcus aureus confirmed it carried toxin genes. Qualitative enzyme-linked immunosorbent assay and enzime linked fluorescent assay methods and quantitative assay identified the enterotoxins in the food leftovers, specifically staphylococcal enterotoxins type A (SEA) and D (SED), respectively 0.49 and 2.04 ng/g. The laboratory results are discussed considering the relation to the fish in oil, survival and heat resistance of S. aureus, and presumptive microbial contamination due to improper handling during home-canning procedures. This is the first reported cluster of foodborne illnesses due to staphylococcal enterotoxins in tuna in Italy. In this study, we reported cases described and analysed for their spa-type. Showing a high heterogeneity of isolates, spa-type t13252 is correlated in a node of the minimum spanning tree and it has never been reported as responsible for foodborne outbreak. This case underlines the importance of risk communication and dissemination of home-canning guidelines to reduce the incidence of foodborne outbreaks caused by homemade conserves. PMID:27800449

  11. Molecular Typing of Staphylococcus Aureus Isolate Responsible for Staphylococcal Poisoning Incident in Homemade Food.

    PubMed

    Macori, Guerrino; Bellio, Alberto; Bianchi, Daniela Manila; Gallina, Silvia; Adriano, Daniela; Zuccon, Fabio; Chiesa, Francesco; Acutis, Pier Luigi; Casalinuovo, Francesco; Decastelli, Lucia

    2016-04-19

    In October 2012, two persons fell ill with symptoms consistent with staphylococcal food poisoning after eating home-canned tuna fish and tomatoes. Laboratory investigation detected the enterotoxins in the home-canned tuna and molecular analysis of the isolated Staphylococcus aureus confirmed it carried toxin genes. Qualitative enzyme-linked immunosorbent assay and enzime linked fluorescent assay methods and quantitative assay identified the enterotoxins in the food leftovers, specifically staphylococcal enterotoxins type A (SEA) and D (SED), respectively 0.49 and 2.04 ng/g. The laboratory results are discussed considering the relation to the fish in oil, survival and heat resistance of S. aureus , and presumptive microbial contamination due to improper handling during home-canning procedures. This is the first reported cluster of foodborne illnesses due to staphylococcal enterotoxins in tuna in Italy. In this study, we reported cases described and analysed for their spa -type. Showing a high heterogeneity of isolates, spa -type t 13252 is correlated in a node of the minimum spanning tree and it has never been reported as responsible for foodborne outbreak. This case underlines the importance of risk communication and dissemination of home-canning guidelines to reduce the incidence of foodborne outbreaks caused by homemade conserves.

  12. Documentation of Production: Allied Medical Publication 8(B), Volume 2, Medical Planning Guide of NBC Battle Casualties (Biological)

    DTIC Science & Technology

    2006-06-01

    causing Venezuelan Equine Encephalitis (VEE). Dose Range Signs/Symptoms of Illness Category Number (organisms) Typical Description Abbreviation 1...98 Table VIII-8. Number of Infected Personnel after a Venezuelean equine encephalitis (VEE) Attack...tactical scenario, agent (anthrax, botulinum neurotoxin, plague, tularemia, Staphylococcal enterotoxin B, Q fever, Venezuelan Equine Encephalitis

  13. Experimental and spontaneous clostridial enteropathies of laboratory and free living lagomorphs.

    PubMed

    Carman, R J; Evans, R H

    1984-10-01

    The enteric diseases of hares, European and cottontail rabbits, which are caused by members of the genus Clostridium are reviewed. Disease caused by C. perfringens Types A and E, C. spiroforme, C. difficile, C. sordellii, C. tympany cuniculi and clostridial enterotoxins are included. Tyzzer's disease also is discussed.

  14. Occurrence of Natural Bacillus thuringiensis Contaminants and Residues of Bacillus thuringiensis-Based Insecticides on Fresh Fruits and Vegetables

    PubMed Central

    Frederiksen, Kristine; Rosenquist, Hanne; Jørgensen, Kirsten; Wilcks, Andrea

    2006-01-01

    A total of 128 Bacillus cereus-like strains isolated from fresh fruits and vegetables for sale in retail shops in Denmark were characterized. Of these strains, 39% (50/128) were classified as Bacillus thuringiensis on the basis of their content of cry genes determined by PCR or crystal proteins visualized by microscopy. Random amplified polymorphic DNA analysis and plasmid profiling indicated that 23 of the 50 B. thuringiensis strains were of the same subtype as B. thuringiensis strains used as commercial bioinsecticides. Fourteen isolates were indistinguishable from B. thuringiensis subsp. kurstaki HD1 present in the products Dipel, Biobit, and Foray, and nine isolates grouped with B. thuringiensis subsp. aizawai present in Turex. The commercial strains were primarily isolated from samples of tomatoes, cucumbers, and peppers. A multiplex PCR method was developed to simultaneously detect all three genes in the enterotoxin hemolysin BL (HBL) and the nonhemolytic enterotoxin (NHE), respectively. This revealed that the frequency of these enterotoxin genes was higher among the strains indistinguishable from the commercial strains than among the other B. thuringiensis and B. cereus-like strains isolated from fruits and vegetables. The same was seen for a third enterotoxin, CytK. In conclusion, the present study strongly indicates that residues of B. thuringiensis-based insecticides can be found on fresh fruits and vegetables and that these are potentially enterotoxigenic. PMID:16672488

  15. Aeromonas caviae strain induces Th1 cytokine response in mouse intestinal tract

    EPA Science Inventory

    Aeromonas caviae has been associated with human gastrointestinal disease. Strains of this species typically lack virulence factors (VFs) such as enterotoxins and hemolysins that are produced by other human pathogens of the Aeromonas genus,. Microarray profiling of...

  16. 2010 Review on the Extension of the AMedP-8(C) Methodology to New Agents, Materials, and Conditions

    DTIC Science & Technology

    2010-12-01

    tularemia ). Applied Research Associates (ARA), under contract to the Defense Threat Reduction Agency (DTRA), has begun the development of similar...x P Included in IDA 2010 Analyses 1 Biological Staphylococcal Enterotoxin B x P Included in IDA 2010 Analyses 1 Biological Tularemia x P

  17. Molecular epidemiology of Vibrio cholerae in Hong Kong.

    PubMed Central

    Yam, W C; Lung, M L; Ng, K Y; Ng, M H

    1989-01-01

    We studied restriction fragment length polymorphism of the enterotoxin genes of isolates of Vibrio cholerae El Tor, indistinguishable by bacteriophage typing, which were collected in Hong Kong since 1978. Using this approach, we could distinguish indigenous and exogenous strains obtained from different sources and epidemiological settings. Images PMID:2570082

  18. Mutation in the S-ribosylhomocysteinase (luxS) gene involved in quorum sensing affects biofilm formation and virulence in a clinical isolate of Aeromonas hydrophila

    EPA Science Inventory

    A diarrheal isolate SSU of Aeromonas hydrophila produces a cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. Our laboratory has characterized from the above Aeromonas strain, in addition to Act, the type 3- and T6-secretion systems and their effec...

  19. 42 CFR 73.3 - HHS select agents and toxins.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Register and will be listed on the CDC Web site at http://www.cdc.gov/. (2) If an excluded attenuated...) Staphylococcal enterotoxins T-2 toxin Tetrodotoxin Tick-borne encephalitis complex (flavi) viruses (Central... listed in paragraph (b) of this section. (2) Recombinant nucleic acids that encode for the functional...

  20. 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

  1. Growth of Staphylococcus aureus in cooked potato and potato salad – A one-step kinetic analysis

    USDA-ARS?s Scientific Manuscript database

    Staphylococcus aureus is a Gram-positive spherically-shaped bacterium capable of producing heat-stable enterotoxins that cause acute gastrointestinal diseases. The growth of this pathogen in food is a major threat to public health worldwide. Potato salad is a frequent vehicle for infection and foo...

  2. 40 CFR 725.421 - Introduced genetic material.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Neurotoxin Staphylococcus aureus Alpha toxin (alpha lysin) Yersinia pestis Murine toxin Snake toxins Bungarus... aeruginosa Proteases Staphylococcus aureus Gamma lysin (Gamma toxin); Enterotoxins (SEA, SEB, SEC, SED SEE); Pyrogenic exotoxins A B; Toxic shock syndrome toxins (TSST-1) Staphylococcus aureus & Pseudomonas aeruginosa...

  3. 40 CFR 725.421 - Introduced genetic material.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Neurotoxin Staphylococcus aureus Alpha toxin (alpha lysin) Yersinia pestis Murine toxin Snake toxins Bungarus... aeruginosa Proteases Staphylococcus aureus Gamma lysin (Gamma toxin); Enterotoxins (SEA, SEB, SEC, SED SEE); Pyrogenic exotoxins A B; Toxic shock syndrome toxins (TSST-1) Staphylococcus aureus & Pseudomonas aeruginosa...

  4. 40 CFR 725.421 - Introduced genetic material.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Neurotoxin Staphylococcus aureus Alpha toxin (alpha lysin) Yersinia pestis Murine toxin Snake toxins Bungarus... aeruginosa Proteases Staphylococcus aureus Gamma lysin (Gamma toxin); Enterotoxins (SEA, SEB, SEC, SED SEE); Pyrogenic exotoxins A B; Toxic shock syndrome toxins (TSST-1) Staphylococcus aureus & Pseudomonas aeruginosa...

  5. 40 CFR 725.421 - Introduced genetic material.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Neurotoxin Staphylococcus aureus Alpha toxin (alpha lysin) Yersinia pestis Murine toxin Snake toxins Bungarus... aeruginosa Proteases Staphylococcus aureus Gamma lysin (Gamma toxin); Enterotoxins (SEA, SEB, SEC, SED SEE); Pyrogenic exotoxins A B; Toxic shock syndrome toxins (TSST-1) Staphylococcus aureus & Pseudomonas aeruginosa...

  6. TCR-Vß8 as alternative to animal testing for quantifying active SEE

    USDA-ARS?s Scientific Manuscript database

    Staphylococcal food poisoning is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by the bacterium Staphylococcus aureus. SEs cause gastroenteritis and also cause activation of T cells and massive cytokine release. A current method for the detection of active SEs relies on its eme...

  7. Volunteer Challenge With Enterotoxigenic Escherichia coli That Express Intestinal Colonization Factor Fimbriae CS17 and CS19

    DTIC Science & Technology

    2011-07-01

    for 18-20 h, bacteria were harvested in sterile saline, and the sus- pension was diluted in phosphate-buffered saline to the ap- propriate...Levine MM, Merson MM. Serologic differentiation between antitoxin responses to infection with Vibrio cholerae and enterotoxin-producing Escherichia coli

  8. Plant compounds enhance assay sensitivity for detection of active bacillus cereus toxin

    USDA-ARS?s Scientific Manuscript database

    Bacillus cereus is an important food pathogen, producing emetic and diarrheal syndromes, the latter mediated by enterotoxins. It has been estimated that there are 84,000 cases of B. cereus food poisoning in the US each year, with an annual cost of USD 36 million. The ability to sensitively trace and...

  9. Prevalence of Toxin Genes among the Clinical Isolates of Staphylococcus aureus and its Clinical Impact

    PubMed Central

    Deodhar, Divya; Varghese, George; Balaji, Veeraraghavan; John, James; Rebekah, Grace; Janardhanan, Jeshina; Jeyaraman, Ranjith; Jasmine, Sudha; Mathews, Prasad

    2015-01-01

    Introduction: Staphylococcus aureus (S. aureus) causes a variety of infections, ranging from a mild skin infection to blood stream infections and deep seated infections. As Stapylococcus aureus bacteremia (SAB) has the tendency to cause endovascular and metastatic infections, complications can occur at almost all sites of the body. Hence, SAB is associated with increased morbidity and mortality in spite of appropriate antimicrobial treatment. The virulence in S. aureus is determined by the presence of adhesins and toxins, which behave like superantigens (SAgs) and leads to a massive release of proinflammatory cytokines causing overwhelming inflammatory response leading to endothelial leakage, hemodynamic shock, multiorgan failure, and possibly death. Materials and Methods: One year prospective study conducted in a tertiary care hospital in southern part of India included all patients with SAB. Clinical details were filled according to. All isolates were subjected to polymerase chain reaction (PCR) for enterotoxin profiling. Results: A total of 101 patients of SAB were identified which comprises of 61 (60.4%) patients with methicillin-susceptible S. aureus (MSSA) and 40 (39.6%) patients with methicillin-resistant S. aureus (MRSA). Most common predictors of mortality were prior hospitalization and antibiotic intake, severe organ dysfunction, shock, tachycardia, and leukocytosis. Two-third of the isolates had at least one enterotoxin, most prevalent was sea; 28% and 27% (P - value = 0.001) MSSA isolates had seg and sei; whereas, 38.6% (P - value < 0.001) of MRSA isolates were found to have sea. The most common enterotoxin associated with mortality was sei, which comprised of 38% of all mortality. Conclusion: In SAB, the significant predictors of mortality were prior hospitalization and antibiotic intake, presence of multiorgan dysfunction, and shock. Although overall significance between the enterotoxin and shock could not be demonstrated, it successfully demonstrated

  10. Prevalence of Toxin Genes among the Clinical Isolates of Staphylococcus aureus and its Clinical Impact.

    PubMed

    Deodhar, Divya; Varghese, George; Balaji, Veeraraghavan; John, James; Rebekah, Grace; Janardhanan, Jeshina; Jeyaraman, Ranjith; Jasmine, Sudha; Mathews, Prasad

    2015-01-01

    Staphylococcus aureus (S. aureus) causes a variety of infections, ranging from a mild skin infection to blood stream infections and deep seated infections. As Stapylococcus aureus bacteremia (SAB) has the tendency to cause endovascular and metastatic infections, complications can occur at almost all sites of the body. Hence, SAB is associated with increased morbidity and mortality in spite of appropriate antimicrobial treatment. The virulence in S. aureus is determined by the presence of adhesins and toxins, which behave like superantigens (SAgs) and leads to a massive release of proinflammatory cytokines causing overwhelming inflammatory response leading to endothelial leakage, hemodynamic shock, multiorgan failure, and possibly death. One year prospective study conducted in a tertiary care hospital in southern part of India included all patients with SAB. Clinical details were filled according to. All isolates were subjected to polymerase chain reaction (PCR) for enterotoxin profiling. A total of 101 patients of SAB were identified which comprises of 61 (60.4%) patients with methicillin-susceptible S. aureus (MSSA) and 40 (39.6%) patients with methicillin-resistant S. aureus (MRSA). Most common predictors of mortality were prior hospitalization and antibiotic intake, severe organ dysfunction, shock, tachycardia, and leukocytosis. Two-third of the isolates had at least one enterotoxin, most prevalent was sea; 28% and 27% (P - value = 0.001) MSSA isolates had seg and sei; whereas, 38.6% (P - value < 0.001) of MRSA isolates were found to have sea. The most common enterotoxin associated with mortality was sei, which comprised of 38% of all mortality. In SAB, the significant predictors of mortality were prior hospitalization and antibiotic intake, presence of multiorgan dysfunction, and shock. Although overall significance between the enterotoxin and shock could not be demonstrated, it successfully demonstrated the difference of enterotoxin between MSSA and MRSA.

  11. Putative Virulence Factor Expression by Clinical and Food Isolates of Bacillus spp. after Growth in Reconstituted Infant Milk Formulae

    PubMed Central

    Rowan, Neil J.; Deans, Karen; Anderson, John G.; Gemmell, Curtis G.; Hunter, Iain S.; Chaithong, Thararat

    2001-01-01

    Forty-seven strains representing 14 different Bacillus species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells. Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion. Of the 21 Bacillus cereus strains examined, 5 (24%) were invasive. A larger percentage of clinically derived Bacillus species (20%) than of similar species tested from the food environment were invasive. Increased invasion occurred after growth of selected Bacillus species in reconstituted IMF containing glucose. While PCR primer studies revealed that many different Bacillus species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF. Of the 47 Bacillus isolates examined, 3 isolates of B. cereus and 1 isolate of B. subtilis produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth. However, eight isolates belonging to the species B. cereus, B. licheniformis, B. circulans, and B. megaterium were found to produce this enterotoxin after growth in reconstituted IMF when assessed with the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit. It is concluded that several Bacillus species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors. PMID:11525980

  12. Spore prevalence and toxigenicity of Bacillus cereus and Bacillus thuringiensis isolates from U.S. retail spices.

    PubMed

    Hariram, Upasana; Labbé, Ronald

    2015-03-01

    Recent incidents of foodborne illness associated with spices as the vehicle of transmission prompted this examination of U.S. retail spices with regard to Bacillus cereus. This study focused on the levels of aerobic-mesophilic spore-forming bacteria and B cereus spores associated with 247 retail spices purchased from five states in the United States. Samples contained a wide range of aerobic-mesophilic bacterial spore counts (< 200 to 8.3 × 10(7) CFU/g), with 19.1% of samples at levels above 10(5) CFU/g. For examples, paprika, allspice, peppercorns, and mixed spices had high levels of aerobic spores (> 10(7) CFU/g). Using a novel chromogenic agar, B. cereus and B. thuringiensis spores were isolated from 77 (31%) and 11 (4%) samples, respectively. Levels of B. cereus were <3 to 1,600 MPN/g. Eighty-eight percent of B. cereus isolates and 91% of B. thuringiensis isolates possessed at least one type of enterotoxin gene: HBL (hemolysin BL) or nonhemolytic enterotoxin (NHE). None of the 88 isolates obtained in this study possessed the emetic toxin gene (ces). Using commercially available immunological toxin detection kits, the toxigenicity of the isolates was confirmed. The NHE enterotoxin was expressed in 98% of B. cereus and 91% of B. thuringiensis isolates that possessed the responsible gene. HBL enterotoxin was detected in 87% of B. cereus and 100% of B. thuringiensis PCR-positive isolates. Fifty-two percent of B. cereus and 54% of B. thuringiensis isolates produced both enterotoxins. Ninety-seven percent of B. cereus isolates grew at 12°C, although only two isolates grew well at 9°C. The ability of these spice isolates to form spores, produce diarrheal toxins, and grow at moderately abusive temperatures makes retail spices an important potential vehicle for foodborne illness caused by B. cereus strains, in particular those that produce diarrheal toxins.

  13. Exotoxin diversity of Staphylococcus aureus isolated from milk of cows with subclinical mastitis in Central Russia.

    PubMed

    Fursova, K K; Shchannikova, M P; Loskutova, I V; Shepelyakovskaya, A O; Laman, A G; Boutanaev, A M; Sokolov, S L; Artem'eva, O A; Nikanova, D A; Zinovieva, N A; Brovko, F A

    2018-05-01

    Mastitis, a major veterinary problem widespread in many regions, is caused mainly by Staphylococcus spp. However, there is no current reliable information about the role of Staphylococcus aureus and their toxins in the development of mastitis in cows in the territory of the Russian Federation. The aim of this investigation was to determine the profile of exotoxins of S. aureus from cow milk from farms of Central Russia. A total of 60 isolates of S. aureus were obtained from milk samples of cows with the subclinical form of mastitis. The exotoxin genes were identified using 2 types of PCR assays. The diversity of enterotoxin genes was studied by multiplex PCR. The percentage occurrence of enterotoxin genes was as follows: sea, 53.3%; seb, 3.3%; sec, 50%; sed, 4%; see, 46.6%; seg, 70%; sei, 10%; selp, 3.3%; and tsst1, 1.6%. The seh gene was not detected. The genes of pore-forming toxins and phenol-soluble modulins were identified by singleplex PCR and consisted of the following: hlA, 70%; lucS, 46.6%; psmA, 81.6%; psmB, 95%; and hld, 78.3%. The most abundant genes were psm (psmB, 95%), which codes for pore-forming toxins, and seg (70%), which codes for enterotoxins. The production of some enterotoxins in bacterial culture medium was detected by ELISA. The level of toxin production was near 1 ng/mL for SEA, SEE, SEG, SEI, SELP, and TSST-1 and reached a maximal level of 18 ng/mL for SEE. In the present work, we show that subclinical mastitis in cows is associated with S. aureus in the central region of the Russian Federation. Most of the isolates containing enterotoxin genes also had cytotoxin genes. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  14. Evaluation of in vitro properties of di-tri-octahedral smectite on clostridial toxins and growth.

    PubMed

    Weese, J S; Cote, N M; deGannes, R V G

    2003-11-01

    Clostridial colitis and endotoxaemia of intestinal origin are significant causes of morbidity and mortality in horses. Intestinal adsorbents are available for treatment of these conditions; however, little information exists supporting their use. To evaluate the ability of di-tri-octahedral smectite to bind to Clostridium difficile toxins A and B, C. perfringens enterotoxin and endotoxin, inhibit clostridial growth and the actions of metronidazole in vitro. Clostridium difficile toxins, C. perfringens enterotoxin and endotoxin were mixed with serial dilutions of di-tri-octahedral smectite, then tested for the presence of clostridial toxins or endotoxin using commercial tests. Serial dilutions of smectite were tested for the ability to inhibit growth of C. perfringens in culture broth, and to interfere with the effect of metronidazole on growth of C. perfringens in culture broth. Clostridium difficile toxins A and B, and C. perfringens enterotoxin were completely bound at dilutions of 1:2 to 1:16. Partial binding of C. difficile toxins occurred at dilutions up to 1:256 while partial binding of C. perfringens enterotoxin occurred up to a dilution of 1:128. Greater than 99% binding of endotoxin occurred with dilutions 1:2 to 1:32. No inhibition of growth of C. difficile or C. perfringens was present at any dilution, and there was no effect on the action of metronidazole. Di-tri-octahedral smectite possesses the ability to bind C. difficile toxins A and B, C. perfringens enterotoxin and endotoxin in vivo while having no effect on bacterial growth or the action of metronidazole. In vivo studies are required to determine whether di-tri-octahedral smectite might be a useful adjunctive treatment of clostridial colitis and endotoxaemia in horses.

  15. Outbreaks of cholera-like diarrhoea caused by enterotoxigenic Escherichia coli in the Brazilian Amazon Rainforest.

    PubMed

    Vicente, Ana C P; Teixeira, Luiz F M; Iniguez-Rojas, L; Luna, M G; Silva, L; Andrade, J R C; Guth, B E C

    2005-09-01

    The relationship between enteropathogens and severe diarrhoea in the Brazilian Amazon is poorly understood. In 1998, outbreaks of acute diarrhoea clinically diagnosed as cholera occurred in two small villages localized far from the main cholera route in the Brazilian rainforest. PCR was performed on some enteropathogens and heat-labile (LT) and/or heat-stable (STh) toxin genes, the virulence determinants of enterotoxigenic Escherichia coli (ETEC), were detected. Further characterization of ETEC isolates revealed the presence of two clones, one from each outbreak. One presenting serotype O167:H5 harboured LT-I and STh toxin genes and expressed the CS5CS6 colonization factor. The other, a non-typeable serotype, was positive for the LT-I gene and expressed the CS7 colonization factor. The current study demonstrates the importance of molecular diagnosis in regions such as the Amazon basin, where the enormous distances and local support conditions make standard laboratory diagnosis difficult. Here we also show that the mis-identified cholera cases were in fact associated with ETEC strains. This is the first report of ETEC, molecularly characterized as the aetiological agent of severe diarrhoea in children and adults in the Brazilian Amazon Rainforest.

  16. Antimicrobial resistance and production of toxins in Escherichia coli strains from wild ruminants and the alpine marmot.

    PubMed

    Caprioli, A; Donelli, G; Falbo, V; Passi, C; Pagano, A; Mantovani, A

    1991-04-01

    Escherichia coli strains isolated from 81 fecal samples from red deer (Cervus elaphus), roe deer (Capreoulus capreoulus), chamois (Rupicapra rupicapra) and alpine marmot (Marmota marmota) living in the Stelvio National Park, Italy, were examined for antimicrobial resistance and production of toxic factors. Direct plating of specimens on media containing antimicrobial drugs allowed us to isolate resistant strains of E. coli from 10 of 59 (17%) specimens examined by this technique. Nine of 31 specimens from red deer (29%) contained resistant strains. Different animals were likely colonized by the same resistant strain of E. coli. Conjugative R plasmids were found in four strains isolated from the marmot, roe deer and chamois. A strain from red deer produced heat-stable enterotoxin and another strain produced both hemolysin and cytotoxic necrotizing factor. A marmot isolate produced hemolysin alone. No strains were found to produce heat-labile enterotoxin or verotoxins.

  17. [Tracing to the source of staphylococcus aureus isolates from ice cream].

    PubMed

    Zhang, Yan-Jun; Xu, Dan-Ge; Fang, Ye-Zhen; Gong, Pu; Zhu, Min; Bao, Fang-Zhen

    2008-07-01

    To investigate the contamination of Staphylococcus aureus isolates in ice cream by phenotypic typing and molecular typing. The Staphylococcus aureus isolates were separated from ice cream, filler, cutter, salves and material. The separated isolates were characterized by drug-resistance, staphylococcal enterotoxin (SEA-E), SE (A-E, G-J) genes and pulsed-field gel electrophoresis (PFGE) types. Two Staphylococcus aureus isolates were separated, one from ice cream, another from cutter. Their characteristics of drug-resistance, staphylococcal enterotoxin (SEA-E), SE (A-E,G-J) genes and PFGE type were the same. The two Staphylococcus aureus isolates were the same clone. The contaminated Staphylococcus aureus isolates could be traced to the contaminated cutters.

  18. Enterotoxigenic Escherichia coli and probiotics in swine: what the bleep do we know?

    PubMed Central

    DUBREUIL, Jean Daniel

    2017-01-01

    The concept of certain microorganisms conferring direct benefits to the host relates to the term “probiotic”. Probiotics are microorganisms, bacteria, or yeast that when administered orally in sufficient quantity can counteract the effect of pathogenic microorganisms. The gastrointestinal (GI) tract is the site where probiotics are believed to play the most important role. The proposed effects of probiotics include antagonism of pathogens, interference with adherence, competition for nutrients, enterotoxin inactivation, modulation of the immune response, and strengthening of the intestinal barrier. From birth to postweaning, piglets are very sensitive to gut colonisation by pathogens. Enterotoxigenic Escherichia coli represents one of the most common agents of swine diarrhoea. The enterotoxins produced by this E. coli virotype are responsible for the loss of electrolytes and water observed following infection. This review addresses more specifically the studies done during the last 10 years deciphering the molecular mechanisms at play between host cell and probiotic interactions in the swine GI tract. PMID:28785529

  19. Incidence of enterotoxigenic staphylococci and their toxins in foods.

    PubMed

    Soriano, J M; Font, G; Rico, H; Moltó, J C; Mañes, J

    2002-05-01

    Of 504 food samples collected from cafeterias, 19 (3.8%) yielded strains of enterotoxigenic staphylococci, and 10 (52.6%), 4 (21.1%), 3 (15.8%), and 2 (10.5%) of these strains produced enterotoxins C (SEC), D (SED), B (SEB), and A (SEA), respectively. Moreover, SEA, SEB, and SEC were isolated from three hamburger samples. Of 181 food samples collected from four restaurants before the implementation of the hazard analysis and critical control point (HACCP) system, 7 (3.9%) were found to contain enterotoxigenic strains, and SED, SEC, and SEA were produced by 4 (57.1%), 2 (28.6%), and 1 (14.3%) of these strains, respectively. One meatball sample with SEC was detected in a restaurant. After the implementation of the HACCP system in four restaurants, neither enterotoxigenic staphylococci nor enterotoxins were detected in 196 studied samples.

  20. Selection of bacteriocin producer strains of lactic acid bacteria from a dairy environment.

    PubMed

    Lasagno, M; Beoleito, V; Sesma, F; Raya, R; Font de Valdez, G; Eraso, A

    2002-01-01

    Two strains showing bacteriocin production were selected from a total of 206 lactic acid bacteria isolated from samples of milk, milk serum, whey and homemade cheeses in Southern Cordoba, Argentina. This property was detected by means of well diffusion assays. The strains were identified as Enterococcus hirae and Enterococcus durans. The protein nature of those substances was proved by showing their sensitivity to type IV and XXV proteases, papaine, trypsin, pepsin and K proteinase. The bacteriocins inhibited the growth of Listeria monocytogenes, Bacillus cereus, Clostridium perfringes and two strains of Staphylococcus aureus, an A-enterotoxin and a B-enterotoxin producers. All of these bacteria are common pathogens usually associated with food borne diseases (ETA). These lactic acid bacteria or their bacteriocins could be suitable candidates for food preservation and specially useful in the our regional dairy industry.

  1. Studies on Pongamia pinnata (L.) Pierre leaves: understanding the mechanism(s) of action in infectious diarrhea*

    PubMed Central

    Brijesh, S.; Daswani, P.G.; Tetali, P.; Rojatkar, S.R.; Antia, N.H.; Birdi, T.J.

    2006-01-01

    While data are available on the effect of medicinal plants on intestinal motility and their antibacterial action, there is a paucity of information on their mode of action on various aspects of diarrheal pathogenicity, namely colonization to intestinal epithelial cells and production/action of enterotoxins. Crude decoction of dried leaves of Pongamia pinnata was evaluated for its antimicrobial (antibacterial, antigiardial and antirotaviral) effect; and its effect on production and action of enterotoxins (cholera toxin, CT; Escherichia coli labile toxin, LT; and E. coli stable toxin, ST); and adherence of enteropathogenic E. coli and invasion of enteroinvasive E. coli and Shigella flexneri to epithelial cells. The decoction had no antibacterial, antigiardial and antirotaviral activity, but reduced production of CT and bacterial invasion to epithelial cells. The observed results indicated that the crude decoction of P. pinnata has selective antidiarrheal action with efficacy against cholera and enteroinvasive bacterial strains causing bloody diarrheal episodes. PMID:16845722

  2. Pathophysiology of Campylobacter Enteritis

    DTIC Science & Technology

    1986-03-01

    variation exists either in individual of adhesion by various agents also suggests a variety of susceptibility to the organism or in the relative...Campylobacter Ill. Public Health Laboratory PERSPECTIVE Service. London. 9. Bloe , M. J., I. D. Berkowitz, F. M. LaForce, J. Cravens, L. B. The related...enterotoxin. p. 128. In A. D. Pearson, M. B. Skirrow. H. Lior, jejuni. Antimicrob. Agents Chemnother. 24-930-935. and B. Rowe (ed.). Campylobacter Ill

  3. Prophylactic Methods in Prevention of Disease Among Army Personnel

    DTIC Science & Technology

    1977-12-01

    E. Coli 1 . Enterotoxin in Infant Dependents. 20. Abstract (Contd) PCoxsackie A 21 and Polio ; and 74.7% by agents that were not determined.) Of the...virus specimens was conducted using Tryptose Phosphate Broth (TPB) as a control. The recovery of Vaccinia, ECHO 9, Coxsackievirus, Polio , Adenovirus...Mycoplasma, Coxsackie A21 and Polio ; and 74.7% by agents that were not determined. Of the Adenovirus isolates (1,073), 87.7% (941) were Adenovirus type 21, 8

  4. Occurrence of clostridia in commercially available curry roux.

    PubMed

    Fujisawa, T; Aikawa, K; Takahashi, T; Yamai, S; Ueda, S

    2001-12-01

    The occurrence of clostridia was investigated in a total of 60 commercially available curry roux samples. Clostridia were isolated from 37 (62%) samples, and Clostridium perfringens was isolated from 7 (12%) samples. The isolates of C. perfringens did not produce enterotoxin. The frequency of occurrence was higher by the enrichment broth culture detection method than by the agar plate or pouch method. These findings suggest that enrichment broth culture is necessary for the detection of clostridia.

  5. Toward Development of an Oral, Plant-Based Vaccine Against Escherichia coli O157:H7

    DTIC Science & Technology

    2004-01-01

    Mason, H. S., Haq, T. A., Clements, J. D., and Arntzen, C. J. (1998). Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT...based Vaccine Against Escherichia coli O157:H7.” beyond brief excerpts is with the permission of the copyright owner, and will save and hold...4. TITLE AND SUBTITLE Toward Development of an Oral, Plant-based Vaccine Against Escherichia coli O157:H7 5a. CONTRACT NUMBER 5b. GRANT

  6. Immunologic Control of Diarrheal Disease Due to Enterotoxigenic Escherichia coli: Reactogenicity, Immunogenicity, and Efficacy Studies of Pili Vaccines

    DTIC Science & Technology

    1981-09-01

    pathogens that lack CFA/I including enteropathogenic E. coli, some ETEC ana Vibrio cholerae (Figure 1). The mean change in net O.D. between the paired...intestine. In a further analogy, we have found that 20% of 50 recipients of a highly ad- hesive non-toxigenic Vibrio cholerae attenuated iaccine strain...and Characteristics of a Vibrio cholerae Mutant Lacking the A (ADP-Ribosylating) Portion of the Cholera Enterotoxin. Proc. Nat. Acad. Sci. USA 76:2052

  7. Infectious Multiple Drug Resistance in the Enterobacteriaceae

    DTIC Science & Technology

    1983-10-01

    producing a form of ST that does not Vibrio cholera and Eichernchia co/i. Infect Immun undergo the same posttranslational modification 1974; 10:320-7... cholera toxin. Although there have been numerous reports of the relationship between E. coli LT toxin (and cholera toxin) and a putative enterotoxin...dissect the fine molecular structure of cholera toxin and thereby create a general strategy by which subunit vaccines might be directly synthesized in the

  8. Seamless Integration of Detection and Therapy for Breast Cancer using Targeted Engineered Nanoparticles

    DTIC Science & Technology

    2007-06-01

    Quantum Electronics Conference, Snowbird, UT, January 2007. 20. “Nanophotonics: the next Big Thing”, Invited talk, CINT Annual Workshop, Los ...affinity, bac- terial, diarrheagenic, heat-stable enterotoxins (STs) and the lower affinity endogenous ligands guanylin and uro - guanylin, which induce...metabolic, and lo - comotor) were compared to explore whether the deficiency of APN altered physiology (Fig. 4). First, activity tests were per- formed in

  9. Review of Chemical, Biological, Radiological, and Nuclear (CBRN) Terminology in Technical Guide 316 (TG 316) and Allied Medical Publication 8(C) (AMedP-8(C))

    DTIC Science & Technology

    2013-05-01

    tularensis causing pneumonic tularemia (Aberdeen Proving Ground, MD: USAPHC, January 2012); USAPHC, Technical Guide 316 Supplement F1; USAPHC, Technical...Phase I preliminary BMEGs are completed for anthrax, plague, tularemia , ricin, and Staphylococcal Enterotoxin B (SEB). The Phase II process has been...Exposure to Specified Biological Agents: Brucellosis, Glanders, Q Fever, SEB and Tularemia , IDA Document D-4132 (Alexandria, VA: Institute for Defense

  10. Occurrence of Salmonella, Listeria monocytogenes, and enterotoxigenic Staphylococcus in goat milk from small and medium-sized farms located in Minas Gerais State, Brazil.

    PubMed

    Cavicchioli, V Q; Scatamburlo, T M; Yamazi, A K; Pieri, F A; Nero, L A

    2015-12-01

    Consumption of goat milk has been increasing due to its nutritional characteristics and health benefits. Therefore, assessment of the presence of foodborne pathogens in this product is critical to ensure its safety to consumers. The present study aimed to identify common foodborne pathogens in raw goat milk. Fifty-three samples of raw goat milk from 11 farms were collected and cultured for the presence of Salmonella spp. and Listeria monocytogenes, as well as for enumeration and isolation of coagulase-positive and coagulase-negative Staphylococcus (CPS and CNS, respectively). All samples tested negative for Salmonella spp. and L. monocytogenes. The CPS counts in raw goat milk samples were predominantly less than 2 log cfu/mL (n=39), and CNS counts were predominantly higher than 3 log cfu/mL (n=42). Based on Staphylococcus counts, 51 isolates were selected (CPS=26; CNS=25) and tested by PCR for the presence of classic enterotoxin-encoding genes (sea, seb, sec, sed, and see). Only 3 isolates (CPS=2, CNS=1) were negative for all enterotoxin-encoding genes tested, and the genotype sec and see was the most frequent (n=16), followed by sea, sec, and see (n=13) and sec (n=13); sed was not detected in any isolate. The frequencies of enterotoxin-encoding genes for CPS and CNS were similar, demonstrating the equivalence of both groups in harboring these virulent markers. These results suggest that Salmonella and L. monocytogenes are not frequent contaminants of raw goat milk, but that Staphylococcus spp. that are capable of producing enterotoxins are prevalent; therefore, consumers of raw goat milk and products made from raw milk are at risk. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  11. Regulation of intestinal mucosa guanylate cyclase by hemin, heme and protoporphyrin IX.

    PubMed

    elDeib, M M; Parker, C D; White, A A

    1987-04-02

    Mg2+-dependent activity of intestinal brush border guanylate cyclase was stimulated 4-5-fold by 50-100 microM hemin. Higher concentrations were inhibitory. In the presence of 25% dimethyl sulfoxide, which stimulated activity 9-times, 50 microM hemin further increased activity 1.7-fold. However, when activity was stimulated 32-fold by the Escherichia coli heat-stable enterotoxin, or 26-fold by Lubrol PX, hemin produced only concentration-dependent inhibition. The first type of activation was more sensitive to hemin than the second. Reduction of hemin by dithiothreitol eliminated stimulation of basal activity, while inhibition of Lubrol PX-stimulated activity remained. Protoporphyrin IX also had no effect on basal activity, however, it inhibited enterotoxin- and Lubrol PX-stimulated activities similarly, but only to half the extent of hemin. Substitution of Mn2+ for Mg2+ elevated basal activity 15-fold, and this Mn2+-dependent activity was inhibited by hemin. Mn2+-dependent activity was stimulated (43%) by enterotoxin, however, the stimulated activity was more sensitive to hemin inhibition than the basal Mn2+-dependent activity and both inhibition curves were congruent above 50 microM hemin. Hemin inhibition of Lubrol PX-stimulated activity was much less with Mn2+ than with Mg2+. These results were interpreted as suggesting two sites of hemin inhibition; on an inhibitory regulator and on the enzyme. We also found that the secretory effect of enterotoxin in the suckling mouse bioassay was reduced 56% by the oral administration of hemin.

  12. Enterotoxigenic coagulase positive Staphylococcus in milk and milk products, lben and jben, in northern Morocco.

    PubMed

    Bendahou, Abdrezzak; Abid, Mohammed; Bouteldoun, Nadine; Catelejine, Dierick; Lebbadi, Mariam

    2009-04-30

    The aim of this research was to determine the prevalence of enterotoxin genes (sea-seo) in Coagulase Positive Staphylococcus (CPS) isolated from unpasteurized milk and milk products. These results were compared with the results obtained by using the detection kit SET-RPLA for the specific detection of staphylococcal enterotoxins (SEA-SED). Eighty-one samples of milk and milk products were analyzed for the presence of Staphylococcus strains. Forty-six coagulase positive Staphylococcus isolates were tested for the production of staphylococcal enterotoxins (SEA-SED) by using the reversed passive latex agglutination method. The strains were also tested for the presence of se genes (sea-seo) by polymerase chain reaction. One or more classical enterotoxin products (SEA-SED) were observed in 39% of the strains tested, while se genes were detected in 56.5%. SEA and sea were most commonly detected. For newly discovered se genes among CPS isolates tested in this study, except the seh gene which was revealed in four isolates (8.7 %), none of the strains harbored any of the other se genes (see, seg, sei, sej, sek, sel, sem, seo and sen). The finding of a pathogen such as staphylococci-producing SEs and containing se genes in milk and milk products in northern Morocco may indicate a problem for public health in this region. The presence of enterotoxigenic strains in food does not always necessarily mean that the toxin will be produced. For that reason, the combination of both methods (RPLA and PCR) is a guarantee for success in diagnostic analysis tests.

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

    PubMed Central

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

    2014-01-01

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

  14. Enteric colonization by staphylococcus delphini in four ferret kits with diarrhoea.

    PubMed

    Gary, J M; Langohr, I M; Lim, A; Bolin, S; Bolin, C; Moore, I; Kiupel, M

    2014-11-01

    Four, 1-to 4-week-old ferret kits were submitted to the Diagnostic Center for Population and Animal Health at Michigan State University for post-mortem examination. Grossly, multiple bowel loops in all ferret kits were distended by mucoid faecal material. Microscopically, there was no evidence of inflammation or notable alteration to the normal mucosal morphology. Gram-positive coccoid bacteria colonized variable segments of the small intestine. These bacteria were identified as Staphylococcus delphini by phenotypic and molecular analyses. Enzyme-linked immunosorbent assay for detection of Staphylococcus enterotoxins was positive and polymerase chain reaction detected the gene for Staphylococcus enterotoxin E in the isolates. The hypersecretory diarrhoea in these ferret kits may have been associated with colonization of the small intestine by S. delphini, cultures of which were shown in vitro to be potentially capable of producing enterotoxin E. The condition described in these ferrets is similar to 'sticky' kit syndrome in mink. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Quantitative Prevalence and Toxin Gene Profile of Bacillus cereus from Ready-to-Eat Vegetables in South Korea.

    PubMed

    Chon, Jung-Whan; Yim, Jin-Hyeok; Kim, Hong-Seok; Kim, Dong-Hyeon; Kim, Hyunsook; Oh, Deog-Hwan; Kim, Soo-Ki; Seo, Kun-Ho

    2015-09-01

    Ready-to-eat (RTE) foods such as prepared vegetables are becoming an increasingly popular food choice. Since RTE vegetables are not commonly sterilized by heat treatment, contamination with foodborne pathogens such as Bacillus cereus (B. cereus) is a major concern. The objective of this study was to assess the quantitative prevalence and toxin gene profiles of B. cereus strains isolated from RTE vegetables. We found that 70 of the 145 (48%) tested retail vegetable salad and sprout samples were positive for B. cereus. The B. cereus isolates harbored at least one enterotoxin gene. The detection rates of nheABC, hblCDA, cytK, and entFM enterotoxin genes among all isolates were 97.1%, 100%, 81.4%, and 98.6%, respectively. No strain carried the emetic toxin genes. Only 4 strains (5.7%) from the 70 isolates were psychrotrophic and were able to grow at 7°C. All of the psychrotrophic isolates possessed at least 1 enterotoxin gene.

  16. Brucella sp. vertebral osteomyelitis with intercurrent fatal Staphylococcus aureus toxigenic enteritis in a bottlenose dolphin (Tursiops truncatus)

    PubMed Central

    Goertz, Caroline E. C.; Frasca, Salvatore; Bohach, Gregory A.; Cowan, Daniel F.; Buck, John D.; French, Richard A.; De Guise, Sylvain; Maratea, Jennifer; Hinckley, Lynn; Ewalt, Darla; Schlievert, Patrick M.; Karst, Sheila M.; Deobald, Claudia F.; St. Aubin, David J.; Dunn, J. Lawrence

    2013-01-01

    A previously beach-stranded, juvenile, male, bottlenose dolphin (Tursiops truncatus) was diagnosed with vertebral osteomyelitis of unknown etiology. Antemortem serological testing suggested past or current Brucella sp. infection; however, this could not be confirmed prior to death despite multiple isolation attempts from aspirates, blood, and biopsies. Systemic antibiotics were administered for over a year to control the suspected infection; however, the animal succumbed peracutely to a highly pathogenic, enterotoxin-secreting Staphylococcus sp. Gross necropsy findings included a fistulous tract leading to locally extensive osteomyelitis of a coccygeal vertebra with sequestra and osteophytes from which a Brucella species was isolated. Histopathological examination of intestine revealed pseudomembranous enteritis with a uniform population of intraluminal Gram-positive cocci. Staphylococcus aureus was isolated in pure culture from the intestine and tested positive for the staphylococcal enterotoxin A gene by polymerase chain reaction analysis. Serum taken shortly before death had endotoxin and elevated antibody titers to staphylococcal enterotoxin A when compared to samples collected during a period of apparent good health eighteen months earlier. The isolation of a pyrogenic toxin superantigen-producing staphylococcal isolate, clinical signs, and diagnostic findings in this animal resembled some of those noted in human toxic shock syndrome. The present case highlights the clinical challenges of treating chronic illnesses, complications of long-term antibiotic use, and promotion of pathogenic strains in cases of prolonged rehabilitation of marine mammals. PMID:21908337

  17. Manufacturing of a novel double-function ssDNA aptamer for sensitive diagnosis and efficient neutralization of SEA.

    PubMed

    Sedighian, Hamid; Halabian, Raheleh; Amani, Jafar; Heiat, Mohammad; Taheri, Ramezan Ali; Imani Fooladi, Abbas Ali

    2018-05-01

    Staphylococcal enterotoxin A (SEA) is an enterotoxin produced mainly by Staphylococcus aureus. In recent years, it has become the most prevalent compound for staphylococcal food poisoning (SFP) around the world. In this study, we isolate new dual-function single-stranded DNA (ssDNA) aptamers by using some new methods, such as the Taguchi method, by focusing on the detection and neutralization of SEA enterotoxin in food and clinical samples. For the asymmetric polymerase chain reaction (PCR) optimization of each round of systematic evolution of ligands by exponential enrichment (SELEX), we use Taguchi L9 orthogonal arrays, and the aptamer mobility shift assay (AMSA) is used for initial evaluation of the protein-DNA interactions on the last SELEX round. In our investigation the dissociation constant (K D ) value and the limit of detection (LOD) of the candidate aptamer were found to be 8.5 ± 0.91 of nM and 5 ng/ml using surface plasmon resonance (SPR). In the current study, the Taguchi and mobility shift assay methods were innovatively harnessed to improve the selection process and evaluate the protein-aptamer interactions. To the best of our knowledge, this is the first report on employing these two methods in aptamer technology especially against bacterial toxin. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. [Cases of menstrual toxic shock syndrome in the Czech Republic in 1997-2011].

    PubMed

    Petrás, P; Machová, I; Rysková, L; Prásil, P

    2011-11-01

    To determine toxigenicity and other basic characteristics of 47 strains of Staphylococcus aureus referred to the National Reference Laboratory for Staphylococci (NRL) as suspected causative agents of menstrual toxic shock syndrome (MTSS). S. aureus strains were collected from 11 administrative regions of the Czech Republic in 1997-2011. The diagnosis was based on phenotypic (reverse latex agglutination test) and genotypic (polymerase chain reaction) methods. Forty-four S. aureus strains were producers of toxic shock syndrome toxin 1 (TSST-1), either alone or in combination with staphylococcal enterotoxin. Three strains only produced enterotoxin (B, C, and H). MTSS is a serious multisystem disease. In this study, MTSS often had a severe course requiring intensive care. All MTSS patients used vaginal tampons that had been identified in the literature as a risk factor for MTSS. The case of MTSS in a 36-year-old woman caused by an enterotoxin H positive strain of S. aureus is probably the first to be reported in the world.

  19. Prevalence of foodborne pathogens in grilled chicken from street vendors and retail outlets in Reynosa, Tamaulipas, Mexico.

    PubMed

    Díaz-López, A; Cantú-Ramírez, R C; Garza-González, E; Ruiz-Tolentino, L; Tellez-Luis, S J; Rivera, G; Bocanegra-García, V

    2011-08-01

    We analyzed a total of 70 grilled chicken samples bought randomly from street vendors and retail outlets in the city of Reynosa, Mexico, to determine the prevalence of Escherichia coli (Shiga toxin producing and enterotoxin producing), Salmonella spp., Staphylococcus aureus, Listeria spp., and Campylobacter spp. using microbiological methods and PCR detection of bacterial sequences. Of the 70 samples, 27 (38.5%) were from retail outlets and 43 (61.4%) from street vendors. All specimens were negative by both microbiological and molecular methods for Listeria monocytogenes, Shiga toxin 2 of Shiga toxin-producing E. coli, lt of enterotoxin-producing E. coli, and st enterotoxin, and all were negative for Salmonella spp. and Campylobacter jejuni by PCR. Of the samples studied, 49 (70%) had undetectable levels of the foodborne pathogens studied with the methods used. In the remaining 21 (30%) specimens, at least one pathogen was isolated or detected, with E. coli being the pathogen most frequently isolated and with two samples bearing the hlyA gene. We found no statistical difference in bacterial prevalence between retail and street vendor samples. The presence of pathogens in grilled chicken is an important public health risk because of the great demand for and daily consumption of this product in this region.

  20. Staphylococcal food poisoning in the United States. New facts and old misconceptions.

    PubMed

    Holmberg, S D; Blake, P A

    1984-01-27

    To determine the current epidemiologic characteristics of staphylococcal food-borne disease (SFD), we reviewed 131 outbreaks reported to the Centers for Disease Control, Atlanta, from 1977 through 1981. Staphylococcal food-borne disease was the second most common cause of reported food-borne illness, affecting more than 7,000 persons during the five-year period; 10% of these patients visited or were admitted to hospitals for their illnesses. The proportion of outbreaks attributable to Staphylococcus aureus enterotoxins A through E has changed, with enterotoxin A being the only toxin incriminated during the last three years of this review. Milk--the most common source of enterotoxin C- and D-producing strains--and commercially packed foods are less common causes of SFD outbreaks now than they were before 1960. However, previously cooked, proteinaceous foods remain preeminent in causing SFD. The presence or absence of fever in infected persons, skin lesions in food handlers, or large numbers of staphylococci in food were unreliable as diagnostic criteria. Thorough epidemiologic investigation remains crucial to identifying SFD and its sources.

  1. Growth of enterotoxigenic Bacillus cereus on salmon (Oncorhynchus nerka).

    PubMed

    Labbé, Ronald; Rahmati, Talat

    2012-06-01

    We previously demonstrated the widespread presence of enterotoxigenic Bacillus cereus in marine foods. In view of the widespread consumption of raw fish, we sought to determine the ability of this organism to grow on the surface of wild Alaskan salmon at abusive temperatures (12, 16, and 20°C), using an isolate able to produce elevated levels of hemolysin BL enterotoxin and nonhemolytic enterotoxin. An incubation temperature of 37°C for colony formation was found to be selective for B. cereus grown on salmon held for up to 24 h at each temperature. A fivefold increase in log CFU per gram was observed after 26 and 22 h at 16 and 20°C, respectively, while a >4-log CFU/g increase occurred on salmon held at 12°C for 48 h. Generation times of 169.7, 53.5, and 45.6 min were observed at 12, 16, and 20°C. Nonhemolytic enterotoxin was detected when levels of B. cereus were in excess of 10(8) CFU/g. Nisin, at concentrations of 1 and 15 m g/g of salmon, reduced levels of B. cereus 2.5- and 25-fold, respectively. Our results indicate that fresh salmon can serve as an excellent substrate for enterotoxigenic B. cereus and that this organism can reach levels associated with foodborne illness following moderate temperature abuse.

  2. A novel bicomponent hemolysin from Bacillus cereus.

    PubMed Central

    Beecher, D J; MacMillan, J D

    1990-01-01

    A procedure combining isoelectric focusing (Sephadex IEF) and fast protein liquid chromatography (Superose 12; Mono-Q) removed hemolytic activity (presumably a contaminant) from partially purified preparations of the multicomponent diarrheal enterotoxin produced by Bacillus cereus. However, when the separated fractions were recombined, hemolytic activity was restored, suggesting that hemolysis is a property of the enterotoxin components. Combined fractions exhibited a unique ring pattern in gel diffusion assays in blood agar. During diffusion of the hemolysin from an agar well, the erythrocytes closest to the well were not lysed initially. After diffusion, hemolysis was observed as a sharp ring beginning several millimeters away from the edge of the well. With time the cells closer to the well were also lysed. This novel hemolysin consists of a protein (component B) which binds to or alters cells, allowing subsequent lysis by a second protein (component L). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and Western blot analysis showed that hemolysin BL has properties similar to those described previously for the enterotoxin and that both components are distinct from cereolysin and cereolysin AB. Images PMID:2114359

  3. 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

  4. Antibiotic resistance and molecular characteristics of Staphylococcus aureus isolated from backyard-raised pigs and pig workers.

    PubMed

    Momoh, Asabe Halimat; Kwaga, Jacob K P; Bello, Mohammed; Sackey, Anthony K B; Larsen, Anders Rhod

    2018-04-19

    Staphylococcus aureus is a commensal and pathogenic bacterium with impact on public health and livestock industry. The study investigated nasal carriage, antibiotic resistance, and molecular characterization of S. aureus in pigs and pig workers. Nasal swabs from 300 backyard-raised pigs and 101 pig workers were used for the study. Resulting isolates were confirmed using MALDI-TOF MS, tested for antibiotic resistance, and three different multiplex PCRs were used to detect enterotoxin, mecA, spaA, scn, and pvl genes. spa typing was used to annotate the isolates into MLST clonal complexes (CC). Structured questionnaire was used to access possible risk factors for S. aureus carriage. The prevalence of S. aureus in pigs and pig workers were 5.3 and 12.9%, respectively. The isolates were resistant to beta-lactams (97%), tetracycline (62%), sulfonamide (52%), aminoglycoside (20.6%), fluoroquinolone (24%), and mupirocin (3.4%). Twenty seven (93%) of the isolates carried scn, 7(24%) pvl, and 12 (41%) enterotoxin genes, respectively. Questionnaire survey showed medical-related occupation of household members was associated (p < 0.5) with S. aureus carriage. This study suggests the presence of human multidrug resistant strains of S. aureus, high carriage of pvl, and enterotoxin genes, and CC5, CC15, and CC152 were the CC-groups shared among pigs and pig workers.

  5. Phenotypic and molecular characterization of Staphylococcus aureus strains of veterinary, dairy and human origin.

    PubMed

    Gonano, M; Hein, I; Zangerl, P; Rammelmayr, A; Wagner, M

    2009-05-01

    Austrian veterinary (n=91), dairy (n=86), and human strains (n=48) of Staphylococcus aureus were tested for various phenotypic properties including clumping factor, egg-yolk reaction, production of thermonuclease and susceptibility to 14 antibiotics. In addition the expression of enterotoxins (A-E), and the presence of enterotoxin genes sea to sej and tst was determined. Significant differences in antimicrobial susceptibility were found with 84.6% of veterinary, 57.0% of dairy, and 20.8% of human strains susceptible to all antibiotics tested (P<0.0005). More human strains produced enterotoxins (41.7%) than veterinary (9.9%) and dairy strains (12.6%) while 40.7% and 38.5% of veterinary, 47.7% and 52.3% of dairy, and 77.1% and 87.5% of human strains were se- and tst-positive, respectively. AFLP analysis revealed nine clusters with over- or under-representation of strains with specific characteristics. Strains clustered according to origin (veterinary, dairy, and human) and/or presence of toxin genes and antimicrobial resistance.

  6. Probiotic Bacillus cereus Strains, a Potential Risk for Public Health in China

    PubMed Central

    Zhu, Kui; Hölzel, Christina S.; Cui, Yifang; Mayer, Ricarda; Wang, Yang; Dietrich, Richard; Didier, Andrea; Bassitta, Rupert; Märtlbauer, Erwin; Ding, Shuangyang

    2016-01-01

    Bacillus cereus is an important cause of foodborne infectious disease and food poisoning. However, B. cereus has also been used as a probiotic in human medicine and livestock production, with low standards of safety assessment. In this study, we evaluated the safety of 15 commercial probiotic B. cereus preparations from China in terms of mislabeling, toxin production, and transferable antimicrobial resistance. Most preparations were incorrectly labeled, as they contained additional bacterial species; one product did not contain viable B. cereus at all. In total, 18 B. cereus group strains—specifically B. cereus and Bacillus thuringiensis—were isolated. Enterotoxin genes nhe, hbl, and cytK1, as well as the ces-gene were assessed by PCR. Enterotoxin production and cytotoxicity were confirmed by ELISA and cell culture assays, respectively. All isolated B. cereus group strains produced the enterotoxin Nhe; 15 strains additionally produced Hbl. Antimicrobial resistance was assessed by microdilution; resistance genes were detected by PCR and further characterized by sequencing, transformation and conjugation assays. Nearly half of the strains harbored the antimicrobial resistance gene tet(45). In one strain, tet(45) was situated on a mobile genetic element—encoding a site-specific recombination mechanism—and was transferable to Staphylococcus aureus and Bacillus subtilis by electro-transformation. In view of the wide and uncontrolled use of these products, stricter regulations for safety assessment, including determination of virulence factors and transferable antimicrobial resistance genes, are urgently needed. PMID:27242738

  7. Organic Electrolytes for Sodium Batteries

    DTIC Science & Technology

    1992-09-01

    3 melts. 3.5.2.3.1.1 Standard potential of copper couples in AICI3 :BuPyCI melts versus aluminum in 3 . 5.3.4.1 acidic 2:1 (molar) A1CI3BuPyCI melt...19 cell. 3 . 5.4 . 2 . 5.2 V LT-i-t Qig~res.z Typical discharge/charge curves for the AI/MEICI:AICI 3 :FcCI 3 /RVC system inan acidic 1.3. 1 methyl...a 3.5.4. 1.2 acidic MEICh:AICI 3 (XAICI3 =0. 6 0) melt. vii List of Figures. Charge/discharge (2 mA) performance of cycle 10 for the Na/Nasi- 3 . 5

  8. Combat Failure: Nightmare of Armored Units Since World War II

    DTIC Science & Technology

    1991-12-18

    Ditribution is Unlimited 92-32409 l ...... i.9 92 REPORT DOCUMENTATION PAGE Inftng frtssfin lot ths. (@(Iftlon of informs lion. "I’ a’ttd to A.If *QE I No u...8217, D ’ IVY04𔃾. IM4Iudinqth lb ifl lt I Of 0 - l .~w~ng nt411`1JClions. 14 Vthing 4 -iiin.s ICII to oul rft M r me-oniemfli the data needed. and to’p4...l.udllng sug"uitrnI lotr redujsrnq thstrurci r rdn to Washington itoidrlujgime, li ",r oiryl r~t lif-otI l lotntr 0oviellons and Iteoorfa. IIIS Jgtmnoin

  9. Image-Based Approach to Mapping, Charting, and Geodesy.

    DTIC Science & Technology

    1982-02-01

    he..( reit- , st the .S Armyv I-npi ner Topographic Laboratories-, hll. Pi."ht inltete;(d inl de term ining the taipabil1i t ic e s nd 8 raotlhacho_...a.ve-ttor- bas-ed tipo I pi calIlIv t i l t ill-( I C I S) I (,r PiPC t; al p I( I i lt i ll (S fai-p i(%’ c t alI . , 1978’) , tr I F :TI, rt it I.,r...rt is to pr i de f 𔃻i : i t i t t he r in I ts of () Po r sei relit inidiui tic-d a t ,IllP. i n dir - ut t \\ Ii lisP Ordr l-P!U, Anit-odment Noe. 125

  10. 3D printing human induced pluripotent stem cells with novel hydroxypropyl chitin bioink: scalable expansion and uniform aggregation.

    PubMed

    Li, Yang; Jiang, Xulin; Li, Ling; Chen, Zhi-Nan; Gao, Ge; Yao, Rui; Sun, Wei

    2018-06-28

    Human induced pluripotent stem cells (hiPSCs) are more likely to successfully avoid the immunological rejection and ethical problems that are often encountered by human embryonic stem cells in various stem cell studies and applications. To transfer hiPSCs from the laboratory to clinical applications, researchers must obtain sufficient cell numbers. In this study, 3D cell printing was used as a novel method for iPSC scalable expansion. Hydroxypropyl chitin (HPCH), utilized as a new type of bioink, and a set of optimized printing parameters were shown to achieve high cell survival (> 90%) after the printing process and high proliferation efficiency (~ 32.3 folds) during subsequent 10-day culture. After the culture, high levels of pluripotency maintenance were recognized by both qualitative and quantitative detections. Compared with static suspension (SS) culture, hiPSC aggregates formed in 3D printed constructs showed a higher uniformity in size. Using novel dual-fluorescent labelling method, hiPSC aggregates in the constructs were found more inclined to form by &lt;i>in situ&lt;/i> proliferation rather than multicellular aggregation. This study revealed unique advantages of non-ionic crosslinking bioink material HPCH, including high gel strength and rapid temperature response in hiPSC printing, and achieved primed state hiPSC printing for the first time. Features achieved in this study, such as high cell yield, high pluripotency maintenance and uniform aggregation provide good foundations for further hiPSC studies on 3D micro-tissue differentiation and drug screening. © 2018 IOP Publishing Ltd.

  11. A study of the Al content impact on the properties of MmNi 4.4- xCo 0.6Al x alloys as precursors for negative electrodes in NiMH batteries

    NASA Astrophysics Data System (ADS)

    Bliznakov, S.; Lefterova, E.; Dimitrov, N.; Petrov, K.; Popov, A.

    AB 5-type hydrogen storage alloys with MmNi 4.4- xCo 0.6Al x (Mm-mischmetal) composition are synthesized, structurally and thermodynamically characterized, and electrochemically tested in 6 M KOH electrolyte. It is shown that an increase of the Al content in the alloy results in expansion of both the alloy lattice cell size and the unit cell volume. These structural changes lead to decrease of the plateau pressure and increase of the plateau width in the pressure-composition-temperature desorption isotherms. Improvement of the specific electrode capacity is also registered with the increase of the cell parameters. In addition to that the higher Al content is found to enhance the stability of the alloy components' hydrides. Maximum discharge capacity of 278 mAh g -1 is measured with an electrode made from a MmNi 3.6Co 0.6Al 0.8 alloy. Cycle life tests of the accordingly prepared electrodes suggest a stability that is comparable to the stability of commercially available hydrogen storage electrodes.

  12. Influence of rare earth content on Mm-based AB 5 metal hydride alloys for Ni-MH batteries-An X-ray fluorescence study

    NASA Astrophysics Data System (ADS)

    Ananth, M. V.; Raju, M.; Manimaran, K.; Balachandran, G.; Nair, Lekshmi M.

    AB 5-type MH alloys with Mm (Misch metal) as the A part (with varied rare earth contents in Mm) were investigated for rare earth by XRF analysis and battery performance by life cycle tests with an objective of understanding the influence of rare earth content on electrochemical hydrogen storage. The La/Ce ratio was found to vary from 0.51 to 18.73. The capacity output varied between 179 and 266 mAh g -1. The results show that the La/Ce ratio has a strong influence on the performance, with the best performance realized with samples having an La/Ce ratio of around 12. La enhancement facilitates easy activation due to refinement in grain size and interstitial dimensions. Also, an orderly influence on crystalline structure could be seen. The study demonstrates that the rare earth content is an essential factor in determining the maximum capacity output because of its influence on crystal orientation as well as an increase in the radius of the interstitials, lattice constants and cell volumes.

  13. Studies on metal hydride electrodes containing no binder additives

    NASA Astrophysics Data System (ADS)

    Rogulski, Z.; Dłubak, J.; Karwowska, M.; Krebs, M.; Pytlik, E.; Schmalz, M.; Gumkowska, A.; Czerwiński, A.

    Electrochemical properties of hydrogen storage alloys (AB 5 type: LaMm-Ni 4.1Al 0.3Mn 0.4Co 0.45) were studied in 6 M KOHaq using Limited Volume Electrode (LVE) method. Working electrodes were prepared by pressing alloy powder (without binding and conducting additives) into a metal net wire serving as a support and as a current collector. Cyclic voltammetry curves reveal well defined hydrogen sorption and desorption peaks which are separated from other faradic processes, such as surface oxidation. Voltammograms of LVE resemble the curves obtained by various authors for single particle metal alloy electrodes. Hydrogen diffusion coefficient calculated at room temperature for LV electrodes and for 100% state of charge reaches a constant value of ca. 3.3 × 10 -9 and 2.1 × 10 -10 cm 2 s -1, for chronoamperometric and chronopotentiometric measurements, respectively. A comparison of the electrodes with average alloy particle sizes of ca. 50 and 4 μm allows us to conclude that at room temperature hydrogen storage capability of AB 5 alloy studied is independent on the alloy particle size. On the other hand, reduction of the particle size increases alloy capacity at temperatures below -10 °C and reduces time of electrochemical activation of the electrode.

  14. Influence of electrolyte composition and temperature on behaviour of AB5 hydrogen storage alloy used as negative electrode in Ni-MH batteries

    NASA Astrophysics Data System (ADS)

    Karwowska, Malgorzata; Jaron, Tomasz; Fijalkowski, Karol J.; Leszczynski, Piotr J.; Rogulski, Zbigniew; Czerwinski, Andrzej

    2014-10-01

    The AB5-type metal alloy (Mm-Ni4.1Al0.2Mn0.4Co0.45) has been investigated in different electrolytes (LiOH, NaOH, KOH, RbOH, CsOH). All of the electrochemical measurements have been performed using limited volume electrode technique (LVE). Thickness of the working electrode is nearly equal to the diameter of the grain (ca. 50 μm). Hydrogen diffusion coefficient has been determined using chronoamperometry. Hydrogen diffusion coefficient calculated for 100% state of charge reaches maximum value in KOH (DH = 4.65·10-10 cm2 s-1). We have obtained the highest value of capacity for the electrode in KOH and the lowest - in CsOH. The temperature influence on alloy capacity has been also tested. The alloy has been also characterised with SEM coupled with EDS, TGA/DSC and powder XRD. The unit cell of MmNi4.1Al0.2Mn0.4Co0.45 have been refined in the Cu5.4Yb0.8 structure type (a modified LaNi5 structure); the structure is unaffected by the electrochemical treatment.

  15. Small polarons and point defects in LaFeO3

    NASA Astrophysics Data System (ADS)

    Zhu, Zhen; Peelaers, Hartwin; van de Walle, Chris G.

    The proton-conductive perovskite-type LaFeO3 is a promising negative-electrode material for Ni/metal-hydride (Ni-MH) batteries. It has a discharge capacity up to 530 mAhg-1 at 333 K, which is significantly higher than commercialized AB5-type alloys. To elucidate the underlying mechanism of this performance, we have investigated the structural and electronic properties of bulk LaFeO3, as well as the effect of point defects, using hybrid density functional methods. LaFeO3 is antiferromagnetic in the ground state with a band gap of 3.54 eV. Small hole and electron polarons can form through self- or point-defect-assisted trapping. We find that La vacancies and Sr substitutional on La sites are shallow acceptors with the induced holes trapped as small polarons, while O and Fe vacancies are deep defect centers. Hydrogen interstitials behave like shallow donors, with the donor electrons localized on nearby iron sites as electron polarons. With a large trapping energy, these polarons can act as electron or hole traps and affect the electrical performance of LaFeO3 as the negative electrode for Ni-MH batteries. We acknowledge DOE for financial support.

  16. Investigation of long term stability in metal hydrides

    NASA Technical Reports Server (NTRS)

    Marmaro, Roger W.; Lynch, Franklin E.; Chandra, Dhanesh; Lambert, Steve; Sharma, Archana

    1991-01-01

    It is apparent from the literature and the results of this study that cyclic degradation of AB(5) type metal hydrides varies widely according to the details of how the specimens are cycled. The Rapid Cycle Apparatus (RCA) used produced less degradation in 5000 to 10000 cycles than earlier work with a Slow Cycle Apparatus (SCA) produced in 1500 cycles. Evidence is presented that the 453 K (356 F) Thermal Aging (TA) time spent in the saturated condition causes hydride degradation. But increasing the cooling (saturation) period in the RCA did not greatly increase the rate of degradation. It appears that TA type degradation is secondary at low temperatures to another degradation mechanism. If rapid cycles are less damaging than slow cycles when the saturation time is equal, the rate of hydriding/dehydriding may be an important factor. The peak temperatures in the RCA were about 30 C lower than the SCA. The difference in peak cycle temperatures (125 C in the SCA, 95 C in RCA) cannot explain the differences in degradation. TA type degradation is similar to cyclic degradation in that nickel peaks and line broadening are observed in X ray diffraction patterns after either form of degradation.

  17. Feasibility analysis of a hydrogen backup power system for Russian telecom market

    NASA Astrophysics Data System (ADS)

    Borzenko, V. I.; Dunikov, D. O.

    2017-11-01

    We performed feasibility analysis of 10 kW hydrogen backup power system (H2BS) consisting of a water electrolyzer, a metal hydride hydrogen storage and a fuel cell. Capital investments in H2BS are mostly determined by the costs of the PEM electrolyzer, the fuel cell and solid state hydrogen storage materials, for single unit or small series manufacture the cost of AB5-type intermetallic compound can reach 50% of total system cost. Today the capital investments in H2BS are 3 times higher than in conventional lead-acid system of the same capacity. Wide distribution of fuel cell hydrogen vehicles, development of hydrogen infrastructure, and mass production of hydrogen power systems will for sure lower capital investments in fuel cell backup power. Operational expenditures for H2BS is only 15% from the expenditures for lead acid systems, and after 4-5 years of exploitation the total cost of ownership will become lower than for batteries.

  18. Methicillin-resistant Staphylococcus aureus: a controversial food-borne pathogen.

    PubMed

    Sergelidis, D; Angelidis, A S

    2017-06-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of severe healthcare-associated (HA) infections. Although during the last decade the incidence of HA invasive infections has dropped, the incidence of community-associated MRSA (CA-MRSA) infections has risen among the general population. Moreover, CA-MRSA, livestock-associated MRSA (LA-MRSA) and HA-MRSA (HA-MRSA) can be found in foods intended for human consumption. Several studies from different geographical areas have reported the presence of enterotoxin genes in several MRSA food isolates. Molecular typing studies have revealed genetic relatedness of these enterotoxigenic isolates with isolates incriminated in human infections. The contamination sources for foods, especially animal-origin foods, may be livestock as well as humans involved in animal husbandry and food-processing. Under favourable environmental conditions for growth and enterotoxin production, enterotoxigenic S. aureus isolates present in foods can cause staphylococcal food poisoning (SFP), irrespective of the contamination origin. Owing to the typically moderate clinical manifestations of SFP, the S. aureus strains responsible for SFP (cases or outbreaks) are frequently either not identified or not further characterized. Antimicrobial susceptibility testing is rarely performed, because administration of antimicrobial therapy is not required in the vast majority of cases. Staphylococcal food poisoning is the result of consumption of foods with preformed enterotoxins. Hence, similar to methicillin-sensitive enterotoxigenic S. aureus, enterotoxigenic MRSA can also act as food-borne pathogens upon favourable conditions for growth and enterotoxin production. The severity of the intoxication is not related to the antimicrobial resistance profile of the causative S. aureus strain and therefore MRSA food-borne outbreaks are not expected to be more severe. This review evaluates the potential of methicillin-resistant Staphylococcus

  19. Enterotoxigenicity and Antimicrobial Resistance of Staphylococcus aureus Isolated from Retail Food in China

    PubMed Central

    Wang, Wei; Baloch, Zulqarnain; Jiang, Tao; Zhang, Cunshan; Peng, Zixin; Li, Fengqin; Fanning, Séamus; Ma, Aiguo; Xu, Jin

    2017-01-01

    Staphylococcus aureus is one of the most common causes of zoonotic agent in the world, which are attributable to the contamination of food with enterotoxins. In this study, a total of 1,150 S. aureus isolates were cultured from 27,000 retail foods items from 203 cities of 24 provinces in China in 2015 and were test for antimicrobial susceptibility. Additionally, the role of the genes responsible for the staphylococcal enterotoxins (SEA to SEE), methicillin resistance (mecA) and the toxigenic capabilities were also assessed. The results showed that 4.3% retail foods were contaminated with S. aureus, and 7.9% retail foods isolates were mecA positive. Some 97.6% of S. aureus isolates were resistant to at least one antimicrobial compound, and 57.5% of these were multi drug resistant (MDR). Resistance to penicillin (83.7%, 963/1,150), was common, followed by linezolid (67.7%, 778/1,150) and erythromycin (52.1%, 599/1,150). The isolates cultured from raw meats showed high levels of resistant to tetracycline (42.8%), ciprofloxacin (17.4%), and chloramphenicol (12.0%) and expressed a MDR phenotype (62.4%). A total of 29.7% S. aureus isolates harbored the classical SEs genes (sea, seb, sec, and sed). The sea and seb genes were the most frequent SEs genes detected. Of note, 22% of the SEs genes positive S. aureus harbored two or three SEs genes, and 16 isolates were confirmed with the capacity to simultaneously produce two or three enterotoxin types. Moreover, nearly 50% of the MRSA isolates were positive for at least one SE gene in this study. Therefore, it is important to monitor the antimicrobial susceptibility and enterotoxigenicity of MDR S. aureus and MRSA in the food chain and to use these data to develop food safety measures, designed to reduce the contamination and transmission of this bacterium. PMID:29209290

  20. Safety hazards in bacteriocinogenic Staphylococcus strains isolated from goat and sheep milk.

    PubMed

    Rahmdel, Samane; Hosseinzadeh, Saeid; Shekarforoush, Seyed Shahram; Torriani, Sandra; Gatto, Veronica; Pashangeh, Safoora

    2018-03-01

    In this study, 28 bacteriocinogenic Staphylococcus strains isolated from goat and sheep milk were subjected to the PCR detection of enterotoxin genes (sea-see), enterotoxin-like toxin Q gene (selq), toxic shock syndrome toxin gene (tst1), and antibiotic resistance genes. They were also evaluated for phenotypic resistance against 10 antibiotics and hemolytic activity. The tyramine and histamine production was investigated using the agar plate assay and capillary zone electrophoretic analysis (CZE). Twenty-five isolates harbored at least one enterotoxin gene. The gene sec was the most frequent (89%). The gene tst1 was found in 84% of sec-positive isolates. The occurrence of antibiotic resistance genes was in the order of blaZ/tetK (100%), mecA/ermB (86%), ermC (50%), and tetM (18%). The genes ermA, aac(6')Ie-aph(2″)Ia, vanA, and vanB were absent in all the isolates. Nineteen isolates were phenotypically susceptible to all the antibiotics. The only isolate with phenotypic resistance to penicillin G and oxacillin was S. epidermidis 4S93 which had a different SmaI-PFGE profile from those of the other S. epidermidis strains. All the S. haemolyticus and S. pseudintermedius isolates were not susceptible to trimethoprim. Twenty-five isolates showed complete or partial hemolytic activity. None of the isolates was able to decarboxylate tyrosine, while CZE analysis revealed histamine formation activity in S. haemolyticus 4S12. The occurrence of safety risks in the isolates reinforces the need for regular monitoring of food-producing animals to mitigate the risks of multidrug resistant and zoonotic pathogens. Moreover, none of the isolates fulfilled the safety criteria to be used as starter cultures or biopreservatives. Copyright © 2018. Published by Elsevier Ltd.

  1. Outbreak of Staphylococcal food poisoning due to SEA-producing Staphylococcus aureus.

    PubMed

    Johler, Sophia; Tichaczek-Dischinger, Petra S; Rau, Jörg; Sihto, Henna-Maria; Lehner, Angelika; Adam, Maja; Stephan, Roger

    2013-09-01

    In 2008, 150 people gathered for a wedding celebration in Baden-Württemberg, Germany. Three hours after ingestion of a variety of foods including pancakes filled with minced chicken, several guests exhibited symptoms of acute gastroenteritis such as vomiting, diarrhea, fever, and ague. Twelve guests were reported to have fallen ill, with nine of these seeking medical care in hospitals. At least four patients were admitted to the hospital and received inpatient treatment, among them a 2-year-old child and a woman in the 4th month of pregnancy. Within 24 h of the event, an investigative team collected a variety of samples including refrigerated leftovers, food in the storage unit of the caterer, nasal swabs of the caterer, as well as 21 environmental swabs. Five stool samples from patients were provided by the hospitals. Staphylococcus aureus isolates were gathered from eight samples, among them nasal swabs of the caterer, food samples, and one stool sample. Fourier transform-infrared spectroscopy was used for species identification and for primary clustering of the isolates in a similarity tree. The isolates were further characterized by spa typing and pulsed-field gel electrophoresis, and a DNA microarray was used to determine the presence/absence of genes involved in virulence and antimicrobial resistance. We were able to match an enterotoxigenic strain from the stool sample of a patient to isolates of the same strain obtained from food and the nasal cavity of a food handler. The strain produced the enterotoxin SEA and the toxic shock syndrome toxin-1, and was also found to exhibit the genes encoding enterotoxins SEG and SEI, as well as the enterotoxin gene cluster egc. This is one of only a few studies that were able to link a staphylococcal food poisoning outbreak to its source.

  2. Staphylococcal poisoning foodborne outbreak: epidemiological investigation and strain genotyping.

    PubMed

    Gallina, S; Bianchi, D M; Bellio, A; Nogarol, C; Macori, G; Zaccaria, T; Biorci, F; Carraro, E; Decastelli, L

    2013-12-01

    In June 2011, an outbreak of Staphylococcus aureus enterotoxin food poisoning gastroenteritis occurred in Turin, Italy, following a catered dinner party at a private home. Within a few hours, 26 of the 47 guests experienced gastrointestinal illness, and 9 were hospitalized. A retrospective cohort study using a standardized questionnaire was carried out, and the risk ratios for each food item were calculated. The analysis indicated consumption of seafood salad as the most probable cause of the outbreak (risk ratio = 11.72; 95 % confidence interval, 1.75 to 78.54). Biological samples were collected from four of the hospitalized guests (stool and vomit), nasal mucosa swabs from three food handlers employed with the caterer, and available food residuals. All stool and vomit samples tested positive for enterotoxigenic S. aureus. As residues of the seafood salad were no longer available for sampling, suspected contamination could not be verified. However, no other food was found contaminated by S. aureus or its enterotoxins. All isolates from the biological samples were characterized at the genomic level by means of two multiplex PCR protocols to determine the presence of genes encoding staphylococcal enterotoxins, pulsed-field gel electrophoresis and staphylococcal protein A gene (spa) typing to describe their genetic profiles. All the isolates presented genes encoding SEA and SEI; the pulsed-field gel electrophoresis genetic profiles revealed the same pulsotype in the microorganism isolated from the hospitalized guests as in one of the isolates from a food handler's nasal mucosa, and the spa typing analysis reported two closely related spa types (t701 and t267), implicating the food handler as the most likely outbreak source.

  3. Diarrhoeal disease through enterocyte secretion: a doctrine untroubled by proof.

    PubMed

    Lucas, Michael L

    2010-04-01

    For almost 40 years, one of the principal causes of diarrhoeal disease has been thought to be fluid secretion emanating from the epithelial cells of the small and large intestine. Given the extremely large fluid losses seen in cholera, where secretion can be up to several litres per day, this seems a plausible hypothesis. The enterocyte (epithelial cell) secretion hypothesis rapidly displaced all other alternatives, such as vasodilatation coupled with enhanced paracellular permeability. An essential mechanism underlying enterocyte secretion has always been assumed to be electrogenic chloride secretion, leading to a localized osmotic imbalance at the mucosal surface of the enterocytes that causes fluid entry into the lumen by osmosis. The chloride secretion basis for enterotoxin-deranged secretion is assumed to be measurable by changes in electrical currents and by altered transport of chloride ion. These can be detected after the small intestine is exposed to a heat-stable enterotoxin (STa) produced by Escherichia coli. However, in vivo, when the recovered volume technique is used, STa is found not to be secretory. The heat-stable enterotoxin is therefore a test case toxin, because the complex techniques used to demonstrate enterocyte secretion after STa exposure show apparent secretion, while the simplest technique based on fluid recovery and genuinely measuring the mass transport of fluid does not. This review scrutinizes the nature of the evidence put forward for enterocyte secretion and reaches the conclusion that there is no evidence for it. Debilitating secretion undoubtedly does take place in severe diarrhoeal disease, but secretion from enterocytes is unlikely to be the cause.

  4. The prevalence of antimicrobial resistance and carriage of virulence genes in Staphylococcus aureus isolated from food handlers in Kuwait City restaurants

    PubMed Central

    Udo, Edet E; Al-Mufti, Siham; Albert, M John

    2009-01-01

    Background Staphylococcus aureus is a major cause of food poisoning due to their ability to produce enterotoxins which if ingested in sufficient amounts results in sickness. Food handlers carrying enterotoxin-producing S. aureus in their noses or hands can contaminate food leading to food poisoning. We characterized 200 S. aureus obtained from food handlers in different restaurants for antibacterial resistance and the carriage of virulence genes. Findings Susceptibility to antibacterial agents was determined by disk diffusion and Etest. PCR was used to detect genes for accessory gene regulator (agr); capsular polysaccharide (cap) 5 and 8, staphylococcal enterotoxins (SE), toxic shock syndrome toxin-1 (TSST-1) and Panton-Valentine leukocidin (PVL). Isolates were typed using pulsed-field gel electrophoresis. In total 185 (92.5%) of the 200 isolates expressed resistance to antibacterial agents. They were resistant to penicillin G (82.0%), tetracycline (19.0%), erythromycin (2.5%), clindamycin (2.0%), trimethoprim (7.5%), kanamycin (2.5%), streptomycin (1.5%), ciprofloxacin (1.5%), fusidic acid (1.0%) and cadmium acetate (68.0%). Seventy-six (38.0%) and 114 (57.0%) isolates had type 5 and type 8 capsular polysaccharides respectively. The agr types I, II and III alleles were detected in 50.5%, 20.0% and 23.5% of the isolates respectively. They contained genes for SEI (38.5%), SEG (24.0%), SEC (23.0%), SEB (12.5%), SEH (21.5%), SEA (11.0), SED (1.5%), SEE (1.5%), TSST-1 (4.0%) and PVL (9.0%). Conclusion This study revealed a high prevalence of antibacterial resistance and virulence determinants in S. aureus from food handlers in Kuwait restaurants justifying the screening of food handlers to detect and treat carriers and protect restaurant customers from staphylococcal food poisoning. PMID:19531224

  5. Genotype and enterotoxigenicity of Staphylococcus epidermidis isolate from ready to eat meat products

    PubMed Central

    Podkowik, Magdalena; Seo, Keun Seok; Schubert, Justyna; Tolo, Isaiah; Robinson, D. Ashley; Bania, Jacek; Bystroń, Jarosław

    2016-01-01

    We have previously shown that potentially pathogenic isolates of Staphylococcus epidermidis occur at high incidence in ready-to-eat food. Now, within 164 samples of ready-to-eat meat products we identified 32 S. epidermidis isolates. In 8 isolates we detected the genes encoding for staphylococcal enterotoxins, but in 7 S. epidermidis isolates these genes were not stable over passages. One isolate designated 4S was shown to stably harbour sec and sel genes.In the genome sequence of S. epidermidis 4S we identified 21,426-bp region flanked by direct-repeats, encompassing sec and sel genes, corresponding to the previously described composite staphylococcal pathogenicity island (SePI) in S. epidermidis FRI909. Alignment of S. epidermidis 4S and S. epidermidis FRI909 SePIs revealed 6 nucleotide mismatches located in 5 of the total of 29 ORFs. Genomic location of S. epidermidis 4S SePI was the same as in FRI909. S. epidermidis 4S is a single locus variant of ST561, being genetically different from FRI909. SECepi was secreted by S. epidermidis 4S to BHI broth ranging from 14 to almost 36 μg/mL, to milk ranging from 6-9 ng/mL, to beef meat juice from 2-3 μg/mL and to pork meat juice from 1-2 μg/mL after 24 and 48 hours of cultivation, respectively. We provide the first evidence that S. epidermidis occurring in food bears an element encoding an orthologue to S. aureus SEC, and that SECepi can be produced in microbial broth, milk and meat juices. Regarding that only enterotoxins produced by S. aureus are officially tracked in food in EU, the ability to produce enterotoxin by S. epidermidis pose real risk for food safety. PMID:27105039

  6. Genotype and enterotoxigenicity of Staphylococcus epidermidis isolate from ready to eat meat products.

    PubMed

    Podkowik, Magdalena; Seo, Keun Seok; Schubert, Justyna; Tolo, Isaiah; Robinson, D Ashley; Bania, Jacek; Bystroń, Jarosław

    2016-07-16

    We have previously shown that potentially pathogenic isolates of Staphylococcus epidermidis occur at high incidence in ready-to-eat food. Now, within 164 samples of ready-to-eat meat products we identified 32 S. epidermidis isolates. In 8 isolates we detected the genes encoding for staphylococcal enterotoxins, but in 7 S. epidermidis isolates these genes were not stable over passages. One isolate designated 4S was shown to stably harbour sec and sel genes. In the genome sequence of S. epidermidis 4S we identified 21,426-bp region flanked by direct-repeats, encompassing sec and sel genes, corresponding to the previously described composite staphylococcal pathogenicity island (SePI) in S. epidermidis FRI909. Alignment of S. epidermidis 4S and S. epidermidis FRI909 SePIs revealed 6 nucleotide mismatches located in 5 of the total of 29 ORFs. Genomic location of S. epidermidis 4S SePI was the same as in FRI909. S. epidermidis 4S is a single locus variant of ST561, being genetically different from FRI909. SECepi was secreted by S. epidermidis 4S to BHI broth ranging from 14 to almost 36μg/mL, to milk ranging from 6 to 9ng/mL, to beef meat juice from 2 to 3μg/mL and to pork meat juice from 1 to 2μg/mL after 24 and 48h of cultivation, respectively. We provide the first evidence that S. epidermidis occurring in food bears an element encoding an orthologue to Staphylococcus aureus SEC, and that SECepi can be produced in microbial broth, milk and meat juices. Regarding that only enterotoxins produced by S. aureus are officially tracked in food in EU, the ability to produce enterotoxin by S. epidermidis pose real risk for food safety. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. The effects of subinhibitory concentrations of costus oil on virulence factor production in Staphylococcus aureus.

    PubMed

    Qiu, J; Wang, J; Luo, H; Du, X; Li, H; Luo, M; Dong, J; Chen, Z; Deng, X

    2011-01-01

    To determine the antimicrobial activity of costus (Saussurea lappa) oil against Staphylococcus aureus, and to evaluate the influence of subinhibitory concentrations of costus oil on virulence-related exoprotein production in staph. aureus. Minimal inhibitory concentrations (MICs) were determined using a broth microdilution method, and the MICs of costus oil against 32 Staph. aureus strains ranged from 0.15 to 0.6 μl ml(-1) . The MIC(50) and MIC(90) were 0.3 and 0.6 μl ml(-1) , respectively. Western blot, haemolytic, tumour necrosis factor (TNF) release and real-time RT-PCR assays were performed to evaluate the effects of subinhibitory concentrations of costus oil on virulence-associated exoprotein production in Staph. aureus. The data presented here show that costus oil dose dependently decreased the production of α-toxin, toxic shock syndrome toxin 1 (TSST-1) and enterotoxins A and B in both methicillin-sensitive Staph. aureus (MSSA) and methicillin-resistant Staph. aureus (MRSA). Costus oil has potent antimicrobial activity against Staph. aureus, and the production of α-toxin, TSST-1 and enterotoxins A and B in Staph. aureus was decreased by costus oil. The data suggest that costus oil may deserve further investigation for its potential therapeutic value in treating Staph. aureus infections. Furthermore, costus oil could be rationally applied in food products as a novel food preservative both to inhibit the growth of Staph. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins. © 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.

  8. Identification of virulence factors in 16S-23S rRNA intergenic spacer genotyped Staphylococcus aureus isolated from water buffaloes and small ruminants.

    PubMed

    Cremonesi, P; Zottola, T; Locatelli, C; Pollera, C; Castiglioni, B; Scaccabarozzi, L; Moroni, P

    2013-01-01

    Staphylococcus aureus is an important human and animal pathogen, and is regarded as an important cause of intramammary infection (IMI) in ruminants. Staphylococcus aureus genetic variability and virulence factors have been well studied in veterinary medicine, especially in cows as support for control and management of IMI. The aim of the present study was to genotype 71 Staph. aureus isolates from the bulk tank and foremilk of water buffaloes (n=40) and from udder tissue (n=7) and foremilk (n=24) from small ruminants. The method used was previously applied to bovine Staph. aureus and is based on the amplification of the 16S-23S rRNA intergenic spacer region. The technique applied was able to identify different Staph. aureus genotypes isolated from dairy species other than the bovine species, and cluster the genotypes according to species and herds. Virulence gene distribution was consistent with genotype differentiation. The isolates were also characterized through determination of the presence of 19 virulence-associated genes by specific PCR. Enterotoxins A, C, D, G, I, J, and L were associated with Staph. aureus isolates from buffaloes, whereas enterotoxins C and L were linked to small ruminants. Genes coding for methicillin resistance, Panton-Valentine leukocidin, exfoliative toxins A and B, and enterotoxins B, E, and H were undetected. These findings indicate that RNA template-specific PCR is a valid technique for typing Staph. aureus from buffaloes and small ruminants and is a useful tool for understanding udder infection epidemiology. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  9. Secreted virulence factor comparison between methicillin-resistant and methicillin-sensitive Staphylococcus aureus, and its relevance to atopic dermatitis.

    PubMed

    Schlievert, Patrick M; Strandberg, Kristi L; Lin, Ying-Chi; Peterson, Marnie L; Leung, Donald Y M

    2010-01-01

    Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains have emerged as serious health threats in the last 15 years. They are associated with large numbers of atopic dermatitis skin and soft tissue infections, but when they originate from skin and mucous membranes, have the capacity to produce sepsis and highly fatal pulmonary infections characterized as necrotizing pneumonia, purpura fulminans, and postviral toxic shock syndrome. This review is a discussion of the emergence of 3 major CA-MRSA organisms, designated CA-MRSA USA400, followed by USA300, and most recently USA200. CA-MRSA USA300 and USA400 isolates and their methicillin-sensitive counterparts (community-associated methicillin-sensitive S aureus) typically produce highly inflammatory cytolysins alpha-toxin, gamma-toxin, delta-toxin (as representative of the phenol soluble modulin family of cytolysins), and Panton Valentine leukocidin. USA300 isolates produce the superantigens enterotoxin-like Q and a highly pyrogenic deletion variant of toxic shock syndrome toxin 1 (TSST-1), whereas USA400 isolates produce the superantigens staphylococcal enterotoxin B or staphylococcal enterotoxin C. USA200 CA-MRSA isolates produce small amounts of cytolysins but produce high levels of TSST-1. In contrast, their methicillin-sensitive S aureus counterparts produce various cytolysins, apparently in part dependent on the niche occupied in the host and levels of TSST-1 expressed. Significant differences seen in production of secreted virulence factors by CA-MRSA versus hospital-associated methicillin-resistant S aureus and community-associated methicillin-sensitive S aureus strains appear to be a result of the need to specialize as the result of energy drains from both virulence factor production and methicillin resistance. Copyright 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  10. “Black holes” and bacterial pathogenicity: A large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli

    PubMed Central

    Maurelli, Anthony T.; Fernández, Reinaldo E.; Bloch, Craig A.; Rode, Christopher K.; Fasano, Alessio

    1998-01-01

    Plasmids, bacteriophages, and pathogenicity islands are genomic additions that contribute to the evolution of bacterial pathogens. For example, Shigella spp., the causative agents of bacillary dysentery, differ from the closely related commensal Escherichia coli in the presence of a plasmid in Shigella that encodes virulence functions. However, pathogenic bacteria also may lack properties that are characteristic of nonpathogens. Lysine decarboxylase (LDC) activity is present in ≈90% of E. coli strains but is uniformly absent in Shigella strains. When the gene for LDC, cadA, was introduced into Shigella flexneri 2a, virulence became attenuated, and enterotoxin activity was inhibited greatly. The enterotoxin inhibitor was identified as cadaverine, a product of the reaction catalyzed by LDC. Comparison of the S. flexneri 2a and laboratory E. coli K-12 genomes in the region of cadA revealed a large deletion in Shigella. Representative strains of Shigella spp. and enteroinvasive E. coli displayed similar deletions of cadA. Our results suggest that, as Shigella spp. evolved from E. coli to become pathogens, they not only acquired virulence genes on a plasmid but also shed genes via deletions. The formation of these “black holes,” deletions of genes that are detrimental to a pathogenic lifestyle, provides an evolutionary pathway that enables a pathogen to enhance virulence. Furthermore, the demonstration that cadaverine can inhibit enterotoxin activity may lead to more general models about toxin activity or entry into cells and suggests an avenue for antitoxin therapy. Thus, understanding the role of black holes in pathogen evolution may yield clues to new treatments of infectious diseases. PMID:9520472

  11. Enterotoxigenicity and Antimicrobial Resistance of Staphylococcus aureus Isolated from Retail Food in China.

    PubMed

    Wang, Wei; Baloch, Zulqarnain; Jiang, Tao; Zhang, Cunshan; Peng, Zixin; Li, Fengqin; Fanning, Séamus; Ma, Aiguo; Xu, Jin

    2017-01-01

    Staphylococcus aureus is one of the most common causes of zoonotic agent in the world, which are attributable to the contamination of food with enterotoxins. In this study, a total of 1,150 S. aureus isolates were cultured from 27,000 retail foods items from 203 cities of 24 provinces in China in 2015 and were test for antimicrobial susceptibility. Additionally, the role of the genes responsible for the staphylococcal enterotoxins (SEA to SEE), methicillin resistance ( mecA ) and the toxigenic capabilities were also assessed. The results showed that 4.3% retail foods were contaminated with S. aureus , and 7.9% retail foods isolates were mecA positive. Some 97.6% of S. aureus isolates were resistant to at least one antimicrobial compound, and 57.5% of these were multi drug resistant (MDR). Resistance to penicillin (83.7%, 963/1,150), was common, followed by linezolid (67.7%, 778/1,150) and erythromycin (52.1%, 599/1,150). The isolates cultured from raw meats showed high levels of resistant to tetracycline (42.8%), ciprofloxacin (17.4%), and chloramphenicol (12.0%) and expressed a MDR phenotype (62.4%). A total of 29.7% S. aureus isolates harbored the classical SEs genes ( sea, seb, sec , and sed ). The sea and seb genes were the most frequent SEs genes detected. Of note, 22% of the SEs genes positive S. aureus harbored two or three SEs genes, and 16 isolates were confirmed with the capacity to simultaneously produce two or three enterotoxin types. Moreover, nearly 50% of the MRSA isolates were positive for at least one SE gene in this study. Therefore, it is important to monitor the antimicrobial susceptibility and enterotoxigenicity of MDR S. aureus and MRSA in the food chain and to use these data to develop food safety measures, designed to reduce the contamination and transmission of this bacterium.

  12. ``Black Holes" and Bacterial Pathogenicity: A Large Genomic Deletion that Enhances the Virulence of Shigella spp. and Enteroinvasive Escherichia coli

    NASA Astrophysics Data System (ADS)

    Maurelli, Anthony T.; Fernandez, Reinaldo E.; Bloch, Craig A.; Rode, Christopher K.; Fasano, Alessio

    1998-03-01

    Plasmids, bacteriophages, and pathogenicity islands are genomic additions that contribute to the evolution of bacterial pathogens. For example, Shigella spp., the causative agents of bacillary dysentery, differ from the closely related commensal Escherichia coli in the presence of a plasmid in Shigella that encodes virulence functions. However, pathogenic bacteria also may lack properties that are characteristic of nonpathogens. Lysine decarboxylate (LDC) activity is present in ≈ 90% of E. coli strains but is uniformly absent in Shigella strains. When the gene for LDC, cadA, was introduced into Shigella flexneri 2a, virulence became attenuated, and enterotoxin activity was inhibited greatly. The enterotoxin inhibitor was identified as cadaverine, a product of the reaction catalyzed by LDC. Comparison of the S. flexneri 2a and laboratory E. coli K-12 genomes in the region of cadA revealed a large deletion in Shigella. Representative strains of Shigella spp. and enteroinvasive E. coli displayed similar deletions of cadA. Our results suggest that, as Shigella spp. evolved from E. coli to become pathogens, they not only acquired virulence genes on a plasmid but also shed genes via deletions. The formation of these ``black holes,'' deletions of genes that are detrimental to a pathogenic lifestyle, provides an evolutionary pathway that enables a pathogen to enhance virulence. Furthermore, the demonstration that cadaverine can inhibit enterotoxin activity may lead to more general models about toxin activity or entry into cells and suggests an avenue for antitoxin therapy. Thus, understanding the role of black holes in pathogen evolution may yield clues to new treatments of infectious diseases.

  13. Clinical and biochemical significance of toxin production by Aeromonas hydrophila.

    PubMed Central

    Kindschuh, M; Pickering, L K; Cleary, T G; Ruiz-Palacios, G

    1987-01-01

    Production of cytotoxin and enterotoxin by Aeromonas strains obtained from stools of 50 children in Mexico and Texas and from blood of 9 children with sepsis was determined. Results were correlated with clinical features of infected children as well as with biochemical traits of Aeromonas strains. Cytotoxin was produced by 40 of 42 Aeromonas strains (95%) isolated from stools of children with diarrhea, by all 8 isolates from stools of well children, and by all 9 isolates from children with sepsis. There was no difference in the quantities (amount of cytotoxin per milligram of protein required to kill 50% of the cells) of cytotoxin produced and in clinical manifestations among the groups. None of the isolates produced a toxin that could be neutralized by antiserum raised against Shiga toxin produced by Shigella dysenteriae 1 60R. Heat-labile-like enterotoxin (LT) was produced by 26 of 42 stool isolates (62%), while only 1 of the 42 isolates (2%) produced enterotoxinlike activity in suckling mice; 65% of the cytotoxin-producing strains also produced an LT-like material. All strains from blood produced LT-like material, and 2 of 6 (33%) produced activity in suckling mice. All strains produced hemolysin; 37 of 57 (65%) were Voges-Proskauer positive; 27 of 57 (47%) were lysine decarboxylase positive by API 20E strips, none were positive for lysine decarboxylose production by lysin-iron agar slants at 24 h, but 17 of 54 (31%) were positive at 48 h. There was no correlation between biochemical reactions and enterotoxin or cytotoxin production. There appears to be no correlation between toxin production by Aeromonas spp. and gastroenteritis. PMID:3584426

  14. Virulence Factors of Staphylococcus aureus Isolates in an Iranian Referral Children's Hospital.

    PubMed

    Sabouni, Farah; Mahmoudi, Shima; Bahador, Abbas; Pourakbari, Babak; Sadeghi, Reihaneh Hosseinpour; Ashtiani, Mohammad Taghi Haghi; Nikmanesh, Bahram; Mamishi, Setareh

    2014-04-01

    The clinical importance of Staphylococcus aureus (S. aureus) is attributed to notable virulence factors, surface proteins, toxins, and enzymes as well as the rapid development of drug resistance. The aim of this study was to compare the occurrence of virulence factors produced by S. aureus strains isolated from children in an Iranian referral children's hospital. The presence of genes encoding for the enterotoxins A (sea), B (seb), C (sec), D (sed), TSST-1 (tsst), exfoliative toxin A (eta), and exfoliative toxin B (etb) were detected by Multiplex polymerase chain reaction (PCR) using specific primers. In addition, the standardized Kirby-Bauer disc-diffusion method was performed on Mueller-Hinton agar. In total, 133 S. aureus isolates were obtained from different patients. Of these S. aureus isolates, 64 (48%) were methicillin-resistant S. aureus (MRSA), and all of these tested positive for the mecA gene. Regarding the classical enterotoxin genes, sea gene (40.6%) was the most prevalent followed by seb (19.6%), tsst (12.8%), eta (11.3%), etb (9%), sed (4.5%), and sec (3%). Among methicillin-susceptible S. aureus (MSSA) isolates, seb and tsst were the more prevalent toxins in comparison with MRSA isolates (p < 0.05), while the frequency of sea, sed, eta, and etb genes were higher among MRSA isolates (p > 0.05). In our study enterotoxin A was produced by 40.6% of the isolates (48% from MRSA and 33% from MSSA isolates) which was higher than in previous reports. According to our results, strict hygiene and preventative measures during food processing are highly recommended.

  15. Modulation of Interleukin-8 and staphylococcal flora by Avène hydrotherapy in patients suffering from chronic inflammatory dermatoses.

    PubMed

    Casas, C; Ribet, V; Alvarez-Georges, S; Sibaud, V; Guerrero, D; Schmitt, A-M; Redoulès, D

    2011-02-01

    A number of studies argue in favour of an important role of microbial colonization, in particular of Staphylococcus aureus, in triggering atopic dermatitis (AD) flare-up and psoriasis, in particular through the superantigenic properties of toxins generated by S. aureus. The aim of this study was to assess the efficacy of a 3-week Avène hydrotherapy on the skin surface of patients suffering from psoriasis or atopic dermatitis. Skin samples were taken from healthy subjects or atopic (n = 18) or psoriatic patients (n = 39) undergoing hydrotherapy at Avène at the beginning (D0) and the end of treatment (D18). The severity of the dermatosis was evaluated according to SCORing Atopic Dermatitis (SCORAD) or Psoriasis Area Severity Index (PASI) scores at D0 and D18. Marker of inflammation interleukin 8 (IL-8), S. aureus colonization (protein A) and enterotoxins were assessed in skin samples using RT-PCR. At D0, significant differences were observed between healthy subjects and atopic or psoriatic patients in all the parameters evaluated (IL-8, protein A). At the end of the hydrotherapy, a significant decrease in SCORAD was associated with a significant reduction of IL-8, S. aureus colonization and enterotoxin D in patients with atopic dermatitis. Similarly, a significant decrease in PASI was associated with a significant reduction of IL-8, S. aureus colonization and enterotoxin N in patients with psoriasis. This study demonstrates the positive effects of Avène hydrotherapy on the skin of patients suffering from chronic dermatosis, with decreased inflammation and reduced colonization by S. aureus. © 2010 The Authors. JEADV © 2010 European Academy of Dermatology and Venereology.

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

    PubMed

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

    2013-01-01

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

  17. [Epidemic of gastroenteritis in Noumea (New Caledonia) caused by an enterotoxinogenic strain of Escherichia coli (0l26:B16) believed to be enteropathogenic].

    PubMed

    Germani, Y; Amat, F; Brethes, B; Begaud, E; Plassart, H

    1985-01-01

    A strain of enteropathogenic Escherichia coli 0126:B16 has been isolated in fifteen children and one adult during a severe outbreak. One infant is dead. The strain produced heat-stable enterotoxin, attach to rabbit enterocytes but did not have colonization factor antigen CFA/I or CFA/II. Its hemagglutination type was the same that the E. coli H10407, CFA/I+. It presented a resistance at eight antibiotics and, with the loss of enterotoxigenicity, there was a loss of resistance at ampicillin and of the capacity to attach to enterocytes.

  18. Targeting of plant-derived vaccine antigens to immunoresponsive mucosal sites.

    PubMed

    Rigano, M Manuela; Sala, Francesco; Arntzen, Charles J; Walmsley, Amanda M

    2003-01-30

    Most pathogenic microorganisms enter their host via the mucosal surfaces lining the digestive, respiratory and urino-reproductive tracts of the body. The most efficient means of protecting these surfaces is through mucosal immunization. Transgenic plants are safe and inexpensive vehicles to produce and mucosally deliver protective antigens. However, the application of this technology is limited by the poor response of the immune system to non-particulate, subunit vaccines. Co-delivery of therapeutic proteins with targeting proteins, such as the B subunit of the Escherichia coli heat labile enterotoxin (LTB), could increase the effectiveness of such antigens.

  19. [Carriage of Staphylococcus aureus among food service workers].

    PubMed

    Alarcón-Lavín, María Paula; Oyarzo, Carolina; Escudero, Carlos; Cerda-Leal, Fabiola; Valenzuela, Francisco J

    2017-12-01

    Background Staphylococcus aureus produces 11 serotypes of endotoxins that may cause food poisoning. Aim To determine the prevalence of type A enterotoxigenic Staphylococcus aureus carriage among food service workers in Chillan, Chile. Material and Methods Pharyngeal swabs were obtained from 100 food service workers and were cultured in Agar plates. After identifying the presence of Staphylococcus aureus, DNA was extracted to identify type A toxin by conventional PCR. Results Thirty eight percent of samples were colonized with Staphylococcus aureus. Among these, 26% were toxin A producers. Conclusions Half of the sampled workers carried Staphylococcus aureus and a quarter of these produced type A enterotoxin.

  20. The Role of Intestinal Bacteria in Acute Diarrheal Diseases

    DTIC Science & Technology

    1981-01-01

    another enteric pathogen, Vibrio cholerae , is present in high number in the cral cavity during acute and convalescent periods. Also, the buccal cells...h42 LT/ST + - - 2 K32&c4 AD) Kenya ... . .- r K32ScT All Kenya - LT - - - NT TD46ZcT ADY Mexfco 06:HT6 LT/ST - - - Z13 TD260cl AD Mexico 06:H16 LT...2 TD514c! AD Mexico .. . . NT TD472cI A Mexico LT/ST - - NT TD514cZ AD Mexico - NT "TABLE 1 CONTINUED 2 1 Enterotoxins MR-HA Pili 3 Human Strains

  1. Staphylococci isolated from ready-to-eat meat - Identification, antibiotic resistance and toxin gene profile.

    PubMed

    Fijałkowski, Karol; Peitler, Dorota; Karakulska, Jolanta

    2016-12-05

    The aim of this study was to analyse the staphylococci isolated from ready-to-eat meat products, including pork ham, chicken cold cuts, pork sausage, salami and pork luncheon meat, sliced in the store to the consumer's specifications, along with species identification and determination of antibiotic resistance. Genes encoding staphylococcal enterotoxins, staphylococcal enterotoxin-like proteins, exfoliative toxins, and toxic shock syndrome toxin 1 were also investigated. From the 41 samples, 75 different staphylococcal isolates were obtained. Based on PCR-RFLP analysis of the gap gene using AluI and HpyCH4V restriction enzymes, the isolates were identified as Staphylococcus equorum (28%), S. vitulinus (16%), S. carnosus (14%), S. succinus (11%), S. xylosus (11%), S. saprophyticus (9%), S. warneri (9%), S. haemolyticus (1%) and S. pasteuri (1%). The incidence and number of resistances to antimicrobials was found to be species but not source of isolation dependent. All S. xylosus, S. saprophyticus, S. haemolyticus and S. pasteuri isolates showed antibiotic resistance. A lower percentage of resistance was recorded for S. warneri (71%) and S. vitulinus (58%), followed by S. equorum (57%), S. carnosus (50%) and S. succinus (50%). The most frequent resistance was observed to fusidic acid (43%). The mecA gene was amplified in 4% of the staphylococci. However, phenotypic resistance to methicillin was not confirmed in any of these isolates. On the other hand, the mecA gene was not detected in any of 9% of the isolates resistant to cefoxitin. It was also found that among 75 isolates, 60 (80%) harbored from 1 to 10 out of 21 analyzed superantigenic toxin genes. The most prevalent genes were: sei (36% isolates) among enterotoxins, seln (32% isolates) among enterotoxin-like proteins and eta encoding exfoliative toxin A (37% isolates). The findings of this study further extend previous observations that, when present in food, not only S. aureus but also other species of

  2. Clostridium perfringens type A fatal acute hemorrhagic gastroenteritis in a dog

    PubMed Central

    Schlegel, Ben J.; Van Dreumel, Tony; Slavić, Durda; Prescott, John F.

    2012-01-01

    The morning after participating in a dog show, a 2-year-old Pomeranian dog was found dead in a pool of bloody feces. Necropsy revealed hemorrhagic gastroenteritis of the entire gastrointestinal tract, with many Gram-positive bacilli on the surface and in the lumen and crypts of the intestine. Enterotoxin-positive type A Clostridium perfringens were isolated in large numbers. This dramatic case of fatal C. perfringens gastroenteritis highlights the need to better understand the role of this bacterium in enteric disease of dogs. PMID:23115371

  3. Clostridium perfringens type A fatal acute hemorrhagic gastroenteritis in a dog.

    PubMed

    Schlegel, Ben J; Van Dreumel, Tony; Slavić, Durda; Prescott, John F

    2012-05-01

    The morning after participating in a dog show, a 2-year-old Pomeranian dog was found dead in a pool of bloody feces. Necropsy revealed hemorrhagic gastroenteritis of the entire gastrointestinal tract, with many Gram-positive bacilli on the surface and in the lumen and crypts of the intestine. Enterotoxin-positive type A Clostridium perfringens were isolated in large numbers. This dramatic case of fatal C. perfringens gastroenteritis highlights the need to better understand the role of this bacterium in enteric disease of dogs.

  4. Serologic test systems development. Progress report, July 1, 1976--September 30, 1977

    SciTech Connect

    Saunders, G.C.; Clinard, E.H.; Bartlett, M.L.

    1978-01-01

    Work has continued on the development and application of the Enzyme-Labeled Antibody (ELA) test to the USDA needs. Results on trichinosis, brucellosis, and staphylococcal enterotoxin A detection are very encouraging. A field test for trichinosis detection is being worked out in cooperation with Food Safety and Quality Service personnel. Work is in progress with the Technicon Instrument Corporation to develop a modification of their equipment to automatically process samples by the ELA procedure. An automated ELA readout instrument for 96-well trays has been completed and is being checked out.

  5. Viability of Staphylococcus xylosus isolated from artisanal sausages for application as starter cultures in meat products

    PubMed Central

    Fiorentini, Ângela Maria; Sawitzki, Maristela Cortez; Bertol, Teresinha Marisa; Sant’Anna, Ernani S.

    2009-01-01

    Viability of Staphylococcus xylosus isolated from artisanal sausages for application as starter cultures in meat products Viability of Staphylococcus xylosus strains AD1 and U5 isolated from natural fermented sausages was investigated as starter cultures in fermented sausages produced in the South Region of Brazil. The study demonstrated that the Staphylococcus xylosus strains AD1 and U5 showed significant growth during fermentation, stability over freeze-dried process, negative reaction for staphylococcal enterotoxins and viability for using as a single-strain culture or associated with lactic acid bacteria for production of fermented sausages. PMID:24031331

  6. Viability of Staphylococcus xylosus isolated from artisanal sausages for application as starter cultures in meat products.

    PubMed

    Fiorentini, Angela Maria; Sawitzki, Maristela Cortez; Bertol, Teresinha Marisa; Sant'anna, Ernani S

    2009-01-01

    Viability of Staphylococcus xylosus isolated from artisanal sausages for application as starter cultures in meat products Viability of Staphylococcus xylosus strains AD1 and U5 isolated from natural fermented sausages was investigated as starter cultures in fermented sausages produced in the South Region of Brazil. The study demonstrated that the Staphylococcus xylosus strains AD1 and U5 showed significant growth during fermentation, stability over freeze-dried process, negative reaction for staphylococcal enterotoxins and viability for using as a single-strain culture or associated with lactic acid bacteria for production of fermented sausages.

  7. In operando neutron diffraction study of LaNdMgNi9H13 as a metal hydride battery anode

    NASA Astrophysics Data System (ADS)

    Nazer, N. S.; Denys, R. V.; Yartys, V. A.; Hu, Wei-Kang; Latroche, M.; Cuevas, F.; Hauback, B. C.; Henry, P. F.; Arnberg, L.

    2017-03-01

    La2MgNi9-related alloys are superior metal hydride battery anodes as compared to the commercial AB5 alloys. Nd-substituted La2-yNdyMgNi9 intermetallics are of particular interest because of increased diffusion rate of hydrogen and thus improved performance at high discharge currents. The present work presents in operando characterization of the LaNdMgNi9 intermetallic as anode for the nickel metal hydride (Ni-MH) battery. We have studied the structural evolution of LaNdMgNi9 during its charge and discharge using in situ neutron powder diffraction. The work included experiments using deuterium gas and electrochemical charge-discharge measurements. The alloy exhibited a high electrochemical discharge capacity (373 mAh/g) which is 20% higher than the AB5 type alloys. A saturated β-deuteride synthesized by solid-gas reaction at PD2 = 1.6 MPa contained 12.9 deuterium atoms per formula unit (D/f.u.) which resulted in a volume expansion of 26.1%. During the electrochemical charging, the volume expansion (23.4%) and D-contents were found to be slightly reduced. The reversible electrochemical cycling is performed through the formation of a two-phase mixture of the α-solid solution and β-hydride phases. Nd substitution contributes to the high-rate dischargeability, while maintaining a good cyclic stability. Electrochemical Impedance Spectroscopy (EIS) was used to characterize the anode electrode on cycling. A mathematical model for the impedance response of a porous electrode was utilized. The EIS showed a decreased hydrogen transport rate during the long-term cycling, which indicated a corresponding slowing down of the electrochemical processes at the surface of the metal hydride anode.

  8. Metrology for hydrogen energy applications: a project to address normative requirements

    NASA Astrophysics Data System (ADS)

    Haloua, Frédérique; Bacquart, Thomas; Arrhenius, Karine; Delobelle, Benoît; Ent, Hugo

    2018-03-01

    Hydrogen represents a clean and storable energy solution that could meet worldwide energy demands and reduce greenhouse gases emission. The joint research project (JRP) ‘Metrology for sustainable hydrogen energy applications’ addresses standardisation needs through pre- and co-normative metrology research in the fast emerging sector of hydrogen fuel that meet the requirements of the European Directive 2014/94/EU by supplementing the revision of two ISO standards that are currently too generic to enable a sustainable implementation of hydrogen. The hydrogen purity dispensed at refueling points should comply with the technical specifications of ISO 14687-2 for fuel cell electric vehicles. The rapid progress of fuel cell technology now requires revising this standard towards less constraining limits for the 13 gaseous impurities. In parallel, optimized validated analytical methods are proposed to reduce the number of analyses. The study aims also at developing and validating traceable methods to assess accurately the hydrogen mass absorbed and stored in metal hydride tanks; this is a research axis for the revision of the ISO 16111 standard to develop this safe storage technique for hydrogen. The probability of hydrogen impurity presence affecting fuel cells and analytical techniques for traceable measurements of hydrogen impurities will be assessed and new data of maximum concentrations of impurities based on degradation studies will be proposed. Novel validated methods for measuring the hydrogen mass absorbed in hydrides tanks AB, AB2 and AB5 types referenced to ISO 16111 will be determined, as the methods currently available do not provide accurate results. The outputs here will have a direct impact on the standardisation works for ISO 16111 and ISO 14687-2 revisions in the relevant working groups of ISO/TC 197 ‘Hydrogen technologies’.

  9. Antibiotic resistance and molecular analysis of Staphylococcus aureus isolated from cow's milk and dairy products in northeast Brazil.

    PubMed

    Silveira-Filho, Vladimir M; Luz, Isabelle S; Campos, Ana Paula F; Silva, Wellington M; Barros, Maria Paloma S; Medeiros, Elizabeth S; Freitas, Manuela F L; Mota, Rinaldo A; Sena, Maria J; Leal-Balbino, Tereza C

    2014-04-01

    This work aimed to assess the clonal distribution among 94 strains of Staphylococcus aureus isolated from cow's milk, raw cheese, and a milking machine in 12 dairy farms in northeast Brazil, by analyzing different typing methods and detecting resistance and toxigenic profiles. For the first time, isolates of this region were assessed simultaneously by the polymorphism of the 3'-end coa gene and 16S-23S rDNA, pulsed-field gel electrophoresis, antibiotic resistance phenotyping, and toxigenic arsenal. Although pulsed-field gel electrophoresis patterns showed a wider variation (discriminatory index 0.83) than the PCR-based methods, the internal transcribed spacer-PCR proved to be a useful and inexpensive procedure for conducting epidemiological surveys of S. aureus on a regional scale. Each dairy farm had its own resistance profile, and in two herds, 63% of the strains were multiresistant, probably due to the indiscriminate use of antibiotics in bovine mastitis treatment. No methicillin-resistant S. aureus strains were detected in this study; however, 93.6% of S. aureus strains harbored variable profiles of staphylococcal enterotoxin genes seg, seh, sei, and sej. Transcriptional analysis revealed that 53.3% of staphylococcal enterotoxin genes actually transcribed, pointing out the food poisoning risk of these dairy products to consumers in the region. Based on the detection of the most prevalent clones in a herd or region, appropriate antibiotic therapy and specific immunization can be used for the treatment and control of staphylococcal mastitis.

  10. Use of the Real Time xCelligence System for Purposes of Medical Microbiology.

    PubMed

    Junka, Adam Feliks; Janczura, Adriana; Smutnicka, Danuta; Mączyńska, Beata; Anna, Secewicz; Nowicka, Joanna; Bartoszewicz, Marzenna; Gościniak, Grażyna

    2012-09-28

    Roche's xCelligence impedance-measuring instrument is one of a few commercially available systems of such type. According to the best knowledge of authors, instrument was tested so far only for eukaryotic cell research. The aim of this work was to estimate xCELLigence suitability for the microbiological tests, including (i) measurement of morphological changes in eukaryotic cells as a result of bacterial toxin activity, (ii) measurement of bacterial biofilm formation and (iii) impact of antiseptics on the biofilm structure. To test the infuence of bacterial LT enterotoxin on eukaryotic cell lines, Chinese Hamster Ovary (CHO) cell line and reference strain Escherichia coli ATTC 35401 were used. To investigate Roche's instrument ability to measure biofilm formation and impact of antiseptics on its development, Staphylococcus aureus ATTC6538 reference strain was used. The data generated during the experiments indicate excellent ability of xCelligence instrument to detect cytopathic effect caused by bacterial LT endotoxin and to detect staphylococcal biofilm formation. However, interpretation of the results obtained during real-time measurement of antiseptic's bactericidal activity against staphylococcal biofilm, caused many difficulties. xCelligence instrument can be used for real-time monitoring of morphological changes in CHO cells treated with bacterial LT enterotoxin and for real-time measurement of staphylococcal biofilm formation in vitro. Further investigation is necessary to confirm suitability of system to analyze antiseptic's antimicrobial activity against biofilm in vitro.

  11. Species diversity and molecular analysis of Staphylococcus in confectioneries of a developing country, Iran.

    PubMed

    Hoveida, Laleh; Ataei, Behrooz; Amirmozafari, Nour; Noormohammadi, Zahra

    2018-06-01

    Confectionery is one of the potential sources of contamination and transmission of gastrointestinal infections to humans. Staphylococcus species, and particularly the coagulase-positive ones, have the remarkable capability to produce high amounts of enterotoxin in food. In the present study, the frequency and diversity of Staphylococcus in confectioneries in Iran were assessed by using a combination of conventional and molecular methods. A total of 55 confection samples were collected from 30 confectioneries of Isfahan. They were analyzed for the presence of Staphylococcus using standard protocols for isolation and characterization of the isolates. The conventional tests were used for primary identification and the sequence analysis of 16S rRNA was used for the species identification. A total of 47 out of 55 samples were gram-positive cocci (85.45%). They belonged to 39 Staphylococcus spp., 7 Macrococcus spp., and one Micrococcus spp. The most prevalent 11 various Staphylococcus species were S. aureus 30.8 %, S. warneri 20.5% and S. succinus 17.9. Identification and characterization of Staphylococcus species can be important for epidemiological investigations and assessment of virulence factors such as enterotoxin production and development of specific management practices to prevent staphylococcal food poisoning.

  12. [Description of a staphylococcal alimentary poisoning outbreak in Las Rosas, Santa Fe Province, Argentina].

    PubMed

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

    2011-01-01

    On February 2008, a suspected foodborne outbreak was reported in Las Rosas (Santa Fe Province, Argentina). The formal procedures indicated that an undetermined number of individuals had experienced food poisoning following consumption of vegetable cannelloni bought at a local shop. The manufacturer establishment was audited. Samples from the suspected food, as well as environmental samples and swabs from food handlers were obtained and involved subjects were interviewed. Remnants of ingested food were also obtained. Routine microbiological procedures of the foodborne outbreak revealed the presence of coagulase positive S. aureus subspecies aureus in samples from ingested and raw food, and from manipulators. Indicator microorganisms did not show significant levels and no other foodborne pathogen was isolated. Presence of staphylococcal enterotoxin-producing genes was subsequently investigated, and a positive result for enterotoxin B was shown in S. aureus strains isolated from a food handler as well as from food linked to the outbreak Moreover, these isolates showed 100% similarity by SmaI-PFGE. Timely notification together with coordinated sanitary measures and the availability of appropriate laboratory tools allowed to interrupt the chain of disease transmission by identifying risk and protective factors.

  13. Investigating an outbreak of staphylococcal food poisoning among travellers across two Australian states.

    PubMed Central

    Boonwaat, Leng; Moore, Terry; Chavada, Ruchir; Conaty, Stephen

    2015-01-01

    Introduction Staphylococcus aureus is a common cause of staphylococcal food poisoning in Australia with several outbreaks associated with foods prepared by commercial caterers. Laboratory testing on cases of gastrointestinal illness caused by enterotoxin-producing S. aureus is not routinely done as this condition is self-limiting. Hence outbreaks of such illness may go undetected. Methods A retrospective cohort study was conducted among a group of tourists who were hospitalized in Sydney shortly after flying from Queensland. The group had consumed food prepared by a restaurant on the Gold Coast before transit. Laboratory analyses on stool specimens were conducted in Sydney. An environmental assessment of the restaurant in the Gold Coast was conducted, and environmental specimens were assessed for contamination. Results Epidemiological investigations linked the outbreak to a restaurant in the Gold Coast where the suspected food was produced. Stool samples from two of the hospitalized cases were confirmed to have enterotoxin-producing S. aureus, and several environmental samples were found to be contaminated with S. aureus as well. Investigations suggested that absence of hand washing and other unhygienic food handling at the implicated restaurant was the likely cause of this outbreak. Conclusion Food poisoning due to toxin-mediated S. aureus is frequently undetected and underreported. Public health units should consider toxin-producing pathogens such as S. aureus when investigating outbreaks where vomiting is the predominant symptom and occurs rapidly after consuming food. PMID:26306211

  14. Staphylococcus aureus and Staphylococcal Food-Borne Disease: An Ongoing Challenge in Public Health

    PubMed Central

    Smith, Tara C.

    2014-01-01

    Staphylococcal food-borne disease (SFD) is one of the most common food-borne diseases worldwide resulting from the contamination of food by preformed S. aureus enterotoxins. It is one of the most common causes of reported food-borne diseases in the United States. Although several Staphylococcal enterotoxins (SEs) have been identified, SEA, a highly heat-stable SE, is the most common cause of SFD worldwide. Outbreak investigations have found that improper food handling practices in the retail industry account for the majority of SFD outbreaks. However, several studies have documented prevalence of S. aureus in many food products including raw retail meat indicating that consumers are at potential risk of S. aureus colonization and subsequent infection. Presence of pathogens in food products imposes potential hazard for consumers and causes grave economic loss and loss in human productivity via food-borne disease. Symptoms of SFD include nausea, vomiting, and abdominal cramps with or without diarrhea. Preventive measures include safe food handling and processing practice, maintaining cold chain, adequate cleaning and disinfection of equipment, prevention of cross-contamination in home and kitchen, and prevention of contamination from farm to fork. This paper provides a brief overview of SFD, contributing factors, risk that it imposes to the consumers, current research gaps, and preventive measures. PMID:24804250

  15. Recombinant expression of in silico identified Bcell epitope of epsilon toxin of Clostridium perfringens in translational fusion with a carrier protein.

    PubMed

    Kaushik, Himani; Deshmukh, Sachin; Mathur, Deepika Dayal; Tiwari, Archana; Garg, Lalit C

    2013-01-01

    Epsilon toxin secreted by Clostridium perfringens types B and D has been directly implicated as the causative agent of fatal enterotoxemia in domestic animals. The aim of the present study is to use in silico approach for identification of B-cell epitope(s) of epsilon toxin, and its expression in fusion with a carrier protein to analyze its potential as vaccine candidate(s). Using different computational analyses and bioinformatics tools, a number of antigenic determinant regions of epsilon toxin were identified. One of the B cell epitopes of epsilon toxin comprising the region (amino acids 40-62) was identified as a promising antigenic determinant. This Etx epitope (Etx40-62) was cloned and expressed as a translational fusion with B-subunit of heat labile enterotoxin (LTB) of E. coli in a secretory expression system. Similar to the native LTB, the recombinant fusion protein retained the ability to pentamerize and bind to GM1 ganglioside receptor of LTB. The rLTB.Etx40-62 could be detected both with anti-Etx and anti-LTB antisera. The rLTB.Etx40-62 fusion protein thus can be evaluated as a potential vaccine candidate against C. perfringens. aa - amino acid(s), Etx - epsilon toxin of Clostridium perfringens, LTB - B-subunit of heat labile enterotoxin of E. coli.

  16. Prevalence and Toxin Characteristics of Bacillus thuringiensis Isolated from Organic Vegetables.

    PubMed

    Kim, Jung-Beom; Choi, Ok-Kyung; Kwon, Sun-Mok; Cho, Seung-Hak; Park, Byung-Jae; Jin, Na Young; Yu, Yong Man; Oh, Deog-Hwan

    2017-08-28

    The prevalence and toxin characteristics of Bacillus thuringiensis isolated from 39 organic vegetables were investigated. B. thuringiensis was detected in 30 out of the 39 organic vegetables (76.9%) with a mean value of 2.60 log CFU/g. Twenty-five out of the 30 B. thuringiensis isolates (83.3%) showed insecticidal toxicity against Spodoptera exigua . The hblCDA, nheABC , and entFM genes were found to be the major toxin genes, but the ces gene was not detected in any of the tested B. thuringiensis isolates. The hemolysin BL enterotoxin was detected in all 30 B. thuringiensis isolates (100%). The non-hemolytic enterotoxin complex was found in 27 out of 30 B. thuringiensis isolates (90.0%). The B. thuringiensis tested in this study had similar toxin gene characteristics to B. cereus , which possessed more than one toxin gene. B. thuringiensis could have the potential risk of foodborne illness based on the toxin genes and toxin-producing ability.

  17. A Chimeric protein of CFA/I, CS6 subunits and LTB/STa toxoid protects immunized mice against enterotoxigenic Escherichia coli.

    PubMed

    Zeinalzadeh, Narges; Salmanian, Ali Hatef; Goujani, Goli; Amani, Jafar; Ahangari, Ghasem; Akhavian, Asal; Jafari, Mahyat

    2017-07-01

    Enterotoxigenic Escherichia Coli (ETEC) strains are the commonest bacteria causing diarrhea in children in developing countries and travelers to these areas. Colonization factors (CFs) and enterotoxins are the main virulence determinants in ETEC pathogenesis. Heterogeneity of CFs is commonly considered the bottleneck to developing an effective vaccine. It is believed that broad spectrum protection against ETEC would be achieved by induced anti-CF and anti-enterotoxin immunity simultaneously. Here, a fusion antigen strategy was used to construct a quadrivalent recombinant protein called 3CL and composed of CfaB, a structural subunit of CFA/I, and CS6 structural subunits, LTB and STa toxoid of ETEC. Its anti-CF and antitoxin immunogenicity was then assessed. To achieve high-level expression, the 3CL gene was synthesized using E. coli codon bias. Female BALB/C mice were immunized with purified recombinant 3CL. Immunized mice developed antibodies that were capable of detecting each recombinant subunit in addition to native CS6 protein and also protected the mice against ETEC challenge. Moreover, sera from immunized mice also neutralized STa toxin in a suckling mouse assay. These results indicate that 3CL can induce anti-CF and neutralizing antitoxin antibodies along with introducing CFA/I as a platform for epitope insertion. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

  18. Clostridium perfringens in London, July 2009: two weddings and an outbreak.

    PubMed

    Eriksen, J; Zenner, D; Anderson, S R; Grant, K; Kumar, D

    2010-06-24

    Food poisoning outbreaks caused by Clostridium perfringens enterotoxin occur occasionally in Europe but have become less common in recent years. This paper presents the microbiological and epidemiological results of a large C. perfringens outbreak occurring simultaneously at two weddings that used the same caterer. The outbreak involved several London locations and required coordination across multiple agencies. A case-control study (n=134) was carried out to analyse possible associations between the food consumed and becoming ill. Food, environmental and stool samples were tested for common causative agents, including enterotoxigenic C. perfringens. The clinical presentation and the epidemiological findings were compatible with C. perfringens food poisoning and C. perfringens enterotoxin was detected in stool samples from two cases. The case-control study found statistically significant associations between becoming ill and eating either a specific chicken or lamb dish prepared by the same food handler of the implicated catering company. A rapid outbreak investigation with preliminary real-time results and the successful collaboration between the agencies and the caterer led to timely identification and rectification of the failures in the food handling practices.

  19. Global analysis of community-associated methicillin-resistant Staphylococcus aureus exoproteins reveals molecules produced in vitro and during infection

    PubMed Central

    Burlak, Christopher; Hammer, Carl H; Robinson, Mary-Ann; Whitney, Adeline R; McGavin, Martin J; Kreiswirth, Barry N; DeLeo, Frank R

    2007-01-01

    Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a threat to human health worldwide. Although progress has been made, mechanisms of CA-MRSA pathogenesis are poorly understood and a comprehensive analysis of CA-MRSA exoproteins has not been conducted. To address that deficiency, we used proteomics to identify exoproteins made by MW2 (USA400) and LAC (USA300) during growth in vitro. Two hundred and fifty unique exoproteins were identified by 2-dimensional gel electrophoresis coupled with automated direct infusion-tandem mass spectrometry (ADI-MS/MS) analysis. Eleven known virulence-related exoproteins differed in abundance between the strains, including alpha-haemolysin (Hla), collagen adhesin (Cna), staphylokinase (Sak), coagulase (Coa), lipase (Lip), enterotoxin C3 (Sec3), enterotoxin Q (Seq), V8 protease (SspA) and cysteine protease (SspB). Mice infected with MW2 or LAC produced antibodies specific for known or putative virulence factors, such as autolysin (Atl), Cna, Ear, ferritin (Ftn), Lip, 1-phosphatidylinositol phosphodiesterase (Plc), Sak, Sec3 and SspB, indicating the exoproteins are made during infection in vivo. We used confocal microscopy to demonstrate aureolysin (Aur), Hla, SspA and SspB are produced following phagocytosis by human neutrophils, thereby linking exoprotein production in vitro with that during host–pathogen interaction. We conclude that the exoproteins identified herein likely account in part for the success of CA-MRSA as a human pathogen. PMID:17217429

  20. Safety Evaluation of the Coagulase-Negative Staphylococci Microbiota of Salami: Superantigenic Toxin Production and Antimicrobial Resistance

    PubMed Central

    Soares Casaes Nunes, Raquel; Mere Del Aguila, Eduardo; Paschoalin, Vânia Margaret Flosi

    2015-01-01

    The risks of contracting staphylococci food poisoning by the consumption of improperly manufactured salami and the possibility of this food being reservoirs for antibiotic resistance were evaluated. Nineteen coagulase-negative staphylococci (CNS) strains were found in commercial and artisanal salami. The species in commercial salami were S. saprophyticus, S. sciuri, S. xylosus, and S. carnosus. Artisanal salami showed S. succinus, S. epidermidis, and S. hominis but no S. carnosus. Phylogenetic analyses grouped the strains into three major staphylococcal species groups, comprised of 4 refined clusters with similarities superior to 90%. Fifteen strains harbored multiple enterotoxin genes, with high incidence of seb/sec and sea, 57% and 50%, respectively, intermediate incidence of sed/seh/selm and sei/seln/tst-H, 33% and 27%, correspondingly, and low incidence of see/selj/selo and seg, of respectively 13% and 1%. Real time RT-PCR and enzyme-linked-immunosorbent assays confirmed the enterotoxigenicity of the strains, which expressed and produced enterotoxins in vitro. The CNS strains showed multiresistance to several antimicrobials of therapeutic importance in both human and veterinarian medicine, such as β-lactams, vancomycin, and linezolid. The effective control of undue staphylococci in fermented meat products should be adopted to prevent or limit the risk of food poisoning and the spread of antimicrobial-resistant strains. PMID:26697486

  1. Vibrio cholerae ACE stimulates Ca(2+)-dependent Cl(-)/HCO(3)(-) secretion in T84 cells in vitro.

    PubMed

    Trucksis, M; Conn, T L; Wasserman, S S; Sears, C L

    2000-09-01

    ACE, accessory cholera enterotoxin, the third enterotoxin in Vibrio cholerae, has been reported to increase short-circuit current (I(sc)) in rabbit ileum and to cause fluid secretion in ligated rabbit ileal loops. We studied the ACE-induced change in I(sc) and potential difference (PD) in T84 monolayers mounted in modified Ussing chambers, an in vitro model of a Cl(-) secretory cell. ACE added to the apical surface alone stimulated a rapid increase in I(sc) and PD that was concentration dependent and immediately reversed when the toxin was removed. Ion replacement studies established that the current was dependent on Cl(-) and HCO(3)(-). ACE acted synergistically with the Ca(2+)-dependent acetylcholine analog, carbachol, to stimulate secretion in T84 monolayers. In contrast, the secretory response to cAMP or cGMP agonists was not enhanced by ACE. The ACE-stimulated secretion was dependent on extracellular and intracellular Ca(2+) but was not associated with an increase in intracellular cyclic nucleotides. We conclude that the mechanism of secretion by ACE involves Ca(2+) as a second messenger and that this toxin stimulates a novel Ca(2+)-dependent synergy.

  2. Mass Spectrometry and Multiplex Antigen Assays to Assess Microbial Quality and Toxin Production of Staphylococcus aureus Strains Isolated from Clinical and Food Samples

    PubMed Central

    Attien, Paul; Sina, Haziz; Moussaoui, Wardi; Zimmermann-Meisse, Gaëlle; Dadié, Thomas; Keller, Daniel; Riegel, Philippe; Edoh, Vincent; Kotchoni, Simeon O.; Djè, Marcellin; Prévost, Gilles

    2014-01-01

    The aim of our study was to investigate the microbial quality of meat products and on some clinical samples in Abidjan focused on Staphylococcus genus and the toxin production profile of Staphylococcus aureus (S. aureus) isolated. Bacteria were collected from 240 samples of three meat products sold in Abidjan and 180 samples issued from clinical infections. The strains were identified by both microbiological and MALDI-TOF-MS methods. The susceptibility to antibiotics was determined by the disc diffusion method. The production of Panton-Valentine Leukocidin, LukE/D, and epidermolysins was screened using radial gel immunodiffusion. The production of staphylococcal enterotoxins and TSST-1 was screened by a Bio-Plex Assay. We observed that 96/240 of meat samples and 32/180 of clinical samples were contaminated by Staphylococcus. Eleven species were isolated from meats and 4 from clinical samples. Forty-two S. aureus strains were isolated from ours samples. Variability of resistance was observed for most of the tested antibiotics but none of the strains displays a resistance to imipenem and quinolones. We observed that 89% of clinical S. aureus were resistant to methicillin against 58% for those issued from meat products. All S. aureus isolates issued from meat products produce epidermolysins whereas none of the clinical strains produced these toxins. The enterotoxins were variably produced by both clinical and meat product samples. PMID:24987686

  3. Potential enterotoxicity and antimicrobial resistance pattern of Aeromonas species isolated from pet turtles and their environment.

    PubMed

    Wimalasena, S H M P; Shin, Gee-Wook; Hossain, Sabrina; Heo, Gang-Joon

    2017-05-23

    To investigate the potential enterotoxicity and antimicrobial resistance of aeromonads from pet turtles as a risk for human infection, one hundred and two Aeromonas spp. were isolated from the feces, skin and rearing environments of pet turtles and identified by biochemical and gyrB sequence analyses. Aeromonas enteropelogenes was the predominant species among the isolates (52.9%) followed by A. hydrophila (32.4%), A. dharkensis (5.9%), A. veronii (4.9%) and A. caviae (3.9%). Their potential enterotoxicities were evaluated by PCR assays for detecting genes encoding cytotoxic enterotoxin (act) and two cytotonic enterotoxins (alt and ast). 75.8% of A. hydrophila isolates exhibited the act + /alt + /ast + genotype, whereas 94.4% of A. enteropelogenes isolates were determined to be act - /alt - /ast - . In an antimicrobial susceptibility test, most isolates were susceptible to all tested antibiotics except amoxicillin, ampicillin, cephalothin, chloramphenicol and tetracycline. Non-susceptible isolates to penicillins (ampicillin and amoxicillin) and fluoroquinolones (ciprofloxacin and norfloxacin) were frequently observed among the A. enteropelogenes isolates. Few isolates were resistant to imipenem, amikacin, ceftriaxone and cefotaxime. Collectively, these results suggest that pet turtles may pose a public health risk of infection by enterotoxigenic and antimicrobial resistant Aeromonas strains.

  4. Antimicrobial resistance levels amongst staphylococci isolated from clinical cases of bovine mastitis in Kosovo.

    PubMed

    Mehmeti, Ibrahim; Behluli, Behlul; Mestani, Mergim; Ademi, Arsim; Nes, Ingolf F; Diep, Dzung B

    2016-10-31

    Mastitis is one of the most frequent and costly disease in cattle. We studied milk samples from cattle with mastitis from farms in Kosovo to identify mastitis-causing pathogens and possible effective antibiotics. Our ultimate goal is to help implement adequate antibiotic management and treatment practices in Kosovo METHODOLOGY: A total of 152 milk samples were collected from cows with clinical mastitis from different farms in Kosovo. After identification of microorganisms, antibiotic susceptibility and the occurrence of enterotoxins was investigated. Staphylococci were found in 89 samples, of which 58 were coagulase negative and 31 coagulase positive. S. aureus was isolated from 27 samples, S. epidermidis from 25, and S. chromogenes from 15, while other species of staphylococci were isolated from the remaining 22 isolates. Interestingly, the bacterial diversity was different between cows in different periods of lactation and among different breeds. Most of the isolates (76/89) were resistant to two or more antibiotics. The highest resistance was to penicillin and ampicillin (> 65%), followed by tetracycline, oxacillin, streptomycin, chloramphenicol (> 23%), and less than 3% to erythromycin. Of the 89 isolates, 40 produced enterotoxins that were most frequently typed as A and C. We detected human bacterial pathogens in the cultures of milk samples from cows with mastitis. The isolates demonstrated resistance to two or more antibiotics, some of which are frequently used to treat animal and human infections. We recommend increased control and more stringent use of antibiotics in veterinary as well as human medicine.

  5. Assessment of hygienic quality of some types of cheese sampled from retail outlets.

    PubMed

    Prencipe, Vincenza; Migliorati, Giacomo; Matteucci, Osvaldo; Calistri, Paolo; Di Giannatale, Elisabetta

    2010-01-01

    The authors evaluated the prevalence of Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp. and staphylococcal enterotoxin, in 2,132 samples selected from six types of cheese on the basis of recorded consumption in Italy in 2004. In L. monocytogenes-positive samples the precise level of contamination was determined. To define the physical-chemical characteristics of the selected natural cheeses, the pH values, water activity and sodium chloride content were determined. The results suggest that blue and soft cheeses (Brie, Camembert, Gorgonzola and Taleggio) are more likely to be contaminated with L. monocytogenes. The mean prevalence of L. monocytogenes in the six types of cheese was 2.4% (from 0.2% in Asiago and Crescenza to 6.5% in Taleggio), with contamination levels of up to 460 MPN/g. No presence of Salmonella spp. and E. coli O157 was found in any sample. Staphylococcus enterotoxin was found in 0.6% of the samples examined. Physical and chemical parameter values confirmed that all types of cheese are considered capable of supporting the growth of L. monocytogenes. The study confirmed the need to apply effective control at production and sales levels to reduce the probability of contamination by L. monocytogenes. This micro-organism can attain high levels of contamination in food products, such as cheeses that have a long shelf-life when associated with difficulties of maintaining appropriate storage temperatures in both sales points and in the home.

  6. Superantigens Modulate Bacterial Density during Staphylococcus aureus Nasal Colonization

    PubMed Central

    Xu, Stacey X.; Kasper, Katherine J.; Zeppa, Joseph J.; McCormick, John K.

    2015-01-01

    Superantigens (SAgs) are potent microbial toxins that function to activate large numbers of T cells in a T cell receptor (TCR) Vβ-specific manner, resulting in excessive immune system activation. Staphylococcus aureus possesses a large repertoire of distinct SAgs, and in the context of host-pathogen interactions, staphylococcal SAg research has focused primarily on the role of these toxins in severe and invasive diseases. However, the contribution of SAgs to colonization by S. aureus remains unclear. We developed a two-week nasal colonization model using SAg-sensitive transgenic mice expressing HLA-DR4, and evaluated the role of SAgs using two well-studied stains of S. aureus. S. aureus Newman produces relatively low levels of staphylococcal enterotoxin A (SEA), and although we did not detect significant TCR-Vβ specific changes during wild-type S. aureus Newman colonization, S. aureus Newman Δsea established transiently higher bacterial loads in the nose. S. aureus COL produces relatively high levels of staphylococcal enterotoxin B (SEB), and colonization with wild-type S. aureus COL resulted in clear Vβ8-specific T cell skewing responses. S. aureus COL Δseb established consistently higher bacterial loads in the nose. These data suggest that staphylococcal SAgs may be involved in regulating bacterial densities during nasal colonization. PMID:26008236

  7. Effect of packaging conditions on the growth of micro-organisms and the quality characteristics of fresh mushrooms (Agaricus bisporus) stored at inadequate temperatures.

    PubMed

    González-Fandos, E; Giménez, M; Olarte, C; Sanz, S; Simón, A

    2000-10-01

    Mushrooms were packed in two polymeric films (perforated and non-perforated PVC) and stored at 17 degrees C and 25 degrees C. The carbon dioxide and oxygen content inside the packages, aerobic mesophiles, Pseudomonas spp., faecal coliforms, Escherichia coli, anaerobic spores and major sensory factors (colour, texture, development stage and presence of moulds) were determined. The non-perforated packages had the highest contents of CO2 (6-7%), the lowest contents of O2 (0.013-0.17%) and the most desirable quality parameters (texture, development stage and absence of moulds). Pseudomonas spp. counts were around 1 logarithmic unit lower in mushrooms packaged in non-perforated film as the O2 concentrations were lower than in perforated film. The mushrooms themselves were inoculated with an enterotoxin A-producing strain of Staphylococcus aureus, packaged in overwrapped trays and stored at 17 and 25 degrees C. Staphylococcus aureus did not grow in the samples stored at 17 degrees C. Only slight growth was observed in mushrooms packaged with non-perforated film after 1 day at 25 degrees C. No enterotoxin was detected in any package. Faecal coliform counts were <2 log cfu g(-1). Escherichia coli was not isolated in any of the samples. At 25 degrees C, counts of anaerobic spores of around 2 log cfu g(-1) were detected in those mushrooms packaged in non-perforated film.

  8. Determination of haemolytic and non haemolytic genes profiles of Bacillus cereus strains isolated from food samples by polymerase chain reaction (pcr) technique

    NASA Astrophysics Data System (ADS)

    Jawad, Nisreen; Ahemd, Asmat; Abdullah, Aminah

    2018-04-01

    The aim of this study was to investigate the presence of Bacillus cereus and detection of enterotoxigenic genes in food samples by utilizing a Polymerase Chain Reaction technique (PCR). In this study the providence of B. cereus was carried out to food samples. The B. cereus isolates were investigated for enterotoxigenic gene. The cooked seafood, and raw milk samples were purchased from several restaurants and market in the area of (Bangi, Kajang, Serdang and UKM) Selangor, Malaysia. A total of 60 samples have been analyzed. B. cereus contamination has been formed between 1.4×105 - 3×105 cfu/mL of cooked seafood and raw milk samples. Five colonies have been detected as B. cereus using biochemical test. All B. cereus isolates named BC1 to BC27, were characterized for haemolytic enterotoxin (HBL) complex encoding genes (hblA), non-haemolytic enterotoxin encoding gene (NheA). 10 isolates have been reported to be positive towards hblA and 12 isolates were positive towards NheA. The presence of B. cereus and their enterotoxigenic genes in cooked seafood and raw milk from to food samples obtained may pose a potential risk for public health.

  9. The 2.3 {angstrom} crystal structure of cholera toxin B subunit pentamer: Choleragenoid

    SciTech Connect

    Zhang, Rong-Guang; Westbrook, M.L.; Maulik, P.R.

    1996-02-01

    Cholera toxin, a heterohexameric AB{sub 5} enterotoxin released by Vibrio cholera, induces a profuse secretory diarrhea in susceptible hosts. Choleragenoid, the B subunit pentamer of cholera toxin, directs the enzymatic A subunit to its target by binding to GM{sub 1} gangliosides exposed on the luminal surface of intestinal epithelial cells. We have solved the crystal structure of choleragenoid at 2.3 {Angstrom} resolution by combining single isomorphous replacement with non-crystallographic symmetry averaging. The structure of the B subunits, and their pentameric arrangement, closely resembles that reported for the intact holotoxin (choleragen), the heat-labile enterotoxin from E. coli, and for a choleragenoid-GM{submore » 1} pentasaccharide complex. In the absence of the A subunit the central cavity of the B pentamer is a highly solvated channel. The binding of the A subunit or the receptor pentasaccharide to choleragenoid has only a modest effect on the local stereochemistry and does not perceptibly alter the subunit interface.« less

  10. Superantigen activates the gp130 receptor on adipocytes resulting in altered adipocyte metabolism.

    PubMed

    Banke, Elin; Rödström, Karin; Ekelund, Mikael; Dalla-Riva, Jonathan; Lagerstedt, Jens O; Nilsson, Staffan; Degerman, Eva; Lindkvist-Petersson, Karin; Nilson, Bo

    2014-06-01

    The bacteria Staphylococcus aureus is part of the normal bacterial flora and produces a repertoire of enterotoxins which can cause food poisoning and toxic shock and might contribute to the pathogenesis of inflammatory diseases. These enterotoxins directly cross-link the T cell receptor with MHC class II, activating large amounts of T cells and are therefore called superantigens. It was recently discovered that the superantigen SEA binds to the cytokine receptor gp130. As obesity and type 2 diabetes are highly associated with inflammation of the adipose tissue and gp130 has been shown to play an important role in adipocytes, we wanted to investigate the effect of SEA on adipocyte signaling and function. Binding of SEA to gp130 was examined using surface plasmon resonance in a cell free system. Effects of SEA on adipocyte signaling, insulin sensitivity and function were studied using western blotting and biological assays for lipolysis, lipogenesis and glucose uptake. We demonstrate that SEA binds to gp130 with a medium affinity. Furthermore, SEA induces phosphorylation of a key downstream target, STAT3, in adipocytes. SEA also inhibits insulin-induced activation of PKB and PKB downstream signaling which was associated with reduced basal and insulin induced glucose uptake, reduced lipogenesis as well as reduced ability of insulin to inhibit lipolysis. SEA inhibits insulin signaling as well as insulin biological responses in adipocytes supporting that bacterial infection might contribute to the development of insulin resistance and type 2 diabetes. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Clostridium perfringens Sporulation and Sporulation-Associated Toxin Production

    PubMed Central

    Li, Jihong; Paredes-Sabja, Daniel; Sarker, Mahfuzur R.; McClane, Bruce A.

    2015-01-01

    The ability of Clostridium perfringens to form spores plays a key role during the transmission of this Gram-positive bacterium to cause disease. Of particular note, the spores produced by food poisoning strains are often exceptionally resistant to food environment stresses such as heat, cold and preservatives, which likely facilitates their survival in temperature-abused foods. The exceptional resistance properties of spores made by most type A food poisoning strains and some type C foodborne disease strains involves their production of a variant small acid soluble protein-4 that binds more tightly to spore DNA compared to the small acid soluble protein-4 made by most other C. perfringens strains. Sporulation and germination by C. perfringens and Bacillus spp. share both similarities and differences. Finally, sporulation is essential for production of C. perfringens enterotoxin, which is responsible for the symptoms of C. perfringens type A food poisoning, the second most common bacterial foodborne disease in the USA. During this foodborne disease, C. perfringens is ingested with food and then, using sporulation-specific alternate sigma factors, this bacterium sporulates and produces the enterotoxin in the intestines. PMID:27337447

  12. Effect of food-related stress conditions and loss of agr and sigB on seb promoter activity in S. aureus.

    PubMed

    Sihto, Henna-Maria; Stephan, Roger; Engl, Christoph; Chen, John; Johler, Sophia

    2017-08-01

    Staphylococcal enterotoxin B (SEB) causes staphylococcal food poisoning and is produced in up to ten times higher quantities than other major enterotoxins. While Staphylococcus aureus growth is often repressed by competing flora, the organism exhibits a decisive growth advantage under some stress conditions. So far, data on the influence of food-related stressors and regulatory mutations on seb expression is limited and largely based on laboratory strains, which were later reported to harbor mutations. Therefore, the aim of this study was to investigate the influence of stress and regulatory mutations on seb promoter activity. To this end, transcriptional fusions were created in two strains, USA300 and HG003, carrying different seb upstream sequences fused to a blaZ reporter. NaCl, nitrite, and glucose stress led to significantly decreased seb promoter activity, while lactic acid stress resulted in significantly increased seb promoter activity. Loss of agr decreased seb promoter activity and loss of sigB increased promoter activity, with the magnitude of change depending on the strain. These results demonstrate that mild stress conditions encountered during food production and preservation can induce significant changes in seb promoter activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Molecular characterization of Clostridium perfringens strains isolated from diseased turkeys in Italy.

    PubMed

    Giovanardi, Davide; Drigo, Ilenia; De Vidi, Beatrice; Agnoletti, Fabrizio; Viel, Laura; Capello, Katia; Berto, Giacomo; Bano, Luca

    2016-06-01

    One hundred and six Clostridium perfringens field strains, isolated from diseased turkeys in Italy between 2006 and 2015, were toxinotyped by polymerase chain reaction. Strains were derived from intestines (87), livers (17) and subcutaneous tissues (2). In addition to the four major toxins, strains were also screened for NetB toxin, enterotoxin and beta2 toxin encoding genes. The intestinal gross lesions of turkeys with enteric disorders were statistically studied with respect to the presence of C. perfringens beta2 toxin encoding gene and coccidia in the gut. All the isolates belonged to the toxinotype A and were netB negative. Enterotoxin (cpe) and beta2 toxin (cpb2) encoding genes were detected in two (2.63%) and 76 (71.69%) strains, respectively. Toxinotype results agree with the few published reports concerning the genetic characterization of C. perfringens of turkey origin. On the contrary, the presence of netB and cpb2 genes differs from the results of a previous study where these genes were detected respectively in 6.6% and in 0.5% of the tested strains. Necrotic enteritis in turkeys was not statistically correlated either to the presence of cpb2 gene, or to the synergistic effect operated by coccidia, even though a high percentage of birds with these protozoa in the gut showed necrotic enteritis lesions (64.29%).

  14. Detection of Clostridium perfringens toxin genes in the gut microbiota of autistic children.

    PubMed

    Finegold, Sydney M; Summanen, Paula H; Downes, Julia; Corbett, Karen; Komoriya, Tomoe

    2017-06-01

    We studied stool specimens from 33 autistic children aged 2-9 years with gastrointestinal (GI) abnormalities and 13 control children without autism and without GI symptoms. We performed quantitative comparison of all Clostridium species and Clostridium perfringens strains from the fecal microbiota by conventional, selective anaerobic culture methods. We isolated C. perfringens strains and performed PCR analysis for the main C. perfringens toxin genes, alpha, beta, beta2, epsilon, iota and C. perfringens enterotoxin gene. Our results indicate that autistic subjects with gastrointestinal disease harbor statistically significantly (p = 0.031) higher counts of C. perfringens in their gut compared to control children. Autistic subjects also harbor statistically significantly (p = 0.015) higher counts of beta2-toxin gene-producing C. perfringens in their gut compared to control children, and the incidence of beta2-toxin gene-producing C. perfringens is significantly higher in autistic subjects compared to control children (p = 0.014). Alpha toxin gene was detected in all C. perfringens strains studied. C. perfringens enterotoxin gene was detected from three autistic and one control subject. Beta, epsilon, and iota toxin genes were not detected from autistic or control subjects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Isolation and identification of Staphylococcus sp. in powdered infant milk

    NASA Astrophysics Data System (ADS)

    Palilu, Prayolga Toban; Budiarso, Tri Yahya

    2017-05-01

    Staphylococcus sp. is one of the most dangerous bacteria that could cause food poisoning. It is a pathogenic bacterium which is able to produce enterotoxin in foods. Milk is an ideal growth medium for Staphylococcus sp., that may cause problem if it is to be consumed, especially by infant. It is the objective of this research to detect the presence of Staphylococcus sp. in powdered infant milk. As many as 14 samples obtained from market were used as samples for bacterial isolation. The isolation were done by employing enrichment step on BHI-broth, continued with Baird-Parker Agar which will produce a typical colony. It is then picked and grown on Mannitol Salt Agar, and gram staining, coagulase assay, and fermentation tests. The confirmation step was done by using API-Staph which gives the identification of Staphylococcus hemoliticus, Staphylococcus aureus and Staphylococcus epidermidis, with a percentage of identity ranging from 65.9-97.7%. Two isolates with the highest identification similarity values were then picked for molecular detection. A PCR primer pair targeting gene coding for enterotoxin A was used, and it gives positive result for the two isolates being tested. It is then concluded that the two isolates belong to Staphylococcus sp., and further research need to be done to correctly identify these isolates.

  16. Sanitary quality, occurrence and identification of Staphylococcus sp. in food services.

    PubMed

    de Mello, Jozi Fagundes; da Rocha, Laura Braga; Lopes, Ester Souza; Frazzon, Jeverson; da Costa, Marisa

    2014-01-01

    Sanitary conditions are essential for the production of meals and control of the presence of pathogensis important to guarantee the health of customers. The aim of this study was to evaluate the sanitary quality of food services by checking the presence of thermotolerant coliforms, Staphylococcus sp. and evaluate the toxigenic potential from the latter. The analysis was performed on water, surfaces, equipment, ready-to-eat foods, hands and nasal cavity of handlers in seven food services. The water used in food services proved to be suitable for the production of meals. Most food, equipment and surfaces showed poor sanitary conditions due to the presence of thermotolerant coliforms (60.6%). Twenty-six Staphylococcus species were identified from the 121 Staphylococcus isolates tested. Staphylococci coagulase-negative species were predominant in the foods, equipment and surfaces. In food handlers and foods, the predominant species was Staphylococcus epidermidis. Twelve different genotypes were found after PCR for the classical enterotoxin genes. The seb gene (19.8%) was the most prevalent among all Staphylococcus sp. Both coagulase-positive and coagulase-negative Staphylococci showed some of the genes of the enterotoxins tested. We conclude that there are hygienic and sanitary deficiencies in the food services analyzed. Although coagulase-positive Staphylococci have not been present in foods there is a wide dispersion of enterotoxigenic coagulase-negative Staphylococci in the environment and in the foods analyzed, indicating a risk to consumer health.

  17. Sanitary quality, occurrence and identification of Staphylococcus sp. in food serv