WorldWideScience

Sample records for antitoxins

  1. Toxin-Antitoxin Battle in Bacteria

    DEFF Research Database (Denmark)

    Cataudella, Ilaria

    This PhD thesis consists of three research projects revolving around the common thread of investigation of the properties and biological functions of Toxin-Antitoxin loci. Toxin-Antitoxin (TA) loci are transcriptionally regulated via an auto-inhibition mechanism called conditional cooperativity, ...

  2. Radioimmunodiffusion technique for determining diphtheria antitoxin

    International Nuclear Information System (INIS)

    Maniar, A.C.

    1977-01-01

    A radioimmunodiffusion technique for detecting low levels of diphtheria antitoxin was developed. Diphtheria toxoid was labelled with 125 I to facilitate detection of lines of precipitation by the use of X-ray film, the lower limit of detection being 0.001 unit per millilitre of diphtheria antitoxin. (author)

  3. 9 CFR 113.451 - Tetanus Antitoxin.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Tetanus Antitoxin. 113.451 Section 113.451 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE... which conforms to the National Institute of Standards and Technology requirements shall be used. The...

  4. Keeping the wolves at bay: antitoxins of prokaryotic type II toxin-antitoxin systems

    Directory of Open Access Journals (Sweden)

    Wai Ting eChan

    2016-03-01

    Full Text Available In their initial stages of discovery, prokaryotic toxin-antitoxin (TA systems were confined to bacterial plasmids where they function to mediate the maintenance and stability of usually low- to medium-copy number plasmids through the post-segregational killing of any plasmid-free daughter cells that developed. Their eventual discovery as nearly ubiquitous and repetitive elements in bacterial chromosomes led to a wealth of knowledge and scientific debate as to their diversity and functionality in the prokaryotic lifestyle. Currently categorized into six different types designated types I – VI, type II TA systems are the best characterized. These generally comprised of two genes encoding a proteic toxin and its corresponding proteic antitoxin, respectively. Under normal growth conditions, the stable toxin is prevented from exerting its lethal effect through tight binding with the less stable antitoxin partner, forming a non-lethal TA protein complex. Besides binding with its cognate toxin, the antitoxin also plays a role in regulating the expression of the type II TA operon by binding to the operator site, thereby repressing transcription from the TA promoter. In most cases, full repression is observed in the presence of the TA complex as binding of the toxin enhances the DNA binding capability of the antitoxin. TA systems have been implicated in a gamut of prokaryotic cellular functions such as being mediators of programmed cell death as well as persistence or dormancy, biofilm formation, as defensive weapons against bacteriophage infections and as virulence factors in pathogenic bacteria. It is thus apparent that these antitoxins, as DNA-binding proteins, play an essential role in modulating the prokaryotic lifestyle whilst at the same time preventing the lethal action of the toxins under normal growth conditions, i.e., keeping the proverbial wolves at bay. In this review, we will cover the diversity and characteristics of various type II TA

  5. Factors influencing Haemagglutination Tests with Tetanus Antitoxin

    Science.gov (United States)

    Fulthorpe, A. J.

    1959-01-01

    The agglutinin titres of tetanus antitoxins tested with preserved sheep cells sensitized with tetanus toxoid have been found to be inversely proportional to the cell concentration used. The agglutinin titres per international unit of antitoxin were widely different among various electrophoretically separated globulin fractions of tetanus antitoxin. There were smaller differences between the agglutinating capacity of γ-globulin fractions of sera from different horses. With haemagglutination inhibition tests there was always a relative increase in test dose between the LA and LA/100 level of test. This relative increase was greatest where the sensitivity of the cell suspensions was high. Increased suspension sensitivity may be the result of treatment of cells with greater quantities of sensitizing antigen or of reduction in the concentration of sensitized cells in the suspension. A greater volume of serum was required to give a standard end point, when mixed with a test dose (LA) of toxin, if the mixtures were allowed to stand for increasing periods of time. These observations have been discussed. PMID:13653730

  6. Mechanisms for Differential Protein Production in Toxin–Antitoxin Systems

    Directory of Open Access Journals (Sweden)

    Heather S. Deter

    2017-07-01

    Full Text Available Toxin–antitoxin (TA systems are key regulators of bacterial persistence, a multidrug-tolerant state found in bacterial species that is a major contributing factor to the growing human health crisis of antibiotic resistance. Type II TA systems consist of two proteins, a toxin and an antitoxin; the toxin is neutralized when they form a complex. The ratio of antitoxin to toxin is significantly greater than 1.0 in the susceptible population (non-persister state, but this ratio is expected to become smaller during persistence. Analysis of multiple datasets (RNA-seq, ribosome profiling and results from translation initiation rate calculators reveal multiple mechanisms that ensure a high antitoxin-to-toxin ratio in the non-persister state. The regulation mechanisms include both translational and transcriptional regulation. We classified E. coli type II TA systems into four distinct classes based on the mechanism of differential protein production between toxin and antitoxin. We find that the most common regulation mechanism is translational regulation. This classification scheme further refines our understanding of one of the fundamental mechanisms underlying bacterial persistence, especially regarding maintenance of the antitoxin-to-toxin ratio.

  7. The DinJ/RelE toxin-antitoxin system suppresses virulence in Xylella fastidiosa

    Science.gov (United States)

    Xylella fastidiosa, the causal agent of a number agriculturally important plant diseases, encodes multiple toxin-antitoxin (TA) systems. TA modules consist of a toxin protein co-expressed with a specific antitoxin, and are often acquired through horizontal gene transfer. Antitoxin molecules (RNA or ...

  8. 9 CFR 113.454 - Clostridium Perfringens Type C Antitoxin.

    Science.gov (United States)

    2010-01-01

    ... Clostridium perfringens Type C. Each serial shall be tested as provided in this section. Any serial found... following words and terms shall mean: (i) International antitoxin unit. (I.U.) That quantity of Beta... chloride in each 100 ml of distilled water; adjusting the pH to 7.2; autoclaving at 250 °F. for 25 minutes...

  9. 9 CFR 113.455 - Clostridium Perfringens Type D Antitoxin.

    Science.gov (United States)

    2010-01-01

    ... Clostridium perfringens Type D. Each serial shall be tested as provided in this section. Any serial found... following words and terms shall mean: (i) International antitoxin unit. (I.U.) That quantity of Epsilon... 0.25 gram of sodium chloride in each 100 ml of distilled water; adjusting the pH to 7.2; autoclaving...

  10. Postbooster Antibodies from Humans as Source of Diphtheria Antitoxin.

    Science.gov (United States)

    Bermejo-Martin, Jesús F; Avila-Alonso, Ana; González-Rivera, Milagros; Tamayo, Eduardo; Eiros, Jose María; Almansa, Raquel

    2016-07-01

    Diphtheria antitoxin for therapeutic use is in limited supply. A potential source might be affinity-purified antibodies originally derived from plasma of adults who received a booster dose of a vaccine containing diphtheria toxoid. These antibodies might be useful for treating even severe cases of diphtheria.

  11. Relationship Between Tetanus Antitoxin Titration Level and Vaccination History

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    Meltem Işıkgöz Taşbakan

    2017-12-01

    Full Text Available Objectives: We aimed to determine tetanus antitoxin levels and to evaluate their relationship with history of vaccination among patients applying to the outpatient clinics of a University hospital. Methods: A questionnaire including socio-demographic characteristics and tetanus vaccination status was applied and blood samples taken from 218 subjects between 1 and 30 June 2015. Participants were classified into five groups according to their vaccination timing. Results: The mean age of participants was 46.7±15.4 years and 134 (61.5% were women. Tetanus antitoxin levels were found weak positive in 54 (24.8% patients, positive in 44 (20.2% and strong positive in 120 (55.0%. Tetanus antitoxin level positivity was significantly associated with vaccination timing according to history. Among 105 participants who did not remember being vaccinated or who knew they were vaccinated but did not remember the date, 16 (15.2% remembered the vaccination time when their injury, military service and pregnancy were questioned specifically. Antitoxin levels decreased with increasing age independent of gender (0.9-fold increase/year. Conclusion: We found that the booster dose recommended every 10 years was not applied sufficiently. Tetanus vaccination history must be questioned in more detail among people who do not remember/know their vaccination history, with specific questions regarding pregnancy, military service and injury histories.

  12. Relationship Between Tetanus Antitoxin Titration Level and Vaccination History

    OpenAIRE

    Işıkgöz Taşbakan, Meltem; Durusoy, Raika; Tosun, Selma

    2017-01-01

    Objectives: We aimed to determine tetanus antitoxin levels and to evaluate their relationship with history of vaccination among patients applying to the outpatient clinics of a University hospital. Methods: A questionnaire including socio-demographic characteristics and tetanus vaccination status was applied and blood samples taken from 218 subjects between 1 and 30 June 2015. Participants were classified into five groups according to their vaccination timing. Results: The mean age of...

  13. Radiosensitivity of antibody responses and radioresistant secondary tetanus antitoxin responses

    International Nuclear Information System (INIS)

    Stoner, R.; Terres, G.; Cottier, H.; Hess, M.

    1976-01-01

    Primary tetanus antitoxin responses were increasingly repressed in mice when gamma radiation doses of 100 to 400 rads were delivered by whole-body exposure prior to immunization with fluid tetanus toxoid (FTT). Nearly normal secondary antitoxin responses were obtained in mice exposed to 600 rads of gamma radiation 4 days after secondary antigenic stimulation with FTT. A rapid transition from radiosensitivity of the antibody-forming system on days 1 to 3 was followed by relative radioresistance on day 4 after the booster injection of toxoid. Studies on lymphoid cellular kinetics in popliteal lymph nodes after injection of 3 H--thymidine ( 3 H--TdR) and incorporation of 3 H--L-histidine into circulating antitoxin were carried out. Analysis of tritium radioactivity in antigen--antibody precipitates of serums 2 hr after injection of the labeled amino acid revealed maximum incorporation into antibody around day 7 after the booster in nonirradiated controls and about day 12, i.e., 8 days after irradiation, in experimental mice. The shift from radiosensitivity to relative radioresistance was attributed to a marked peak of plasma-cell proliferation in the medulla of lymph nodes on day 3. Many medullary plasma cells survived and continued to proliferate after exposure to radiation. Germinal centers were destroyed by radiation within 1 day. Since antibody formation continued after exposure to radiation and after the loss of germinal centers, this supports the view that germinal-center cells were involved more in the generation of memory cells than in antibody synthesis

  14. Crystallization of the HigBA2 toxin-antitoxin complex from Vibrio cholerae

    DEFF Research Database (Denmark)

    Hadǽi, San; Garcia-Pino, Abel; Martinez-Rodriguez, Sergio

    2013-01-01

    The genome of Vibrio cholerae encodes two higBA toxin-antitoxin (TA) modules that are activated by amino-acid starvation. Here, the TA complex of the second module, higBA2, as well as the C-terminal domain of the corresponding HigA2 antitoxin, have been purified and crystallized. The HigBA2 complex...

  15. Higher-Order Structure in Bacterial VapBC Toxin-Antitoxin Complexes

    DEFF Research Database (Denmark)

    Bendtsen, Kirstine L; Brodersen, Ditlev E

    2017-01-01

    that allow auto-regulation of transcription by direct binding to promoter DNA. In this chapter, we review our current understanding of the structural characteristics of type II toxin-antitoxin complexes in bacterial cells, with a special emphasis on the staggering variety of higher-order architecture...... conditions, type II toxins are inhibited through tight protein-protein interaction with a cognate antitoxin protein. This toxin-antitoxin complex associates into a higher-order macromolecular structure, typically heterotetrameric or heterooctameric, exposing two DNA binding domains on the antitoxin...... observed among members of the VapBC family. This structural variety is a result of poor conservation at the primary sequence level and likely to have significant and functional implications on the way toxin-antitoxin expression is regulated....

  16. The antitoxin protein of a toxin-antitoxin system from Xylella fastidiosa is secreted via outer membrane vesicles.

    Directory of Open Access Journals (Sweden)

    André Santiago

    2016-12-01

    Full Text Available The Xylella fastidiosa subsp pauca strain 9a5c is a Gram-negative, xylem-limited bacterium that is able to form a biofilm and affects citrus crops in Brazil. Some genes are considered to be involved in biofilm formation, but the specific mechanisms involved in this process remain unknown. This limited understanding of how some bacteria form biofilms is a major barrier to our comprehension of the progression of diseases caused by biofilm-producing bacteria. Several investigations have shown that the toxin-antitoxin (TA operon is related to biofilm formation. This operon is composed of a toxin with RNAse activity and its cognate antitoxin. Previous reports have indicated that the antitoxin is able to inhibit toxin activity and modulate the expression of the operon as well as other target genes involved in oxidative stress and mobility. In this study, we characterize a toxin-antitoxin system consisting of XfMqsR and XfYgiT, respectively, from X. fastidiosa subsp pauca strain 9a5c. These proteins display a high similarity to their homologues in X. fastidiosa strain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT from Escherichia coli. The characterization was performed using in vitro assays such as analytical ultracentrifugation (AUC, size exclusion chromatography, isothermal titration calorimetry and western blotting. Using a fluorometric assay to detect RNAses, we demonstrated that XfMqsR is thermostable and can degrade RNA. XfMqsR is inhibited by XfYgiT, which interacts with its own promoter. XfYgiT is known to be localized in the intracellular compartment; however, we provide strong evidence that X. fastidiosa secretes wild-type XfYgiT into the extracellular environment via outer membrane vesicles, as confirmed by western blotting and specific immunofluorescence labeling visualized by fluorescence microscopy. Taken together, our results characterize the TA system from X. fastidiosa strain 9a5c, and we also discuss

  17. The Antitoxin Protein of a Toxin-Antitoxin System fromXylella fastidiosaIs Secreted via Outer Membrane Vesicles.

    Science.gov (United States)

    Santiago, André da Silva; Mendes, Juliano S; Dos Santos, Clelton A; de Toledo, Marcelo A S; Beloti, Lilian L; Crucello, Aline; Horta, Maria A C; Favaro, Marianna T de Pinho; Munar, Duber M M; de Souza, Alessandra A; Cotta, Mônica A; de Souza, Anete P

    2016-01-01

    The Xylella fastidiosa subsp pauca strain 9a5c is a Gram-negative, xylem-limited bacterium that is able to form a biofilm and affects citrus crops in Brazil. Some genes are considered to be involved in biofilm formation, but the specific mechanisms involved in this process remain unknown. This limited understanding of how some bacteria form biofilms is a major barrier to our comprehension of the progression of diseases caused by biofilm-producing bacteria. Several investigations have shown that the toxin-antitoxin (TA) operon is related to biofilm formation. This operon is composed of a toxin with RNAse activity and its cognate antitoxin. Previous reports have indicated that the antitoxin is able to inhibit toxin activity and modulate the expression of the operon as well as other target genes involved in oxidative stress and mobility. In this study, we characterize a toxin-antitoxin system consisting of XfMqsR and XfYgiT, respectively, from X. fastidiosa subsp. pauca strain 9a5c. These proteins display a high similarity to their homologs in X. fastidiosa strain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT from Escherichia coli . The characterization was performed using in vitro assays such as analytical ultracentrifugation (AUC), size exclusion chromatography, isothermal titration calorimetry, and Western blotting. Using a fluorometric assay to detect RNAses, we demonstrated that XfMqsR is thermostable and can degrade RNA. XfMqsR is inhibited by XfYgiT, which interacts with its own promoter. XfYgiT is known to be localized in the intracellular compartment; however, we provide strong evidence that X. fastidiosa secretes wild-type XfYgiT into the extracellular environment via outer membrane vesicles, as confirmed by Western blotting and specific immunofluorescence labeling visualized by fluorescence microscopy. Taken together, our results characterize the TA system from X. fastidiosa strain 9a5c, and we also discuss the possible

  18. Emerging Roles of Toxin-Antitoxin Modules in Bacterial Pathogenesis

    Directory of Open Access Journals (Sweden)

    Barbara Kędzierska

    2016-06-01

    Full Text Available Toxin-antitoxin (TA cassettes are encoded widely by bacteria. The modules typically comprise a protein toxin and protein or RNA antitoxin that sequesters the toxin factor. Toxin activation in response to environmental cues or other stresses promotes a dampening of metabolism, most notably protein translation, which permits survival until conditions improve. Emerging evidence also implicates TAs in bacterial pathogenicity. Bacterial persistence involves entry into a transient semi-dormant state in which cells survive unfavorable conditions including killing by antibiotics, which is a significant clinical problem. TA complexes play a fundamental role in inducing persistence by downregulating cellular metabolism. Bacterial biofilms are important in numerous chronic inflammatory and infectious diseases and cause serious therapeutic problems due to their multidrug tolerance and resistance to host immune system actions. Multiple TAs influence biofilm formation through a network of interactions with other factors that mediate biofilm production and maintenance. Moreover, in view of their emerging contributions to bacterial virulence, TAs are potential targets for novel prophylactic and therapeutic approaches that are required urgently in an era of expanding antibiotic resistance. This review summarizes the emerging evidence that implicates TAs in the virulence profiles of a diverse range of key bacterial pathogens that trigger serious human disease.

  19. Wake me when it's over - Bacterial toxin-antitoxin proteins and induced dormancy.

    Science.gov (United States)

    Coussens, Nathan P; Daines, Dayle A

    2016-06-01

    Toxin-antitoxin systems are encoded by bacteria and archaea to enable an immediate response to environmental stresses, including antibiotics and the host immune response. During normal conditions, the antitoxin components prevent toxins from interfering with metabolism and arresting growth; however, toxin activation enables microbes to remain dormant through unfavorable conditions that might continue over millions of years. Intense investigations have revealed a multitude of mechanisms for both regulation and activation of toxin-antitoxin systems, which are abundant in pathogenic microorganisms. This minireview provides an overview of the current knowledge regarding type II toxin-antitoxin systems along with their clinical and environmental implications. © 2016 by the Society for Experimental Biology and Medicine.

  20. Discovery of Functional Toxin/Antitoxin Systems in Bacteria by Shotgun Cloning

    Energy Technology Data Exchange (ETDEWEB)

    Sberro, Hila; Leavitt, Azita; Kiro, Ruth; Koh, Eugene; Peleg, Yoav; Qimron, Udi; Sorek, Rotem

    2013-04-01

    Toxin-antitoxin (TA) modules, composed of a toxic protein and a counteracting antitoxin, play important roles in bacterial physiology. We examined the experimental insertion of 1.5 million genes from 388 microbial genomes into an Escherichia coli host using over 8.5 million random clones. This revealed hundreds of genes (toxins) that could only be cloned when the neighboring gene (antitoxin) was present on the same clone. Clustering of these genes revealed TA families widespread in bacterial genomes, some of which deviate from the classical characteristics previously described for such modules. Introduction of these genes into E. coli validated that the toxin toxicity is mitigated by the antitoxin. Infection experiments with T7 phage showed that two of the new modules can provide resistance against phage. Moreover, our experiments revealed an 'anti-defense' protein in phage T7 that neutralizes phage resistance. Our results expose active fronts in the arms race between bacteria and phage.

  1. Wake me when it’s over – Bacterial toxin–antitoxin proteins and induced dormancy

    OpenAIRE

    Coussens, Nathan P; Daines, Dayle A

    2016-01-01

    Toxin–antitoxin systems are encoded by bacteria and archaea to enable an immediate response to environmental stresses, including antibiotics and the host immune response. During normal conditions, the antitoxin components prevent toxins from interfering with metabolism and arresting growth; however, toxin activation enables microbes to remain dormant through unfavorable conditions that might continue over millions of years. Intense investigations have revealed a multitude of mechanisms for bo...

  2. Effect of biotherapeutics on antitoxin IgG in experimentally induced Clostridium difficile infection

    Directory of Open Access Journals (Sweden)

    S Kaur

    2012-01-01

    Full Text Available Purpose: Recurrent diarrhoea after successful treatment of primary Clostridium difficile associated disease (CDAD occurs due to bowel flora alterations and failure to mount an effective antibody response. Apart from antibiotics, risk factors include immunosuppressive and acid-suppressive drug administration. Biotherapeutics such as probiotic and epidermal growth factor (EGF may offer potential effective therapy for CDAD. Materials and Methods: The effect of biotherapeutics in mounting an antibody response against C. difficile toxins was studied in BALB/c mice challenged with C. difficile after pre-treatment with ampicillin, lansoprazole or cyclosporin. Sera from sacrificed animals were estimated for antitoxin IgG by enzyme linked immunosorbent assay. Results: Antitoxin IgG was significantly higher (P0.05 in animals in which C. difficile was given after pre-treatment with cyclosporin compared to those without any pre-treatment, or pre-treatment with antibiotic or lansoprazole. In inter-subgroup comparisons also significant anomaly in production of antitoxin IgG was found. The antitoxin IgG levels were raised in animals administered C. difficile after pre-treatment with ampicillin, but lower in animals administered cyclosporin. High levels of antitoxin IgG were also found in the serum samples of animals receiving lansoprazole and C. difficile. Conclusions: Probiotics showed their beneficial effect by boosting the immune response as seen by production of antitoxin IgG. Oral administration of EGF did not affect the immune response to C. difficile toxins as significant increase was not observed in the serum antitoxin IgG levels in any of the groups investigated.

  3. Access to diphtheria antitoxin for therapy and diagnostics.

    Science.gov (United States)

    Both, L; White, J; Mandal, S; Efstratiou, A

    2014-06-19

    The most effective treatment for diphtheria is swift administration of diphtheria antitoxin (DAT) with conjunct antibiotic therapy. DAT is an equine immunoglobulin preparation and listed among the World Health Organization Essential Medicines. Essential Medicines should be available in functioning health systems at all times in adequate amounts, in appropriate dosage forms, with assured quality, and at prices individuals and the community can afford. However, DAT is in scarce supply and frequently unavailable to patients because of discontinued production in several countries, low economic viability, and high regulatory requirements for the safe manufacture of blood-derived products. DAT is also a cornerstone of diphtheria diagnostics but several diagnostic reference laboratories across the European Union (EU) and elsewhere routinely face problems in sourcing DAT for toxigenicity testing. Overall, global access to DAT for both therapeutic and diagnostic applications seems inadequate. Therefore--besides efforts to improve the current supply of DAT--accelerated research and development of alternatives including monoclonal antibodies for therapy and molecular-based methods for diagnostics are required. Given the rarity of the disease, it would be useful to organise a small stockpile centrally for all EU countries and to maintain an inventory of DAT availability within and between countries.

  4. Regulating Toxin-Antitoxin Expression: Controlled Detonation of Intracellular Molecular Timebombs

    Directory of Open Access Journals (Sweden)

    Finbarr Hayes

    2014-01-01

    Full Text Available Genes for toxin-antitoxin (TA complexes are widely disseminated in bacteria, including in pathogenic and antibiotic resistant species. The toxins are liberated from association with the cognate antitoxins by certain physiological triggers to impair vital cellular functions. TAs also are implicated in antibiotic persistence, biofilm formation, and bacteriophage resistance. Among the ever increasing number of TA modules that have been identified, the most numerous are complexes in which both toxin and antitoxin are proteins. Transcriptional autoregulation of the operons encoding these complexes is key to ensuring balanced TA production and to prevent inadvertent toxin release. Control typically is exerted by binding of the antitoxin to regulatory sequences upstream of the operons. The toxin protein commonly works as a transcriptional corepressor that remodels and stabilizes the antitoxin. However, there are notable exceptions to this paradigm. Moreover, it is becoming clear that TA complexes often form one strand in an interconnected web of stress responses suggesting that their transcriptional regulation may prove to be more intricate than currently understood. Furthermore, interference with TA gene transcriptional autoregulation holds considerable promise as a novel antibacterial strategy: artificial release of the toxin factor using designer drugs is a potential approach to induce bacterial suicide from within.

  5. Doc of prophage P1 is inhibited by its antitoxin partner Phd through fold complementation

    DEFF Research Database (Denmark)

    Garcia-Pino, Abel; Christensen-Dalsgaard, Mikkel; Wyns, Lode

    2008-01-01

    Prokaryotic toxin-antitoxin modules are involved in major physiological events set in motion under stress conditions. The toxin Doc (death on curing) from the phd/doc module on phage P1 hosts the C-terminal domain of its antitoxin partner Phd (prevents host death) through fold complementation....... This Phd domain is intrinsically disordered in solution and folds into an alpha-helix upon binding to Doc. The details of the interactions reveal the molecular basis for the inhibitory action of the antitoxin. The complex resembles the Fic (filamentation induced by cAMP) proteins and suggests a possible...... evolutionary origin for the phd/doc operon. Doc induces growth arrest of Escherichia coli cells in a reversible manner, by targeting the protein synthesis machinery. Moreover, Doc activates the endogenous E. coli RelE mRNA interferase but does not require this or any other known chromosomal toxin...

  6. Purification and crystallization of Phd, the antitoxin of the phd/doc operon

    International Nuclear Information System (INIS)

    Garcia-Pino, Abel; Sterckx, Yann; Vandenbussche, Guy; Loris, Remy

    2010-01-01

    The antitoxin Phd from the phd/doc operon of bacteriophage P1 was crystallized in two distinct crystal forms. The antitoxin Phd from the phd/doc module of bacteriophage P1 was crystallized in two distinct crystal forms. Crystals of His-tagged Phd contain a C-terminally truncated version of the protein and diffract to 2.20 Å resolution. Crystals of untagged Phd purified from the Phd–Doc complex diffract to 2.25 Å resolution. These crystals are partially merohedrally twinned and contain the full-length version of the protein

  7. The interaction of the antitoxin DM43 with a snake venom metalloproteinase analyzed by mass spectrometry and surface plasmon resonance

    DEFF Research Database (Denmark)

    Brand, Guilherme D; Salbo, Rune; Jørgensen, Thomas J D

    2012-01-01

    DM43 is a circulating dimeric antitoxin isolated from Didelphis aurita, a South American marsupial naturally immune to snake envenomation. This endogenous inhibitor binds non-covalently to jararhagin, the main hemorrhagic metalloproteinase from Bothrops jararaca snake venom, and efficiently...

  8. Crystallization of two operator complexes from the Vibrio cholerae HigBA2 toxin-antitoxin module

    DEFF Research Database (Denmark)

    Hadzi, San; Garcia-Pino, Abel; Gerdes, Kenn

    2015-01-01

    The HigA2 antitoxin and the HigBA2 toxin-antitoxin complex from Vibrio cholerae were crystallized in complex with their operator box. Screening of 22 different DNA duplexes led to two crystal forms of HigA2 complexes and one crystal form of a HigBA2 complex. Crystals of HigA2 in complex with a 17...

  9. Rapid Curtailing of the Stringent Response by Toxin-Antitoxin Encoded mRNases

    DEFF Research Database (Denmark)

    Tian, Chengzhe; Roghanian, Mohammad; Jørgensen, Mikkel Girke

    2016-01-01

    Escherichia coli regulates its metabolism to adapt to changes in the environment, in particular to stressful downshifts in nutrient quality. Such shifts elicit the so-called stringent response coordinated by the alarmone guanosine tetra- and pentaphosphate [(p)ppGpp]. At sudden amino-acid (aa......RNase-encoding TA modules present in the wt strain. This observation suggested that toxins are part of the negative feedback to control the (p)ppGpp level during early stringent response. We built a ribosome trafficking model to evaluate the fold of increase in the RelA activity just after the onset of aa...... %. IMPORTANCE: The early stringent response elicited by amino-acid starvation is controlled by a sharp increase of the cellular (p)ppGpp level. Toxin-antitoxin encoded mRNases are activated by (p)ppGpp through enhanced degradation of antitoxins. The present work shows that this activation happens at a very...

  10. Bacterial toxin-antitoxin gene system as containment control in yeast cells

    DEFF Research Database (Denmark)

    Kristoffersen, P.; Jensen, G. B.; Gerdes, K.

    2000-01-01

    The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae, Expression of the relE gene was highly toxic to yeast cells. However, expression...... fermentation processes in which the escape of genetically modified cells would be considered highly risky....

  11. Regulation of the vapBC-1 toxin-antitoxin locus in nontypeable Haemophilus influenzae.

    Directory of Open Access Journals (Sweden)

    Susan D Cline

    Full Text Available Nontypeable Haemophilus influenzae (NTHi are human-adapted commensal bacteria that can cause a number of chronic mucosal infections, including otitis media and bronchitis. One way for these organisms to survive antibiotic therapy and cause recurrent disease is to stop replicating, as most antimicrobials target essential biosynthetic pathways. Toxin-antitoxin (TA gene pairs have been shown to facilitate entry into a reversible bacteriostatic state. Characteristically, these operons encode a protein toxin and an antitoxin that associate following translation to form a nontoxic complex, which then binds to and regulates the cognate TA promoter. Under stressful conditions, the labile antitoxin is degraded and the complex disintegrates, freeing the stable toxin to facilitate growth arrest. How these events affected the regulation of the TA locus, as well as how the transcription of the operon was subsequently returned to its normal state upon resumption of growth, was not fully understood. Here we show that expression of the NTHi vapBC-1 TA locus is repressed by a complex of VapB-1 and VapC-1 under conditions favorable for growth, and activated by the global transactivator Factor for Inversion Stimulation (Fis upon nutrient upshift from stationary phase. Further, we demonstrate for the first time that the VapC-1 toxin alone can bind to its cognate TA locus control region and that the presence of VapB-1 directs the binding of the VapBC-1 complex in the transcriptional regulation of vapBC-1.

  12. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

    Directory of Open Access Journals (Sweden)

    Chew Chieng Yeo

    2016-02-01

    Full Text Available Toxin-antitoxin (TA systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  13. Structural characterizations of phage antitoxin Dmd and its interactions with bacterial toxin RnlA.

    Science.gov (United States)

    Wei, Yong; Gao, Zengqiang; Zhang, Heng; Dong, Yuhui

    2016-04-15

    Toxin-antitoxin (TA) loci are widespread in bacteria plasmids and chromosomes, and target various cellular functions to regulate cell growth and death. A type II TA system RnlA-RnlB from Escherichia coli is associated with phage-resistance. After the infection of bacteriophage T4 with Dmd defection, RnlA is activated by the disappearance of RnlB, resulting in the rapid degradation of T4 mRNAs. Dmd can bind to RnlA directly and neutralize RnlA toxicity to allow phage reproduction. Dmd represent a heterogenous antitoxin of RnlA replacing antitoxin RnlB. Here, we reported two structures of Dmd from T4 phage and RB69 phage. Both Dmd structures are high similar with a compacted domain composed of a four-stranded anti-parallel β-sheet and an α-helix. Chromatography and SAXS suggest Dmd forms a dimer in solution consistent with that in crystal. Structure-based mutagenesis of Dmd reveals key residues involved in RnlA-binding. Possibility cavities in Dmd used for compounds design were modeled. Our structural study revealed the recognition and inhibition mechanism of RnlA by Dmd and providing a potential laboratory phage prevention target for drug design. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications.

    Science.gov (United States)

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-02-19

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  15. A Novel Rabbit Spirometry Model of Type E Botulism and its Use for the Evaluation of Post-symptom Antitoxin Efficacy.

    Science.gov (United States)

    Diamant, Eran; Pass, Avi; Rosen, Osnat; Ben David, Alon; Torgeman, Amram; Barnea, Ada; Tal, Arnon; Rosner, Amir; Zichel, Ran

    2018-02-05

    Botulinum neurotoxins (BoNTs), the most poisonous substances known in nature, pose significant concern to health authorities. The only approved therapeutic for botulism is antitoxin. While administered to patients only after symptom onset, antitoxin efficacy is evaluated in animals mostly in relation to time post-intoxication regardless of symptoms. This is most likely due to the difficulty to measure early symptoms of botulism in animals. In the current study, a rabbit spirometry model was developed to quantify early respiratory symptoms of type E botulism that were further used as trigger for treatment. Impaired respiration, in the form of reduced minute volume, was detected as early as 18.1±2.9 hours post-intramuscular exposure to 2 rabbit lethal dose fifty (LD 50 ) of BoNT/E, preceding any visible symptoms. All rabbits treated with antitoxin immediately following symptom onset survived. Post-symptom antitoxin efficacy was further evaluated in relation to toxin and antitoxin dosage as well as to delayed antitoxin administration. Our system enabled us to demonstrate, for the first time, full antitoxin protection of animals treated with antitoxin after the onset of objective and quantitative type E botulism symptoms. This model may be utilized to evaluate the efficacy of antitoxins in additional serotypes of BoNT as well as that of next generation anti-BoNT drugs. Copyright © 2018 American Society for Microbiology.

  16. Crystal Structure of the VapBC Toxin–Antitoxin Complex from Shigella flexneri Reveals a Hetero-Octameric DNA-Binding Assembly

    DEFF Research Database (Denmark)

    Dienemann, Christian; Bøggild, Andreas; Winther, Kristoffer S.

    2011-01-01

    Toxin–antitoxin (TA) loci are common in archaea and prokaryotes and allow cells to rapidly adapt to changing environmental conditions through release of active regulators of metabolism. Many toxins are endonucleases that target cellular mRNA and tRNAs, while the antitoxins tightly wrap around...

  17. The Intrinsically Disordered Domain of the Antitoxin Phd Chaperones the Toxin Doc against Irreversible Inactivation and Misfolding*

    Science.gov (United States)

    De Gieter, Steven; Konijnenberg, Albert; Talavera, Ariel; Butterer, Annika; Haesaerts, Sarah; De Greve, Henri; Sobott, Frank; Loris, Remy; Garcia-Pino, Abel

    2014-01-01

    The toxin Doc from the phd/doc toxin-antitoxin module targets the cellular translation machinery and is inhibited by its antitoxin partner Phd. Here we show that Phd also functions as a chaperone, keeping Doc in an active, correctly folded conformation. In the absence of Phd, Doc exists in a relatively expanded state that is prone to dimerization through domain swapping with its active site loop acting as hinge region. The domain-swapped dimer is not capable of arresting protein synthesis in vitro, whereas the Doc monomer is. Upon binding to Phd, Doc becomes more compact and is secured in its monomeric state with a neutralized active site. PMID:25326388

  18. Identification of the first functional toxin-antitoxin system in Streptomyces.

    Directory of Open Access Journals (Sweden)

    Laura Sevillano

    Full Text Available Toxin-antitoxin (TA systems are widespread among the plasmids and genomes of bacteria and archaea. This work reports the first description of a functional TA system in Streptomyces that is identical in two species routinely used in the laboratory: Streptomyces lividans and S. coelicolor. The described system belongs to the YefM/YoeB family and has a considerable similarity to Escherichia coli YefM/YoeB (about 53% identity and 73% similarity. Lethal effect of the S. lividans putative toxin (YoeBsl was observed when expressed alone in E. coli SC36 (MG1655 ΔyefM-yoeB. However, no toxicity was obtained when co-expression of the antitoxin and toxin (YefM/YoeBsl was carried out. The toxic effect was also observed when the yoeBsl was cloned in multicopy in the wild-type S. lividans or in a single copy in a S. lividans mutant, in which this TA system had been deleted. The S. lividans YefM/YoeBsl complex, purified from E. coli, binds with high affinity to its own promoter region but not to other three random selected promoters from Streptomyces. In vivo experiments demonstrated that the expression of yoeBsl in E. coli blocks translation initiation processing mRNA at three bases downstream of the initiation codon after 2 minutes of induction. These results indicate that the mechanism of action is identical to that of YoeB from E. coli.

  19. A viral nanoparticle with dual function as an anthrax antitoxin and vaccine.

    Directory of Open Access Journals (Sweden)

    Darly J Manayani

    2007-10-01

    Full Text Available The recent use of Bacillus anthracis as a bioweapon has stimulated the search for novel antitoxins and vaccines that act rapidly and with minimal adverse effects. B. anthracis produces an AB-type toxin composed of the receptor-binding moiety protective antigen (PA and the enzymatic moieties edema factor and lethal factor. PA is a key target for both antitoxin and vaccine development. We used the icosahedral insect virus Flock House virus as a platform to display 180 copies of the high affinity, PA-binding von Willebrand A domain of the ANTXR2 cellular receptor. The chimeric virus-like particles (VLPs correctly displayed the receptor von Willebrand A domain on their surface and inhibited lethal toxin action in in vitro and in vivo models of anthrax intoxication. Moreover, VLPs complexed with PA elicited a potent toxin-neutralizing antibody response that protected rats from anthrax lethal toxin challenge after a single immunization without adjuvant. This recombinant VLP platform represents a novel and highly effective, dually-acting reagent for treatment and protection against anthrax.

  20. A Viral Nanoparticle with Dual Function as an Anthrax Antitoxin and Vaccine

    Science.gov (United States)

    Manayani, Darly J; Thomas, Diane; Dryden, Kelly A; Reddy, Vijay; Siladi, Marc E; Marlett, John M; Rainey, G. Jonah A; Pique, Michael E; Scobie, Heather M; Yeager, Mark; Young, John A. T; Manchester, Marianne; Schneemann, Anette

    2007-01-01

    The recent use of Bacillus anthracis as a bioweapon has stimulated the search for novel antitoxins and vaccines that act rapidly and with minimal adverse effects. B. anthracis produces an AB-type toxin composed of the receptor-binding moiety protective antigen (PA) and the enzymatic moieties edema factor and lethal factor. PA is a key target for both antitoxin and vaccine development. We used the icosahedral insect virus Flock House virus as a platform to display 180 copies of the high affinity, PA-binding von Willebrand A domain of the ANTXR2 cellular receptor. The chimeric virus-like particles (VLPs) correctly displayed the receptor von Willebrand A domain on their surface and inhibited lethal toxin action in in vitro and in vivo models of anthrax intoxication. Moreover, VLPs complexed with PA elicited a potent toxin-neutralizing antibody response that protected rats from anthrax lethal toxin challenge after a single immunization without adjuvant. This recombinant VLP platform represents a novel and highly effective, dually-acting reagent for treatment and protection against anthrax. PMID:17922572

  1. Structural insights into the inhibition mechanism of bacterial toxin LsoA by bacteriophage antitoxin Dmd.

    Science.gov (United States)

    Wan, Hua; Otsuka, Yuichi; Gao, Zeng-Qiang; Wei, Yong; Chen, Zhen; Masuda, Michiaki; Yonesaki, Tetsuro; Zhang, Heng; Dong, Yu-Hui

    2016-09-01

    Bacteria have obtained a variety of resistance mechanisms including toxin-antitoxin (TA) systems against bacteriophages (phages), whereas phages have also evolved to overcome bacterial anti-phage mechanisms. Dmd from T4 phage can suppress the toxicities of homologous toxins LsoA and RnlA from Escherichia coli, representing the first example of a phage antitoxin against multiple bacterial toxins in known TA systems. Here, the crystal structure of LsoA-Dmd complex showed Dmd is inserted into the deep groove between the N-terminal repeated domain (NRD) and the Dmd-binding domain (DBD) of LsoA. The NRD shifts significantly from a 'closed' to an 'open' conformation upon Dmd binding. Site-directed mutagenesis of Dmd revealed the conserved residues (W31 and N40) are necessary for LsoA binding and the toxicity suppression as determined by pull-down and cell toxicity assays. Further mutagenesis identified the conserved Dmd-binding residues (R243, E246 and R305) of LsoA are vital for its toxicity, and suggested Dmd and LsoB may possess different inhibitory mechanisms against LsoA toxicity. Our structure-function studies demonstrate Dmd can recognize LsoA and inhibit its toxicity by occupying the active site possibly via substrate mimicry. These findings have provided unique insights into the defense and counter-defense mechanisms between bacteria and phages in their co-evolution. © 2016 John Wiley & Sons Ltd.

  2. Structural studies of the toxin-antitoxin proteins RelE and RelB from E. coli

    DEFF Research Database (Denmark)

    Andersen, Kasper Røjkjær; Overgaard, Martin; Gerdes, Kenn

    the special tRNA-mRNA mimic, tmRNA [1]. Questions to be addressed Many questions remain to be answered in the bacterial toxin-antitoxin system. The crystal structure of RelBE from Pyrococcus horikoshii OT3 was previously solved at 2.3Å [2]. This structure shows the molecule in an inactive state, but OT3......The bacterial toxin-antitoxin system The relBE operon in E. coli encodes two small proteins: A toxin, RelE (12 kDa) and an antitoxin, RelB (9 kDa). RelE is activated under nutritional stress and is able to inhibit protein synthesis by cleaving the mRNA in the ribosomal A-site. This stress response...... serves to down-regulate metabolism in the cell when growth conditions are limited. RelB is expressed in excess over RelE during balanced growth, and inhibits the toxicity of RelE by forming an extremely stable toxin-antitoxin complex. The activation of RelE is induced when the labile RelB protein...

  3. Early and enhanced antitoxin responses elicited with complexes of tetanus toxoid and specific mouse and human antibodies

    International Nuclear Information System (INIS)

    Stoner, R.D.; Terres, G.; Hess, M.W.

    1975-01-01

    Primary tetanus antitoxin responses were early and enhanced in mice when tetanus toxoid was administered in complex with specific isologous antitoxin or specific mouse γ-globulin. Antitoxin responses were enhanced when fluid tetanus toxoid was complexed in vitro in antigen-to-antibody ratios of equivalence or antigen excess; responses to complexed toxoid in antibody excess were comparatively repressed. Primary responses were greatly inhibited in mice immunized with the same amount of toxoid complexed at equivalence or in antibody excess with specific human γ-globulin. Although primary responses were totally repressed, a primed state developed; a second injection of fluid toxoid within a few days produced excellent antitoxin responses. Separate injections of antigen and antibody at different sites produced an excellent in vivo primed state for early and high responses. Antibody production after stimulation with complexed toxoid was also enhanced in mice irradiated with 400 rads, a dose that ordinarily completely suppresses primary responses with fluid toxoid alone. These data provide evidence for the efficacy of antigen-antibody complexes in early and active immunization. (U.S.)

  4. The DinJ/RelE Toxin-Antitoxin System Suppresses Bacterial Proliferation and Virulence of Xylella fastidiosa in Grapevine.

    Science.gov (United States)

    Burbank, Lindsey P; Stenger, Drake C

    2017-04-01

    Xylella fastidiosa, the causal agent of Pierce's disease of grapes, is a slow-growing, xylem-limited, bacterial pathogen. Disease progression is characterized by systemic spread of the bacterium through xylem vessel networks, causing leaf-scorching symptoms, senescence, and vine decline. It appears to be advantageous to this pathogen to avoid excessive blockage of xylem vessels, because living bacterial cells are generally found in plant tissue with low bacterial cell density and minimal scorching symptoms. The DinJ/RelE toxin-antitoxin system is characterized here for a role in controlling bacterial proliferation and population size during plant colonization. The DinJ/RelE locus is transcribed from two separate promoters, allowing for coexpression of antitoxin DinJ with endoribonuclease toxin RelE, in addition to independent expression of RelE. The ratio of antitoxin/toxin expressed is dependent on bacterial growth conditions, with lower amounts of antitoxin present under conditions designed to mimic grapevine xylem sap. A knockout mutant of DinJ/RelE exhibits a hypervirulent phenotype, with higher bacterial populations and increased symptom development and plant decline. It is likely that DinJ/RelE acts to prevent excessive population growth, contributing to the ability of the pathogen to spread systemically without completely blocking the xylem vessels and increasing probability of acquisition by the insect vector.

  5. Proteomic profiling of Mycobacterium tuberculosis identifies nutrient-starvation-responsive toxin-antitoxin systems

    DEFF Research Database (Denmark)

    Albrethsen, Jakob; Agner, Jeppe; Piersma, Sander R

    2013-01-01

    In order to successfully enter the latent stage, Mycobacterium tuberculosis must adapt to conditions such as nutrient limitation and hypoxia. In vitro models that mimic latent infection are valuable tools for describing the changes in metabolism that occur when the bacterium exists in a non......-growing form. We used two complementary proteomic approaches, label-free LC-MS/MS analysis and two-dimensional difference gel electrophoresis, to determine the proteome profile of extracellular proteins from M. tuberculosis cultured under nutrient starvation. Through the label-free LC-MS/MS analysis......, significant differences in the overall metabolism during nutrient starvation were detected. Notably, members of the toxin-antitoxin systems were present in larger quantities in nutrient-starved cultures, supporting a role for these global modules as M. tuberculosis switches its metabolism into dormancy...

  6. Structural and thermodynamic characterization of the Escherichia coli RelBE toxin-antitoxin system: indication for a functional role of differential stability

    DEFF Research Database (Denmark)

    Cherny, Izhack; Overgaard, Martin; Borch, Jonas

    2007-01-01

    The RelE and RelB proteins constitute the RNA interferase (toxin) and its cognate inhibitor (antitoxin) components of the Escherichia coli relBE toxin-antitoxin system. Despite the well-described functionality and physiological activity of this system in E. coli, no structural study was performed...... on the folding and stability of the protein pair in solution. Here we structurally and thermodynamically characterize the RelBE system components from E. coli in solution, both separately and in their complexed state. The RelB antitoxin, an alpha-helical protein according to circular dichroism and infrared......, our results support that RelB protein from E. coli is similar to ParD and CcdA antitoxins in both fold and thermodynamic properties. The differential folding state of the proteins is discussed in the context of their physiological activities....

  7. The intrinsically disordered domain of the antitoxin Phd chaperones the toxin Doc against irreversible inactivation and misfolding.

    Science.gov (United States)

    De Gieter, Steven; Konijnenberg, Albert; Talavera, Ariel; Butterer, Annika; Haesaerts, Sarah; De Greve, Henri; Sobott, Frank; Loris, Remy; Garcia-Pino, Abel

    2014-12-05

    The toxin Doc from the phd/doc toxin-antitoxin module targets the cellular translation machinery and is inhibited by its antitoxin partner Phd. Here we show that Phd also functions as a chaperone, keeping Doc in an active, correctly folded conformation. In the absence of Phd, Doc exists in a relatively expanded state that is prone to dimerization through domain swapping with its active site loop acting as hinge region. The domain-swapped dimer is not capable of arresting protein synthesis in vitro, whereas the Doc monomer is. Upon binding to Phd, Doc becomes more compact and is secured in its monomeric state with a neutralized active site. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Crystal structure of the antitoxin-toxin protein complex RelB-RelE from Methanococcus jannaschii.

    Science.gov (United States)

    Francuski, Djordje; Saenger, Wolfram

    2009-11-06

    Here we present the crystal structure of the Methanococcus jannaschii RelE-RelB (RelBE) toxin-antitoxin (TA) protein complex determined by the MIRAS (multiple isomorphous replacement with anomalous signal) method. The genes encoding this TA system are located in the chromosome of this archaeon and involved in stress response. RelE acts as an endoribonuclease that cleaves mRNA on the ribosome, and we compare the RelBE complex to the known structures of other TA systems belonging to this group and to endoribonucleases. M. jannaschii RelBE forms a heterotetramer with the antitoxin in the centre of the complex, a configuration that differs vastly from the heterotetramer structure of the previously published RelBE from another archaeon, Pyrococcus horikoshii. The long N-terminal alpha-helix of the tightly bound M. jannaschii antitoxin RelB covers the presumed active site of the toxin RelE that is formed by a central beta-sheet, a loop on one side and a C-terminal alpha-helix on the other side. The active site of the M. jannaschii toxin RelE harbours positive charges that are thought to neutralize the negative charges of the substrate mRNA, including Arg62 that was changed to Ser62 by the Escherichia coli expression system, thereby leading to inactive toxin RelE. Comparative studies suggest that Asp43 and His79 are also involved in the activity of the toxin.

  9. Toxin-antitoxin vapBC locus participates in formation of the dormant state in Mycobacterium smegmatis.

    Science.gov (United States)

    Demidenok, Oksana I; Kaprelyants, Arseny S; Goncharenko, Anna V

    2014-03-01

    Toxin-antitoxin (TA) loci are widely spread in bacterial plasmids and chromosomes. Toxins affect important functions of bacterial cells such as translation, replication and cell-wall synthesis, whereas antitoxins are toxin inhibitors. Participation in formation of the dormant state in bacteria is suggested to be a possible function of toxins. Here we show that overexpression of VapC toxin in Mycobacterium smegmatis results in development of morphologically distinct ovoid cells. The ovoid cells were nonreplicating and revealed a low level of uracil incorporation and respiration that indicated their dormant status. To validate the role of VapBC in dormancy formation, we used a model of dormant, 'nonculturable' (NC) M. smegmatis cells obtained in potassium-limited conditions. Overexpression of VapB antitoxin prevented transition to dormancy, presumably due to a decreased level of the free VapC protein. Indeed, this effect of the VapB was neutralized by coexpression of the cognate VapC as a part of the vapBC operon. In summary, these findings reveal participation of vapBC products in formation of the dormant state in M. smegmatis. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  10. Crystallization of Doc and the Phd-Doc toxin-antitoxin complex.

    Science.gov (United States)

    Garcia-Pino, Abel; Dao-Thi, Minh-Hoa; Gazit, Ehud; Magnuson, Roy David; Wyns, Lode; Loris, Remy

    2008-11-01

    The phd/doc addiction system is responsible for the stable inheritance of lysogenic bacteriophage P1 in its plasmidic form in Escherichia coli and is the archetype of a family of bacterial toxin-antitoxin modules. The His66Tyr mutant of Doc (Doc(H66Y)) was crystallized in space group P2(1), with unit-cell parameters a = 53.1, b = 198.0, c = 54.1 A, beta = 93.0 degrees . These crystals diffracted to 2.5 A resolution and probably contained four dimers of Doc in the asymmetric unit. Doc(H66Y) in complex with a 22-amino-acid C-terminal peptide of Phd (Phd(52-73Se)) was crystallized in space group C2, with unit-cell parameters a = 111.1, b = 38.6, c = 63.3 A, beta = 99.3 degrees , and diffracted to 1.9 A resolution. Crystals of the complete wild-type Phd-Doc complex belonged to space group P3(1)21 or P3(2)21, had an elongated unit cell with dimensions a = b = 48.9, c = 354.9 A and diffracted to 2.4 A resolution using synchrotron radiation.

  11. Crystallization of Doc and the Phd–Doc toxin–antitoxin complex

    International Nuclear Information System (INIS)

    Garcia-Pino, Abel; Dao-Thi, Minh-Hoa; Gazit, Ehud; Magnuson, Roy David; Wyns, Lode; Loris, Remy

    2008-01-01

    Crystals of bacteriophage P1 Doc were grown in the free state, in complex with a 22-amino-acid C-terminal peptide of Phd and in complex with full-length Phd. The phd/doc addiction system is responsible for the stable inheritance of lysogenic bacteriophage P1 in its plasmidic form in Escherichia coli and is the archetype of a family of bacterial toxin–antitoxin modules. The His66Tyr mutant of Doc (Doc H66Y ) was crystallized in space group P2 1 , with unit-cell parameters a = 53.1, b = 198.0, c = 54.1 Å, β = 93.0°. These crystals diffracted to 2.5 Å resolution and probably contained four dimers of Doc in the asymmetric unit. Doc H66Y in complex with a 22-amino-acid C-terminal peptide of Phd (Phd 52-73Se ) was crystallized in space group C2, with unit-cell parameters a = 111.1, b = 38.6, c = 63.3 Å, β = 99.3°, and diffracted to 1.9 Å resolution. Crystals of the complete wild-type Phd–Doc complex belonged to space group P3 1 21 or P3 2 21, had an elongated unit cell with dimensions a = b = 48.9, c = 354.9 Å and diffracted to 2.4 Å resolution using synchrotron radiation

  12. Studies of the distribution of intrathecally injected 125I-tetanus antitoxin-F(ab')2

    International Nuclear Information System (INIS)

    Hanauske, A.R.

    1981-01-01

    Overall F(ab') 2 and antitetanus-f(ab') 2 - fragments were labelled with 125 I and injected i.th. into normal juvenile cats and adult rats. One group of rats was normal; in the other, unilateral local tetanus had been induced by injection of tetanus toxin into a M. gastrocnemius. The animals were sacrificed 24 h after the i.th. injection, and tissue samples were taken for histoautoradiography. 125 I-antitetanus-F(ab') 2 permeated into the extracellular space of the spinal cord, roots, and ganglia but not into the neuronal intracellular space. 125 I-overall-F(ab') showed identical permeation behaviour. 125 I-antitetanus-F(ab') 2 reacted with tetanus toxin issuing from the motoneurons after i.th. injection, forming an immunocomplex around the motorneurons. The immunocomplex was not formed around pseudo-unipolar ganglian cells in the spinal ganglia even though some of the ganglian cells contained tetanus toxin, and 125 I-antitetanus-F(ab') 2 was present in the extracellular space. As an explanation, it was suggested that tetanus toxin does not permeate into the extracellular space through the membrane of the pseudo-unipolar ganglian cells so that immune reactions will not occur. These findings help to explain the widely divergent results of tetanus therapy by means of i.th. injection of tetanus antitoxin. Recommendations for future therapy measures are derived from the findings. (orig./MG) [de

  13. Identification of a functional toxin-antitoxin system located in the genomic island PYG1 of piezophilic hyperthermophilic archaeon Pyrococcus yayanosii.

    Science.gov (United States)

    Li, Zhen; Song, Qinghao; Wang, Yinzhao; Xiao, Xiang; Xu, Jun

    2018-01-15

    Toxin-antitoxin (TA) system is bacterial or archaeal genetic module consisting of toxin and antitoxin gene that be organized as a bicistronic operon. TA system could elicit programmed cell death, which is supposed to play important roles for the survival of prokaryotic population under various physiological stress conditions. The phage abortive infection system (AbiE family) belongs to bacterial type IV TA system. However, no archaeal AbiE family TA system has been reported so far. In this study, a putative AbiE TA system (PygAT), which is located in a genomic island PYG1 in the chromosome of Pyrococcus yayanosii CH1, was identified and characterized. In Escherichia coli, overexpression of the toxin gene pygT inhibited its growth while the toxic effect can be suppressed by introducing the antitoxin gene pygA in the same cell. PygAT also enhances the stability of shuttle plasmids with archaeal plasmid replication protein Rep75 in E. coli. In P. yayanosii, disruption of antitoxin gene pygA cause a significantly growth delayed under high hydrostatic pressure (HHP). The antitoxin protein PygA can specifically bind to the PygAT promoter region and regulate the transcription of pygT gene in vivo. These results show that PygAT is a functional TA system in P. yayanosii, and also may play a role in the adaptation to HHP environment.

  14. Identification of a human monoclonal antibody to replace equine diphtheria antitoxin for treatment of diphtheria intoxication.

    Science.gov (United States)

    Sevigny, Leila M; Booth, Brian J; Rowley, Kirk J; Leav, Brett A; Cheslock, Peter S; Garrity, Kerry A; Sloan, Susan E; Thomas, William; Babcock, Gregory J; Wang, Yang

    2013-11-01

    Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (hMAbs) directly from antibody-secreting cells in the circulation of immunized human volunteers. We isolated a panel of diverse hMAbs that recognized diphtheria toxoid, as well as a variety of recombinant protein fragments of diphtheria toxin. Forty-five unique hMAbs were tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays with a 50% effective concentration of 0.65 ng/ml for the lead candidate hMAb, 315C4. In addition, 25 μg of 315C4 completely protected guinea pigs from intoxication in an in vivo lethality model, yielding an estimated relative potency of 64 IU/mg. In comparison, 1.6 IU of DAT was necessary for full protection from morbidity and mortality in this model. We further established that our lead candidate hMAb binds to the receptor-binding domain of diphtheria toxin and physically blocks the toxin from binding to the putative receptor, heparin-binding epidermal growth factor-like growth factor. The discovery of a specific and potent human neutralizing antibody against diphtheria toxin holds promise as a potential therapeutic.

  15. Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test

    Directory of Open Access Journals (Sweden)

    M.H. Sonobe

    2007-01-01

    Full Text Available A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test. In this study, the serum titers (values between 1.0 and 19.5 IU measured by a modified TOBI test (Modi-TOBI test and toxin neutralization assays were correlated (P < 0.0001. Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r² = 0.95, P < 0.0001 and for diphtheria (r² = 0.93, P < 0.0001 between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

  16. Toxin-Antitoxin Systems in Estuarine Synechococcus Strain CB0101 and Their Transcriptomic Responses to Environmental Stressors

    Directory of Open Access Journals (Sweden)

    David Marsan

    2017-07-01

    Full Text Available Bacterial toxin–antitoxin (TA systems are genetic elements composed of a toxin gene and its cognate antitoxin, with the ability to regulate growth. TA systems have not previously been reported in marine Synechococcus or Prochlorococcus. Here we report the finding of seven TA system pairs (Type II in the estuarine Synechococcus CB0101, and their responses of these TA genes to under different stress conditions, which include; nitrogen and phosphate starvation, phage infection, zinc toxicity, and photo-oxidation. Database searches discovered that eight other marine Synechococcus strains also contain at least one TA pair but none were found in Prochlorococcus. We demonstrate that the relB/relE TA pair was active and resulted in RNA degradation when CB0101 was under oxidative stress caused by either zinc toxicity or high light intensities, but the growth inhibition was released when the stress was removed. Having TA systems allows Synechococcus CB0101 to adapt to the low light and highly variable environments in the Chesapeake Bay. We propose that TA systems could be more important for picocyanobacteria living in the freshwater and estuarine environments compared to those living in the open ocean.

  17. A Toxin-Antitoxin System VapBC15 from Synechocystis sp. PCC 6803 Shows Distinct Regulatory Features

    Directory of Open Access Journals (Sweden)

    Qian Fei

    2018-03-01

    Full Text Available Type II toxin–antitoxin (TA systems play important roles in bacterial stress survival by regulating cell growth or death. They are highly abundant in cyanobacteria yet remain poorly characterized. Here, we report the identification and regulation of a putative type II TA system from Synechocystis PCC6803, VapBC15. The VapBC15 system is encoded by the chromosomal operon vapBC15. Exogenous expression of VapC15 dramatically arrested cell growth of Escherichia coli and reduced the numbers of colony-forming units (CFU. The VapC15 toxicity could be which was counteracted neutralized by simultaneous or delayed production of VapB15. Biochemical analysis demonstrated the formation of VapB15-VapC15 complexes by the physical interaction between VapB15 and VapC15. Notably, the VapB15 antitoxin up-regulated the transcription of the vapBC15 operon by directly binding to the promoter region, and the VapC15 toxin abolished the up-regulatory effect by destabilizing the binding. Moreover, VapB15 can be degraded by the proteases Lons and ClpXP2s from Synechocystis PCC6803, thus activating the latent toxicity of VapBC15. These findings suggest that VapBC15 represents a genuine TA system that utilizes a distinct mechanism to regulate toxin activity.

  18. A toxin antitoxin system promotes the maintenance of the IncA/C-mobilizable Salmonella Genomic Island 1.

    Science.gov (United States)

    Huguet, Kevin T; Gonnet, Mathieu; Doublet, Benoît; Cloeckaert, Axel

    2016-08-31

    The multidrug resistance Salmonella Genomic Island 1 (SGI1) is an integrative mobilizable element identified in several enterobacterial pathogens. This chromosomal island requires a conjugative IncA/C plasmid to be excised as a circular extrachromosomal form and conjugally mobilized in trans. Preliminary observations suggest stable maintenance of SGI1 in the host chromosome but paradoxically also incompatibility between SGI1 and IncA/C plasmids. Here, using a Salmonella enterica serovar Agona clonal bacterial population as model, we demonstrate that a Toxin-Antitoxin (TA) system encoded by SGI1 plays a critical role in its stable host maintenance when an IncA/C plasmid is concomitantly present. This system, designated sgiAT for Salmonella genomic island 1 Antitoxin and Toxin respectively, thus seems to play a stabilizing role in a situation where SGI1 is susceptible to be lost through plasmid IncA/C-mediated excision. Moreover and for the first time, the incompatibility between SGI1 and IncA/C plasmids was experimentally confirmed.

  19. A bacterial toxin-antitoxin module is the origin of inter-bacterial and inter-kingdom effectors of Bartonella.

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    Alexander Harms

    2017-10-01

    Full Text Available Host-targeting type IV secretion systems (T4SS evolved from conjugative T4SS machineries that mediate interbacterial plasmid transfer. However, the origins of effectors secreted by these virulence devices have remained largely elusive. Previous work showed that some effectors exhibit homology to toxins of bacterial toxin-antitoxin modules, but the evolutionary trajectories underlying these ties had not been resolved. We previously reported that FicT toxins of FicTA toxin-antitoxin modules disrupt cellular DNA topology via their enzymatic FIC (filamentation induced by cAMP domain. Intriguingly, the FIC domain of the FicT toxin VbhT of Bartonella schoenbuchensis is fused to a type IV secretion signal-the BID (Bep intracellular delivery domain-similar to the Bartonella effector proteins (Beps that are secreted into eukaryotic host cells via the host-targeting VirB T4SS. In this study, we show that the VbhT toxin is an interbacterial effector protein secreted via the conjugative Vbh T4SS that is closely related to the VirB T4SS and encoded by plasmid pVbh of B. schoenbuchensis. We therefore propose that the Vbh T4SS together with its effector VbhT represent an evolutionary missing link on a path that leads from a regular conjugation system and FicTA toxin-antitoxin modules to the VirB T4SS and the Beps. Intriguingly, phylogenetic analyses revealed that the fusion of FIC and BID domains has probably occurred independently in VbhT and the common ancestor of the Beps, suggesting parallel evolutionary paths. Moreover, several other examples of TA module toxins that are bona fide substrates of conjugative T4SS indicate that their recruitment as interbacterial effectors is prevalent and serves yet unknown biological functions in the context of bacterial conjugation. We propose that the adaptation for interbacterial transfer favors the exaptation of FicT and other TA module toxins as inter-kingdom effectors and may thus constitute an important stepping

  20. A bacterial toxin-antitoxin module is the origin of inter-bacterial and inter-kingdom effectors of Bartonella.

    Science.gov (United States)

    Harms, Alexander; Liesch, Marius; Körner, Jonas; Québatte, Maxime; Engel, Philipp; Dehio, Christoph

    2017-10-01

    Host-targeting type IV secretion systems (T4SS) evolved from conjugative T4SS machineries that mediate interbacterial plasmid transfer. However, the origins of effectors secreted by these virulence devices have remained largely elusive. Previous work showed that some effectors exhibit homology to toxins of bacterial toxin-antitoxin modules, but the evolutionary trajectories underlying these ties had not been resolved. We previously reported that FicT toxins of FicTA toxin-antitoxin modules disrupt cellular DNA topology via their enzymatic FIC (filamentation induced by cAMP) domain. Intriguingly, the FIC domain of the FicT toxin VbhT of Bartonella schoenbuchensis is fused to a type IV secretion signal-the BID (Bep intracellular delivery) domain-similar to the Bartonella effector proteins (Beps) that are secreted into eukaryotic host cells via the host-targeting VirB T4SS. In this study, we show that the VbhT toxin is an interbacterial effector protein secreted via the conjugative Vbh T4SS that is closely related to the VirB T4SS and encoded by plasmid pVbh of B. schoenbuchensis. We therefore propose that the Vbh T4SS together with its effector VbhT represent an evolutionary missing link on a path that leads from a regular conjugation system and FicTA toxin-antitoxin modules to the VirB T4SS and the Beps. Intriguingly, phylogenetic analyses revealed that the fusion of FIC and BID domains has probably occurred independently in VbhT and the common ancestor of the Beps, suggesting parallel evolutionary paths. Moreover, several other examples of TA module toxins that are bona fide substrates of conjugative T4SS indicate that their recruitment as interbacterial effectors is prevalent and serves yet unknown biological functions in the context of bacterial conjugation. We propose that the adaptation for interbacterial transfer favors the exaptation of FicT and other TA module toxins as inter-kingdom effectors and may thus constitute an important stepping stone in the

  1. A bacterial toxin-antitoxin module is the origin of inter-bacterial and inter-kingdom effectors of Bartonella

    Science.gov (United States)

    Liesch, Marius

    2017-01-01

    Host-targeting type IV secretion systems (T4SS) evolved from conjugative T4SS machineries that mediate interbacterial plasmid transfer. However, the origins of effectors secreted by these virulence devices have remained largely elusive. Previous work showed that some effectors exhibit homology to toxins of bacterial toxin-antitoxin modules, but the evolutionary trajectories underlying these ties had not been resolved. We previously reported that FicT toxins of FicTA toxin-antitoxin modules disrupt cellular DNA topology via their enzymatic FIC (filamentation induced by cAMP) domain. Intriguingly, the FIC domain of the FicT toxin VbhT of Bartonella schoenbuchensis is fused to a type IV secretion signal–the BID (Bep intracellular delivery) domain—similar to the Bartonella effector proteins (Beps) that are secreted into eukaryotic host cells via the host-targeting VirB T4SS. In this study, we show that the VbhT toxin is an interbacterial effector protein secreted via the conjugative Vbh T4SS that is closely related to the VirB T4SS and encoded by plasmid pVbh of B. schoenbuchensis. We therefore propose that the Vbh T4SS together with its effector VbhT represent an evolutionary missing link on a path that leads from a regular conjugation system and FicTA toxin-antitoxin modules to the VirB T4SS and the Beps. Intriguingly, phylogenetic analyses revealed that the fusion of FIC and BID domains has probably occurred independently in VbhT and the common ancestor of the Beps, suggesting parallel evolutionary paths. Moreover, several other examples of TA module toxins that are bona fide substrates of conjugative T4SS indicate that their recruitment as interbacterial effectors is prevalent and serves yet unknown biological functions in the context of bacterial conjugation. We propose that the adaptation for interbacterial transfer favors the exaptation of FicT and other TA module toxins as inter-kingdom effectors and may thus constitute an important stepping stone in the

  2. Antiradiation Antitoxin IgG : Immunological neutralization of Radiation Toxins at Acute Radiation Syndromes.

    Science.gov (United States)

    Popov, Dmitri; Maliev, Slava

    radiation toxins to induce hyperimmune serum: Group A -Toxoid form of CV ARS toxins ( SRD-1); Group B-Toxoid form of CR ARS (SRD-2)toxins ; Group C -Toxoid form of GI ARS (SRD-3); Group D -Toxoid form of HP ARS (SRD-4). After the hyperimmune serum was pooled from several animals, purified, and concentrated, the IgG fraction was separated. Enzyme-linked immunosorbent assays of the hyper-immune serum had revealed high titers of IgG with specific binding to radi-ation toxins. The antiradiation IgG preparation was injected into laboratory animals one hour before and three hours after irradiation, and was evaluated for its ability to protect inoculated animals against the development of acute radiation syndromes. Results: Animals that were inoculated with specific antiradiation antibodies before and after receiving lethal irradiation at LD 100/30 exhibited 60-75% survival rate within 30 days. Also, these animals inoculated with the Antiradiation Antitoxin had exhibited markedly reduced clinical symptoms of the ARS, even those ones that did not survive irradiation. Discussion: The results of our experiments have demonstrated that the rabbit hyperimmune IgG preparations directed against SRD toxins provide a significant protection against high doses of radiation. In comparison, the mortality rate of irradiated control animals was 100% in the same time period. The mortality rates of animals treated by the hyperimmune IgG antidote have varied in the different groups of ani-mals and different forms of the ARS. However, significant radioprotection was observed in each group treated with the IgGs. The specific antiradiation antidote IGg isolated from hyperim-mune serum of immunized horses is under study. The specific antiradiation antidote contains antibodies to neurotoxins -SAAN IgG includes 50% IgG to Cv ARS, 25% IgG to Cr ARS and 25 % IgG to Gi ARS. The other type of the Specific antiradiation antidote containes antibodies to hematotoxins -SAAH IgG -100%. A combined variant is under

  3. Is the mazEF toxin-antitoxin system responsible for vancomycin resistance in clinical isolates of Enterococcus faecalis?

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    Sadeghifard, Nourkhoda

    2014-03-01

    Full Text Available [english] The current study was conducted to investigate the relationship between vancomycin-resistant (VRE and the presence of toxin-antitoxin (TA system, which may be useful as target for novel antimicrobial therapy concepts. The susceptibility of was determined by MIC, and the presence of the TA system was evaluated by PCR. Among 200 isolates 39.5% showed resistance to vancomycin (VRE, while 60.5% were susceptible strains (VSE. The TA system was positive in all VRE isolates (100%, but less prevalent (38/121, 31.4% among the 121 VSE strains. In conclusion, our study demonstrated a positive relationship between the presence of vancomycin resistance and TA system. This observation may introduce therapeutic options against a novel antimicrobial target in enterococci.

  4. The Influence of the Toxin/Antitoxin mazEF on Growth and Survival of Listeria monocytogenes under Stress

    DEFF Research Database (Denmark)

    Curtis, Thomas; Takeuchi, Ippei; Gram, Lone

    2017-01-01

    A major factor in the resilience of Listeria monocytogenes is the alternative sigma factor B (σB). Type II Toxin/Antitoxin (TA) systems are also known to have a role in the bacterial stress response upon activation via the ClpP or Lon proteases. Directly upstream of the σB operon in L....... monocytogenes is the TA system mazEF, which can cleave mRNA at UACMU sites. In this study, we showed that the mazEF TA locus does not affect the level of persister formation during treatment with antibiotics in lethal doses, but exerts different effects according to the sub-inhibitory stress added. Growth...... it is not analogous to the system of S. aureus, suggesting a novel mode of action for MazEF in L. monocytogenes....

  5. Wound Botulism in Injection Drug Users: Time to Antitoxin Correlates with Intensive Care Unit Length of Stay

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    Offerman, Steven R

    2009-11-01

    Full Text Available Objectives: We sought to identify factors associated with need for mechanical ventilation (MV, length of intensive care unit (ICU stay, length of hospital stay, and poor outcome in injection drug users (IDUs with wound botulism (WB.Methods: This is a retrospective review of WB patients admitted between 1991-2005. IDUs were included if they had symptoms of WB and diagnostic confirmation. Primary outcome variables were the need for MV, length of ICU stay, length of hospital stay, hospital-related complications, and death.Results: Twenty-nine patients met inclusion criteria. Twenty-two (76% admitted to heroin use only and seven (24% admitted to heroin and methamphetamine use. Chief complaints on initial presentation included visual changes, 13 (45%; weakness, nine (31%; and difficulty swallowing, seven (24%. Skin wounds were documented in 22 (76%. Twenty-one (72% patients underwent mechanical ventilation (MV. Antitoxin (AT was administered to 26 (90% patients but only two received antitoxin in the emergency department (ED. The time from ED presentation to AT administration was associated with increased length of ICU stay (Regression coefficient = 2.5; 95% CI 0.45, 4.5. The time from ED presentation to wound drainage was also associated with increased length of ICU stay (Regression coefficient = 13.7; 95% CI = 2.3, 25.2. There was no relationship between time to antibiotic administration and length of ICU stay.Conclusion: MV and prolonged ICU stays are common in patients identified with WB. Early AT administration and wound drainage are recommended as these measures may decrease ICU length of stay.[West J Emerg Med. 2009;10(4:251-256.

  6. Cryptic Streptococcus mutans 5.6-kb plasmids encode a toxin–antitoxin system for plasmid stabilization

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    Anke Rheinberg

    2013-01-01

    Full Text Available Background: In all Streptococcus mutans strains, 5–13% carry a 5.6-kb plasmid. Despite its frequency, little is known about its mediated functions with most of the information coming from a single study focussing on plasmid pUA140. Objective: Here, we describe the sequence and genetic organization of two S. mutans 5.6-kb plasmids, pDC09 and pNC101. Results: Based on PicoGreen dsDNA quantification and Real-Time quantitative PCR (RTQ-PCR, the plasmid copy number was found to range between 10 and 74, depending on the strain tested. In contrast to literature, we identified six instead of five open reading frames (ORFs. While the putative gene products of ORF1 (as a Rep-protein and ORF2 (as a Mob-protein could be confirmed as being identical to those from pUA140, the functions of ORF3 (unknown and ORF 4 (possibly AtpE homologue could not be further revealed. However, the product of ORF5 showed a fairly high identity (38–50% and structural similarity (58–74% to RelE of Streptococcus pneumoniae, Streptococcus equi, and Streptococcus downei. In addition, we identified a functionally corresponding ORF6 encoding a protein with 61–68% identity (81–86% similarity to the S. equi and S. downei antitoxin of the RelB family. RelE and RelB together form a plasmid-encoded toxin-antitoxin (TA system, RelBEplas. Despite its rather limited sequence similarity with chromosomal TA systems in S. mutans (RelBEchro, MazEF, HicBA, we found similar tertiary structures applying I-Tasser protein prediction analysis. Conclusion: Type II-toxins, as the plasmid-encoded RelE, are RNA endonucleases. Depending on their mRNA cleavage activity, they might 1 kill every plasmid-free progeny, thereby stabilizing plasmid transfer at the expense of the host and/or 2 help S. mutans enter a dormant state and survive unfavourable environmental conditions. Whilst a function in plasmid stabilization has been confirmed, a function in persistence under nutritional stress, tested here

  7. Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection.

    Science.gov (United States)

    Burbank, Lindsey P; Stenger, Drake C

    2016-08-01

    The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention.

  8. Toxin-antitoxin loci vapBC-1 and vapXD contribute to survival and virulence in nontypeable Haemophilus influenzae

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    Ren Dabin

    2012-11-01

    Full Text Available Abstract Background Nontypeable Haemophilus influenzae (NTHi is a significant human pathogen responsible for respiratory tract infections and the most common cause of recurrent otitis media. Type II toxin-antitoxin (TA systems are genetic elements that code for a stable protein toxin and a labile antitoxin that are thought to be involved in metabolic regulation of bacteria by enabling a switch to a dormant state under stress conditions. The contribution to infection persistence of the NTHi TA loci vapBC-1 and vapXD was examined in this study. Results Deletions in vapBC-1, vapXD and vapBC-1 vapXD significantly decreased the survival of NTHi co-cultured with primary human respiratory tissue at the air-liquid interface and in the chinchilla model of otitis media. The TA deletions did not affect the growth dynamics of the mutants in rich media, their ultra-structural morphology, or display appreciable synergy during NTHi infections. The toxin and antitoxin proteins of both pairs heterodimerized in vivo. Consistent with our previous findings regarding the VapC-1 toxin, the NTHi VapD toxin also displayed ribonuclease activity. Conclusions We conclude that the vapBC-1 and vapXD TA loci enhance NTHi survival and virulence during infection in vitro and in vivo using a mechanism of mRNA cleavage, and that these conserved TA pairs represent new targets for the prophylaxis and therapy of otitis media and other NTHi-caused mucosal diseases.

  9. Transcriptional profiling of type II toxin-antitoxin genes of Helicobacter pylori under different environmental conditions: identification of HP0967-HP0968 system

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    MIGUEL A DE LA CRUZ

    2016-11-01

    Full Text Available Helicobacter pylori is a Gram-negative bacterium that colonizes the human gastric mucosa and is responsible for causing peptic ulcers and gastric carcinoma. The expression of virulence factors allows the persistence of H. pylori in the stomach, which results in a chronic, sometimes uncontrolled inflammatory response. Type II toxin-antitoxin systems have emerged as important virulence factors in many pathogenic bacteria. Three type II toxin-antitoxin (TA systems have previously been identified in the genome of H. pylori 26695: HP0315-HP0316, HP0892-HP0893, and HP0894-HP0895. Here we characterized a heretofore undescribed type II TA system in H. pylori, HP0967-HP0968, which is encoded by the bicistronic operon hp0968-hp0967 and belongs to the Vap family. The predicted HP0967 protein is a toxin with ribonuclease activity whereas HP0968 is an antitoxin that binds to its own regulatory region. We found that all type II TA systems were expressed in H. pylori during early stationary growth phase, and differentially expressed in the presence of urea, nickel, and iron, although the hp0968-hp0967 pair was the most affected under these environmental conditions. Transcription of hp0968-hp0967 was strongly induced in a mature H. pylori biofilm and when the bacteria interacted with AGS epithelial cells. Kanamycin and chloramphenicol considerably boosted transcription levels of all the four type II TA systems. The hp0968-hp0967 TA system was the most frequent among 317 H. pylori strains isolated from all over the world. This study is the first report on the transcription of type II TA genes in H. pylori under different environmental conditions. Our data show that the HP0967 and HP0968 proteins constitute a bona fide type II TA system in H. pylori, whose expression is regulated by environmental cues, which are relevant in the context of infection of the human gastric mucosa.

  10. Potency of a human monoclonal antibody to diphtheria toxin relative to equine diphtheria anti-toxin in a guinea pig intoxication model.

    Science.gov (United States)

    Smith, Heidi L; Cheslock, Peter; Leney, Mark; Barton, Bruce; Molrine, Deborah C

    2016-08-17

    Prompt administration of anti-toxin reduces mortality following Corynebacterium diphtheriae infection. Current treatment relies upon equine diphtheria anti-toxin (DAT), with a 10% risk of serum sickness and rarely anaphylaxis. The global DAT supply is extremely limited; most manufacturers have ceased production. S315 is a neutralizing human IgG1 monoclonal antibody to diphtheria toxin that may provide a safe and effective alternative to equine DAT and address critical supply issues. To guide dose selection for IND-enabling pharmacology and toxicology studies, we dose-ranged S315 and DAT in a guinea pig model of diphtheria intoxication based on the NIH Minimum Requirements potency assay. Animals received a single injection of antibody premixed with toxin, were monitored for 30 days, and assigned a numeric score for clinical signs of disease. Animals receiving ≥ 27.5 µg of S315 or ≥ 1.75 IU of DAT survived whereas animals receiving ≤ 22.5 µg of S315 or ≤ 1.25 IU of DAT died, yielding a potency estimate of 17 µg S315/IU DAT (95% CI 16-21) for an endpoint of survival. Because some surviving animals exhibited transient limb weakness, likely a systemic sign of toxicity, DAT and S315 doses required to prevent hind limb paralysis were also determined, yielding a relative potency of 48 µg/IU (95% CI 38-59) for this alternate endpoint. To support advancement of S315 into clinical trials, potency estimates will be used to evaluate the efficacy of S315 versus DAT in an animal model with antibody administration after toxin exposure, more closely modeling anti-toxin therapy in humans.

  11. Functional validation of putative toxin-antitoxin genes from the Gram-positive pathogen Streptococcus pneumoniae: phd-doc is the fourth bona-fide operon.

    Science.gov (United States)

    Chan, Wai Ting; Yeo, Chew Chieng; Sadowy, Ewa; Espinosa, Manuel

    2014-01-01

    Bacterial toxin-antitoxin (TAs) loci usually consist of two genes organized as an operon, where their products are bound together and inert under normal conditions. However, under stressful circumstances the antitoxin, which is more labile, will be degraded more rapidly, thereby unleashing its cognate toxin to act on the cell. This, in turn, causes cell stasis or cell death, depending on the type of TAs and/or time of toxin exposure. Previously based on in silico analyses, we proposed that Streptococcus pneumoniae, a pathogenic Gram-positive bacterium, may harbor between 4 and 10 putative TA loci depending on the strains. Here we have chosen the pneumococcal strain Hungary(19A)-6 which contains all possible 10 TA loci. In addition to the three well-characterized operons, namely relBE2, yefM-yoeB, and pezAT, we show here the functionality of a fourth operon that encodes the pneumococcal equivalent of the phd-doc TA. Transcriptional fusions with gene encoding Green Fluorescent Protein showed that the promoter was slightly repressed by the Phd antitoxin, and exhibited almost background values when both Phd-Doc were expressed together. These findings demonstrate that phd-doc shows the negative self-regulatory features typical for an authentic TA. Further, we also show that the previously proposed TAs XreA-Ant and Bro-XreB, although they exhibit a genetic organization resembling those of typical TAs, did not appear to confer a functional behavior corresponding to bona fide TAs. In addition, we have also discovered new interesting bioinformatics results for the known pneumococcal TAs RelBE2 and PezAT. A global analysis of the four identified toxins-antitoxins in the pneumococcal genomes (PezAT, RelBE2, YefM-YoeB, and Phd-Doc) showed that RelBE2 and Phd-Doc are the most conserved ones. Further, there was good correlation among TA types, clonal complexes and sequence types in the 48 pneumococcal strains analyzed.

  12. Construction of a novel, stable, food-grade expression system by engineering the endogenous toxin-antitoxin system in Bacillus subtilis.

    Science.gov (United States)

    Yang, Sen; Kang, Zhen; Cao, Wenlong; Du, Guocheng; Chen, Jian

    2016-02-10

    Bacillus subtilis as an important workhorse that has been widely used to produce enzymes and metabolites. To broaden its applications, especially in the food and feed industry, we constructed a novel, stable, food-grade expression system by engineering its type II toxin-antitoxin system. The expression of the toxin EndoA, encoded by the chromosomal ydcE gene, was regulated by an endogenous, xylose-inducible promoter, while the ydcD gene, which encodes the unstable antitoxin EndoB, was inserted into a food-grade vector backbone, where its expression was driven by the native, constitutive promoter PylxM. By maintaining the xylose concentration above 2.0 g L(-1), this auto-regulated expression system was absolutely stable after 100 generations. Compared with traditional antibiotic-dependent expression systems, this novel expression system resulted in greater biomass and higher titers of desired products (enzymes or metabolites). Our results demonstrate that this stable, food-grade expression system is suitable for enzyme production and pathway engineering, especially for the production of food-grade enzymes and metabolites. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. The mazEF toxin-antitoxin system as an attractive target in clinical isolates of Enterococcus faecium and Enterococcus faecalis.

    Science.gov (United States)

    Soheili, Sara; Ghafourian, Sobhan; Sekawi, Zamberi; Neela, Vasantha Kumari; Sadeghifard, Nourkhoda; Taherikalani, Morovat; Khosravi, Afra; Ramli, Ramliza; Hamat, Rukman Awang

    2015-01-01

    The toxin-antitoxin (TA) system is a regulatory system where two sets of genes encode the toxin and its corresponding antitoxin. In this study, the prevalence of TA systems in independently isolated clinical isolates of Enterococcus faecium and Enterococcus faecalis was determined, the dominant TA system was identified, different virulence genes in E. faecium and E. faecalis were surveyed, the level of expression of the virulence and TA genes in normal and stress conditions was determined, and finally their associations with the TA genes were defined. Remarkably, the analysis demonstrated higBA and mazEF in all clinical isolates, and their locations were on chromosomes and plasmids, respectively. On the other hand, a quantitative analysis of TA and virulence genes revealed that the expression level in both genes is different under normal and stress conditions. The results obtained by anti-mazF peptide nucleic acids demonstrated that the expression level of virulence genes had decreased. These findings demonstrate an association between TA systems and virulence factors. The mazEF on the plasmids and the higBA TA genes on the chromosomes of all E. faecium and E. faecalis strains were dominant. Additionally, there was a decrease in the expression of virulence genes in the presence of anti-mazF peptide nucleic acids. Therefore, it is suggested that mazEF TA systems are potent and sensitive targets in all E. faecium and E. faecalis strains.

  14. The MqsRA Toxin-Antitoxin System from Xylella fastidiosa Plays a Key Role in Bacterial Fitness, Pathogenicity, and Persister Cell Formation

    Science.gov (United States)

    Merfa, Marcus V.; Niza, Bárbara; Takita, Marco A.; De Souza, Alessandra A.

    2016-01-01

    Through the formation of persister cells, bacteria exhibit tolerance to multidrug and other environmental stresses without undergoing genetic changes. The toxin-antitoxin (TA) systems are involved in the formation of persister cells because they are able to induce cell dormancy. Among the TA systems, the MqsRA system has been observed to be highly induced in persister cells of Xylella fastidiosa (causal agent of citrus variegated chlorosis—CVC) activated by copper stress, and has been described in Escherichia coli as related to the formation of persister cells and biofilms. Thus, we evaluated the role of this TA system in X. fastidiosa by overexpressing the MqsR toxin, and verified that the toxin positively regulated biofilm formation and negatively cell movement, resulting in reduced pathogenicity in citrus plants. The overexpression of MqsR also increased the formation of persister cells under copper stress. Analysis of the gene and protein expression showed that this system likely has an autoregulation mechanism to express the toxin and antitoxin in the most beneficial ratio for the cell to oppose stress. Our results suggest that this TA system plays a key role in the adaptation and survival of X. fastidiosa and reveal new insights into the physiology of phytopathogen-host interactions. PMID:27375608

  15. The monoclonal antitoxin antibodies (actoxumab-bezlotoxumab treatment facilitates normalization of the gut microbiota of mice with Clostridium difficile infection

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    Mária Džunková

    2016-10-01

    detected by the end of the experiments. In conclusion, MK-3415A (actoxumab-bezlotoxumab treatment facilitates normalization of the gut microbiota in CDI mice. It remains to be examined whether or not the prevention of recurrent CDI by the antitoxin antibodies observed in clinical trials occurs through modulation of microbiota.

  16. Three dimensional structure of the MqsR:MqsA complex: a novel TA pair comprised of a toxin homologous to RelE and an antitoxin with unique properties.

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    Breann L Brown

    2009-12-01

    Full Text Available One mechanism by which bacteria survive environmental stress is through the formation of bacterial persisters, a sub-population of genetically identical quiescent cells that exhibit multidrug tolerance and are highly enriched in bacterial toxins. Recently, the Escherichia coli gene mqsR (b3022 was identified as the gene most highly upregulated in persisters. Here, we report multiple individual and complex three-dimensional structures of MqsR and its antitoxin MqsA (B3021, which reveal that MqsR:MqsA form a novel toxin:antitoxin (TA pair. MqsR adopts an alpha/beta fold that is homologous with the RelE/YoeB family of bacterial ribonuclease toxins. MqsA is an elongated dimer that neutralizes MqsR toxicity. As expected for a TA pair, MqsA binds its own promoter. Unexpectedly, it also binds the promoters of genes important for E. coli physiology (e.g., mcbR, spy. Unlike canonical antitoxins, MqsA is also structured throughout its entire sequence, binds zinc and coordinates DNA via its C- and not N-terminal domain. These studies reveal that TA systems, especially the antitoxins, are significantly more diverse than previously recognized and provide new insights into the role of toxins in maintaining the persister state.

  17. Toxin-antitoxin loci as stress-response-elements: ChpAK/MazF and ChpBK cleave translated RNAs and are counteracted by tmRNA

    DEFF Research Database (Denmark)

    Christensen, S.K.; Pedersen, K.; Hansen, Flemming G.

    2003-01-01

    Prokaryotic chromosomes encode toxin-antitoxin loci, often in multiple copies. In most cases, the function of these genes is not known. The chpA (mazEF) locus of Escherichia coli has been described as a cell killing module that induces bacterial apoptosis during nutritional stress. However, we...... found recently that ChpAK (MazF) does not confer cell killing but rather, induces a bacteriostatic condition from which the cells could be resuscitated. Results presented here yield a mechanistic explanation for the detrimental effect on cell growth exerted by ChpAK and the homologous ChpBK protein of E......AK cleaved tmRNA in its coding region. Thus, ChpAK and ChpBK inhibit translation by a mechanism very similar to that of E. coli RelE. On the basis of these results, we propose a model that integrates TA loci into general prokaryotic stress physiology....

  18. Environmental T4-Family Bacteriophages Evolve to Escape Abortive Infection via Multiple Routes in a Bacterial Host Employing "Altruistic Suicide" through Type III Toxin-Antitoxin Systems.

    Science.gov (United States)

    Chen, Bihe; Akusobi, Chidiebere; Fang, Xinzhe; Salmond, George P C

    2017-01-01

    Abortive infection is an anti-phage mechanism employed by a bacterium to initiate its own death upon phage infection. This reduces, or eliminates, production of viral progeny and protects clonal siblings in the bacterial population by an act akin to an "altruistic suicide." Abortive infection can be mediated by a Type III toxin-antitoxin system called ToxIN Pa consisting of an endoribonuclease toxin and RNA antitoxin. ToxIN Pa is a heterohexameric quaternary complex in which pseudoknotted RNA inhibits the toxicity of the toxin until infection by certain phages causes destabilization of ToxIN Pa , leading to bacteriostasis and, eventually, lethality. However, it is still unknown why only certain phages are able to activate ToxIN Pa . To try to address this issue we first introduced ToxIN Pa into the Gram-negative enterobacterium, Serratia sp. ATCC 39006 ( S 39006) and then isolated new environmental S 39006 phages that were scored for activation of ToxIN Pa and abortive infection capacity. We isolated three T4-like phages from a sewage treatment outflow point into the River Cam, each phage being isolated at least a year apart. These phages were susceptible to ToxIN Pa -mediated abortive infection but produced spontaneous "escape" mutants that were insensitive to ToxIN Pa . Analysis of these resistant mutants revealed three different routes of escaping ToxIN Pa , namely by mutating asiA (the product of which is a phage transcriptional co-activator); by mutating a conserved, yet functionally unknown, orf84 ; or by deleting a 6.5-10 kb region of the phage genome. Analysis of these evolved escape mutants may help uncover the nature of the corresponding phage product(s) involved in activation of ToxIN Pa .

  19. Workgroup Report by the Joint Task Force Involving American Academy of Allergy, Asthma & Immunology (AAAAI); Food Allergy, Anaphylaxis, Dermatology and Drug Allergy (FADDA) (Adverse Reactions to Foods Committee and Adverse Reactions to Drugs, Biologicals, and Latex Committee); and the Centers for Disease Control and Prevention Botulism Clinical Treatment Guidelines Workgroup-Allergic Reactions to Botulinum Antitoxin: A Systematic Review.

    Science.gov (United States)

    Schussler, Edith; Sobel, Jeremy; Hsu, Joy; Yu, Patricia; Meaney-Delman, Dana; Grammer, Leslie C; Nowak-Wegrzyn, Anna

    2017-12-27

    Naturally occurring botulism is rare, but a large number of cases could result from unintentional or intentional contamination of a commercial food. Despeciated, equine-derived, heptavalent botulinum antitoxin (HBAT) is licensed in the United States. Timely treatment reduces morbidity and mortality, but concerns that botulinum antitoxin can induce anaphylaxis exist. We sought to quantify the allergy risk of botulinum antitoxin treatment and the usefulness of skin testing to assess this risk. We conducted a systematic review of (1) allergic reactions to botulinum antitoxin and (2) the predictive value of skin testing (ST) before botulinum antitoxin administration. We searched 5 scientific literature databases, reviewed articles' references, and obtained data from the HBAT manufacturer and from the Centers for Disease Control and Prevention. Anaphylaxis incidence was determined for HBAT and previously employed botulinum antitoxins. We calculated the positive predictive value (PPV) and negative predictive value (NPV) of ST for anaphylaxis related to HBAT and other botulinum antitoxins. Seven articles were included. Anaphylaxis incidence was 1.64% (5/305 patients) for HBAT and 1.16% (8/687 patients) for all other botulinum antitoxins (relative risk, 1.41 [95% confidence interval, .47-4.27]; P = .5). Observed values for both PPV and NPV for HBAT-ST (33 patients) were 100%. Observed PPVs and NPVs of ST for other botulinum antitoxins (302 patients) were 0-56% and 50%-100%, respectively. There were no reports of fatal anaphylaxis. Considering the <2 % rate of anaphylaxis, fatal outcomes, modest predictive value of ST, resource requirements for ST, and the benefits of early treatment, data do not support delaying HBAT administration to perform ST in a mass botulinum toxin exposure. Anaphylactic reactions may occur among 1%-2% of botulinum antitoxin recipients and will require epinephrine and antihistamine treatment and, possibly, intensive care. Published by Oxford

  20. VapC toxins from Mycobacterium tuberculosis are ribonucleases that differentially inhibit growth and are neutralized by cognate VapB antitoxins.

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    Bintou Ahmadou Ahidjo

    Full Text Available The chromosome of Mycobacterium tuberculosis (Mtb encodes forty seven toxin-antitoxin modules belonging to the VapBC family. The role of these modules in the physiology of Mtb and the function(s served by their expansion are unknown. We investigated ten vapBC modules from Mtb and the single vapBC from M. smegmatis. Of the Mtb vapCs assessed, only Rv0549c, Rv0595c, Rv2549c and Rv2829c were toxic when expressed from a tetracycline-regulated promoter in M. smegmatis. The same genes displayed toxicity when conditionally expressed in Mtb. Toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that the VapC level must exceed a threshold for toxicity to be observed. In addition, the level of Rv2456 protein induced in M. smegmatis was markedly lower than Rv2549c, which may account for the lack of toxicity of this and other VapCs scored as 'non-toxic'. The growth inhibitory effects of toxic VapCs were neutralized by expression of the cognate VapB as part of a vapBC operon or from a different chromosomal locus, while that of non-cognate antitoxins did not. These results demonstrated a specificity of interaction between VapCs and their cognate VapBs, a finding corroborated by yeast two-hybrid analyses. Deletion of selected vapC or vapBC genes did not affect mycobacterial growth in vitro, but rendered the organisms more susceptible to growth inhibition following toxic VapC expression. However, toxicity of 'non-toxic' VapCs was not unveiled in deletion mutant strains, even when the mutation eliminated the corresponding cognate VapB, presumably due to insufficient levels of VapC protein. Together with the ribonuclease (RNase activity demonstrated for Rv0065 and Rv0617--VapC proteins with similarity to Rv0549c and Rv3320c, respectively--these results suggest that the VapBC family potentially provides an abundant source of RNase activity in Mtb, which may profoundly impact the physiology of the organism.

  1. Dominant negative mutants of Bacillus thuringiensis Cry1Ab toxin function as anti-toxins: demonstration of the role of oligomerization in toxicity.

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    Claudia Rodríguez-Almazán

    Full Text Available Bacillus thuringiensis Cry toxins, that are used worldwide in insect control, kill insects by a mechanism that depends on their ability to form oligomeric pores that insert into the insect-midgut cells. These toxins are being used worldwide in transgenic plants or spray to control insect pests in agriculture. However, a major concern has been the possible effects of these insecticidal proteins on non-target organisms mainly in ecosystems adjacent to agricultural fields.We isolated and characterized 11 non-toxic mutants of Cry1Ab toxin affected in different steps of the mechanism of action namely binding to receptors, oligomerization and pore-formation. These mutant toxins were analyzed for their capacity to block wild type toxin activity, presenting a dominant negative phenotype. The dominant negative phenotype was analyzed at two levels, in vivo by toxicity bioassays against susceptible Manduca sexta larvae and in vitro by pore formation activity in black lipid bilayers. We demonstrate that some mutations located in helix alpha-4 completely block the wild type toxin activity at sub-stoichiometric level confirming a dominant negative phenotype, thereby functioning as potent antitoxins.This is the first reported case of a Cry toxin dominant inhibitor. These data demonstrate that oligomerization is a fundamental step in Cry toxin action and represent a potential mechanism to protect special ecosystems from the possible effect of Cry toxins on non-target organisms.

  2. Effect of the abortive infection mechanism and type III toxin/antitoxin system AbiQ on the lytic cycle of Lactococcus lactis phages.

    Science.gov (United States)

    Samson, Julie E; Bélanger, Maxime; Moineau, Sylvain

    2013-09-01

    To survive in phage-containing environments, bacteria have evolved an array of antiphage systems. Similarly, phages have overcome these hurdles through various means. Here, we investigated how phages are able to circumvent the Lactococcus lactis AbiQ system, a type III toxin-antitoxin with antiviral activities. Lactococcal phage escape mutants were obtained in the laboratory, and their genomes were sequenced. Three unrelated genes of unknown function were mutated in derivatives of three distinct lactococcal siphophages: orf38 of phage P008, m1 of phage bIL170, and e19 of phage c2. One-step growth curve experiments revealed that the phage mutations had a fitness cost while transcriptional analyses showed that AbiQ modified the early-expressed phage mRNA profiles. The L. lactis AbiQ system was also transferred into Escherichia coli MG1655 and tested against several coliphages. While AbiQ was efficient against phages T4 (Myoviridae) and T5 (Siphoviridae), escape mutants of only phage 2 (Myoviridae) could be isolated. Genome sequencing revealed a mutation in gene orf210, a putative DNA polymerase. Taking these observations together, different phage genes or gene products are targeted or involved in the AbiQ phenotype. Moreover, this antiviral system is active against various phage families infecting Gram-positive and Gram-negative bacteria. A model for the mode of action of AbiQ is proposed.

  3. Structure of the Escherichia coli Antitoxin MqsA (YgiT/b3021) Bound to Its Gene Promoter Reveals Extensive Domain Rearrangements and the Specificity of Transcriptional Regulation

    Energy Technology Data Exchange (ETDEWEB)

    B Brown; T Wood; W Peti; R Page

    2011-12-31

    Bacterial cultures, especially biofilms, produce a small number of persister cells, a genetically identical subpopulation of wild type cells that are metabolically dormant, exhibit multidrug tolerance, and are highly enriched in bacterial toxins. The gene most highly up-regulated in Escherichia coli persisters is mqsR, a ribonuclease toxin that, along with mqsA, forms a novel toxin-antitoxin (TA) system. Like all known TA systems, both the MqsR-MqsA complex and MqsA alone regulate their own transcription. Despite the importance of TA systems in persistence and biofilms, very little is known about how TA modules, and antitoxins in particular, bind and recognize DNA at a molecular level. Here, we report the crystal structure of MqsA bound to a 26-bp fragment from the mqsRA promoter. We show that MqsA binds DNA predominantly via its C-terminal helix-turn-helix domain, with direct binding of recognition helix residues Asn{sup 97} and Arg{sup 010} to the DNA major groove. Unexpectedly, the structure also revealed that the MqsA N-terminal domain interacts with the DNA phosphate backbone. This results in a more than 105{sup o} rotation of the N-terminal domains between the free and complexed states, an unprecedented rearrangement for an antitoxin. The structure also shows that MqsA bends the DNA by more than 55{sup o} in order to achieve symmetrical binding. Finally, using a combination of biochemical and NMR studies, we show that the DNA sequence specificity of MqsA is mediated by direct readout.

  4. AvrRxo1 Is a Bifunctional Type III Secreted Effector and Toxin-Antitoxin System Component with Homologs in Diverse Environmental Contexts.

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    Lindsay R Triplett

    Full Text Available Toxin-antitoxin (TA systems are ubiquitous bacterial systems that may function in genome maintenance and metabolic stress management, but are also thought to play a role in virulence by helping pathogens survive stress. We previously demonstrated that the Xanthomonas oryzae pv. oryzicola protein AvrRxo1 is a type III-secreted virulence factor that has structural similarities to the zeta family of TA toxins, and is toxic to plants and bacteria in the absence of its predicted chaperone Arc1. In this work, we confirm that AvrRxo1 and its binding partner Arc1 function as a TA system when expressed in Escherichia coli. Sequences of avrRxo1 homologs were culled from published and newly generated phytopathogen genomes, revealing that avrRxo1:arc1 modules are rare or frequently inactivated in some species and highly conserved in others. Cloning and functional analysis of avrRxo1 from Acidovorax avenae, A. citrulli, Burkholderia andropogonis, Xanthomonas translucens, and Xanthomonas euvesicatoria showed that some AvrRxo1 homologs share the bacteriostatic and Rxo1-mediated cell death triggering activities of AvrRxo1 from X. oryzae. Additional distant putative homologs of avrRxo1 and arc1 were identified in genomic or metagenomic sequence of environmental bacteria with no known pathogenic role. One of these distant homologs was cloned from the filamentous soil bacterium Cystobacter fuscus. avrRxo1 from C. fuscus caused watersoaking and triggered Rxo1-dependent cell collapse in Nicotiana benthamiana, but no growth suppression in E. coli was observed. This work confirms that a type III effector can function as a TA system toxin, and illustrates the potential of microbiome data to reveal new environmental origins or reservoirs of pathogen virulence factors.

  5. The relBE2Spn toxin-antitoxin system of Streptococcus pneumoniae: role in antibiotic tolerance and functional conservation in clinical isolates.

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    Concha Nieto

    2010-06-01

    Full Text Available Type II (proteic chromosomal toxin-antitoxin systems (TAS are widespread in Bacteria and Archaea but their precise function is known only for a limited number of them. Out of the many TAS described, the relBE family is one of the most abundant, being present in the three first sequenced strains of Streptococcus pneumoniae (D39, TIGR4 and R6. To address the function of the pneumococcal relBE2Spn TAS in the bacterial physiology, we have compared the response of the R6-relBE2Spn wild type strain with that of an isogenic derivative, Delta relB2Spn under different stress conditions such as carbon and amino acid starvation and antibiotic exposure. Differences on viability between the wild type and mutant strains were found only when treatment directly impaired protein synthesis. As a criterion for the permanence of this locus in a variety of clinical strains, we checked whether the relBE2Spn locus was conserved in around 100 pneumococcal strains, including clinical isolates and strains with known genomes. All strains, although having various types of polymorphisms at the vicinity of the TA region, contained a functional relBE2Spn locus and the type of its structure correlated with the multilocus sequence type. Functionality of this TAS was maintained even in cases where severe rearrangements around the relBE2Spn region were found. We conclude that even though the relBE2Spn TAS is not essential for pneumococcus, it may provide additional advantages to the bacteria for colonization and/or infection.

  6. Insight into the specific virulence related genes and toxin-antitoxin virulent pathogenicity islands in swine streptococcosis pathogen Streptococcus equi ssp. zooepidemicus strain ATCC35246.

    Science.gov (United States)

    Ma, Zhe; Geng, Jianing; Yi, Li; Xu, Bin; Jia, Ruoyu; Li, Yue; Meng, Qingshu; Fan, Hongjie; Hu, Songnian

    2013-06-07

    Streptococcus equi ssp. zooepidemicus (S. zooepidemicus) is an important pathogen causing swine streptococcosis in China. Pathogenicity islands (PAIs) of S. zooepidemicus have been transferred among bacteria through horizontal gene transfer (HGT) and play important roles in the adaptation and increased virulence of S. zooepidemicus. The present study used comparative genomics to examine the different pathogenicities of S. zooepidemicus. Genome of S. zooepidemicus ATCC35246 (Sz35246) comprises 2,167,264-bp of a single circular chromosome, with a GC content of 41.65%. Comparative genome analysis of Sz35246, S. zooepidemicus MGCS10565 (Sz10565), Streptococcus equi. ssp. equi. 4047 (Se4047) and S. zooepidemicus H70 (Sz70) identified 320 Sz35246-specific genes, clustered into three toxin-antitoxin (TA) systems PAIs and one restriction modification system (RM system) PAI. These four acquired PAIs encode proteins that may contribute to the overall pathogenic capacity and fitness of this bacterium to adapt to different hosts. Analysis of the in vivo and in vitro transcriptomes of this bacterium revealed differentially expressed PAI genes and non-PAI genes, suggesting that Sz35246 possess mechanisms for infecting animals and adapting to a wide range of host environments. Analysis of the genome identified potential Sz35246 virulence genes. Genes of the Fim III operon were presumed to be involved in breaking the host-restriction of Sz35246. Genome wide comparisons of Sz35246 with three other strains and transcriptome analysis revealed novel genes related to bacterial virulence and breaking the host-restriction. Four specific PAIs, which were judged to have been transferred into Sz35246 genome through HGT, were identified for the first time. Further analysis of the TA and RM systems in the PAIs will improve our understanding of the pathogenicity of this bacterium and could lead to the development of diagnostics and vaccines.

  7. Environmental T4-Family Bacteriophages Evolve to Escape Abortive Infection via Multiple Routes in a Bacterial Host Employing “Altruistic Suicide” through Type III Toxin-Antitoxin Systems

    Science.gov (United States)

    Chen, Bihe; Akusobi, Chidiebere; Fang, Xinzhe; Salmond, George P. C.

    2017-01-01

    Abortive infection is an anti-phage mechanism employed by a bacterium to initiate its own death upon phage infection. This reduces, or eliminates, production of viral progeny and protects clonal siblings in the bacterial population by an act akin to an “altruistic suicide.” Abortive infection can be mediated by a Type III toxin-antitoxin system called ToxINPa consisting of an endoribonuclease toxin and RNA antitoxin. ToxINPa is a heterohexameric quaternary complex in which pseudoknotted RNA inhibits the toxicity of the toxin until infection by certain phages causes destabilization of ToxINPa, leading to bacteriostasis and, eventually, lethality. However, it is still unknown why only certain phages are able to activate ToxINPa. To try to address this issue we first introduced ToxINPa into the Gram-negative enterobacterium, Serratia sp. ATCC 39006 (S 39006) and then isolated new environmental S 39006 phages that were scored for activation of ToxINPa and abortive infection capacity. We isolated three T4-like phages from a sewage treatment outflow point into the River Cam, each phage being isolated at least a year apart. These phages were susceptible to ToxINPa-mediated abortive infection but produced spontaneous “escape” mutants that were insensitive to ToxINPa. Analysis of these resistant mutants revealed three different routes of escaping ToxINPa, namely by mutating asiA (the product of which is a phage transcriptional co-activator); by mutating a conserved, yet functionally unknown, orf84; or by deleting a 6.5–10 kb region of the phage genome. Analysis of these evolved escape mutants may help uncover the nature of the corresponding phage product(s) involved in activation of ToxINPa. PMID:28620370

  8. Environmental T4-Family Bacteriophages Evolve to Escape Abortive Infection via Multiple Routes in a Bacterial Host Employing “Altruistic Suicide” through Type III Toxin-Antitoxin Systems

    Directory of Open Access Journals (Sweden)

    Bihe Chen

    2017-05-01

    Full Text Available Abortive infection is an anti-phage mechanism employed by a bacterium to initiate its own death upon phage infection. This reduces, or eliminates, production of viral progeny and protects clonal siblings in the bacterial population by an act akin to an “altruistic suicide.” Abortive infection can be mediated by a Type III toxin-antitoxin system called ToxINPa consisting of an endoribonuclease toxin and RNA antitoxin. ToxINPa is a heterohexameric quaternary complex in which pseudoknotted RNA inhibits the toxicity of the toxin until infection by certain phages causes destabilization of ToxINPa, leading to bacteriostasis and, eventually, lethality. However, it is still unknown why only certain phages are able to activate ToxINPa. To try to address this issue we first introduced ToxINPa into the Gram-negative enterobacterium, Serratia sp. ATCC 39006 (S 39006 and then isolated new environmental S 39006 phages that were scored for activation of ToxINPa and abortive infection capacity. We isolated three T4-like phages from a sewage treatment outflow point into the River Cam, each phage being isolated at least a year apart. These phages were susceptible to ToxINPa-mediated abortive infection but produced spontaneous “escape” mutants that were insensitive to ToxINPa. Analysis of these resistant mutants revealed three different routes of escaping ToxINPa, namely by mutating asiA (the product of which is a phage transcriptional co-activator; by mutating a conserved, yet functionally unknown, orf84; or by deleting a 6.5–10 kb region of the phage genome. Analysis of these evolved escape mutants may help uncover the nature of the corresponding phage product(s involved in activation of ToxINPa.

  9. Novel Clostridium difficile Anti-Toxin (TcdA and TcdB Humanized Monoclonal Antibodies Demonstrate In Vitro Neutralization across a Broad Spectrum of Clinical Strains and In Vivo Potency in a Hamster Spore Challenge Model.

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    Hongyu Qiu

    Full Text Available Clostridium difficile (C. difficile infection (CDI is the main cause of nosocomial antibiotic-associated colitis and increased incidence of community-associated diarrhea in industrialized countries. At present, the primary treatment of CDI is antibiotic administration, which is effective but often associated with recurrence, especially in the elderly. Pathogenic strains produce enterotoxin, toxin A (TcdA, and cytotoxin, toxin B (TcdB, which are necessary for C. difficile induced diarrhea and gut pathological changes. Administration of anti-toxin antibodies provides an alternative approach to treat CDI, and has shown promising results in preclinical and clinical studies. In the current study, several humanized anti-TcdA and anti-TcdB monoclonal antibodies were generated and their protective potency was characterized in a hamster infection model. The humanized anti-TcdA (CANmAbA4 and anti-TcdB (CANmAbB4 and CANmAbB1 antibodies showed broad spectrum in vitro neutralization of toxins from clinical strains and neutralization in a mouse toxin challenge model. Moreover, co-administration of humanized antibodies (CANmAbA4 and CANmAbB4 cocktail provided a high level of protection in a dose dependent manner (85% versus 57% survival at day 22 for 50 mg/kg and 20 mg/kg doses, respectively in a hamster gastrointestinal infection (GI model. This study describes the protective effects conferred by novel neutralizing anti-toxin monoclonal antibodies against C. difficile toxins and their potential as therapeutic agents in treating CDI.

  10. Síntese, caracterização e estudos de interação de um análogo da antitoxina CcdA empregando fluorescência no estado estacionário Synthesis, characterization and interaction studies of an analog of CcdA antitoxin by steady state fluorescence

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    Camila Aparecida Cotrim

    2010-01-01

    Full Text Available Toxin-antitoxin (TA systems contribute to plasmid stability by a mechanism called post-segregational killing. The ccd was the first TA system to be discovered with CcdB being the toxin and CcdA the antitoxin. CcdA, an 8.3 kDa protein, interacts with CcdB (11.7 kDa, preventing the cytotoxic activity of CcdB on the DNA gyrase. As an approach to understanding this interaction, CcdA41, a polypeptide derived from CcdA, was synthesized by solid-phase methodology and its interaction with CcdB was analyzed by steady state fluorescence. CcdA41 formed a stable complex with CcdBET2, a peptide based on CcdB, the more recently described bacterial topoisomerase inhibitor.

  11. Characterization of plasmids carrying the blaOXA-24/40 carbapenemase gene and the genes encoding the AbkA/AbkB proteins of a toxin/antitoxin system.

    Science.gov (United States)

    Mosqueda, Noraida; Gato, Eva; Roca, Ignasi; López, María; de Alegría, Carlos Ruíz; Fernández Cuenca, Felipe; Martínez-Martínez, Luis; Pachón, Jerónimo; Cisneros, José Miguel; Rodríguez-Baño, Jesús; Pascual, Alvaro; Vila, Jordi; Bou, Germán; Tomás, María

    2014-10-01

    Carbapenem-resistant Acinetobacter baumannii (CRAb) is a major source of nosocomial infections in Spain associated with the production of OXA-58-like or OXA-24/40-like β-lactamase enzymes. We analysed the plasmids carrying the bla(OXA-24/40)-like gene in CRAb isolates obtained a decade apart. The presence of β-lactamases was screened for by PCR (metallo-β-lactamases, carbapenem-hydrolysing class D β-lactamases, GES and KPC) in 101 CRAb isolates obtained in two multicentre studies (GEIH/REIPI-Ab-2000 and GEIH/REIPI-Ab-2010; n = 493 Acinetobacter spp). We analysed the distribution and characterization of the plasmids carrying the bla(OXA-24/40)-like gene and sequenced two plasmids, AbATCC223p (2000) and AbATCC329p (2010) from A. baumannii ATCC 17978 transformants. Acquisition of the bla(OXA-24/40)-like gene was the main mechanism underlying resistance to carbapenems (48.7% in 2000 compared with 51.6% in 2010). This gene was mainly isolated in ST2 A. baumannii strains in both studies, although some novel STs (ST79 and ST80) appeared in 2010. The gene was located in plasmids (8-12 kbp) associated with the repAci2 or repAci2/repGR12 types. The sequences of AbATCC223p (8840 bp) and AbATCC329p (8842 bp) plasmids were similar, particularly regarding the presence of the genes encoding the AbkA/AbkB proteins associated with the toxin/antitoxin system. Moreover, the abkA/abkB gene sequences (>96% identity) were also located in plasmids harbouring the bla(OXA-58)-like gene. The action of OXA-24/40 and OXA-58 β-lactamase-like enzymes represents the main mechanism underlying resistance to carbapenems in Spain in the last decade. AbkA/AbkB proteins in the toxin/antitoxin system may be involved in the successful dissemination of plasmids carrying the bla(OXA-24/40)-like gene, and probably also the bla(OXA-58)-like gene, thus contributing to the plasmid stability. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial

  12. Genetic fusions of a CFA/I/II/IV MEFA (multiepitope fusion antigen and a toxoid fusion of heat-stable toxin (STa and heat-labile toxin (LT of enterotoxigenic Escherichia coli (ETEC retain broad anti-CFA and antitoxin antigenicity.

    Directory of Open Access Journals (Sweden)

    Xiaosai Ruan

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

  13. Genetic fusions of a CFA/I/II/IV MEFA (multiepitope fusion antigen) and a toxoid fusion of heat-stable toxin (STa) and heat-labile toxin (LT) of enterotoxigenic Escherichia coli (ETEC) retain broad anti-CFA and antitoxin antigenicity.

    Science.gov (United States)

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

    2015-01-01

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

  14. Decline of Tetanus Antitoxin Level with Age in Taiwan

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    Chi-Jung Wu

    2009-05-01

    Conclusion: Waning immunity to tetanus was observed after primary tetanus vaccination or toxoid booster. The public health policy that one dose of toxoid booster after primary vaccination should be emphasized for continuing protection against tetanus.

  15. Proteção do recém-nascido contra o tétano pela imunização ativa da gestante com antitoxina tetânica: estudo original de 1953 Protection of newborn infants against tetanus by active immunization of the pregnant women with tetanus antitoxin: the 1953 original study

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    Augusto Gomes Mattos

    2008-12-01

    Full Text Available OBJETIVO: Determinar, em cobaias prenhes e em gestantes, a produção de antitoxina tetânica induzida pela aplicação da anatoxina tetânica e estudar a sua passagem para o recém-nascido. MÉTODOS: Na primeira fase, em estudo experimental, cobaias prenhes foram vacinadas com duas doses de toxóide tetânico em um intervalo de 15 dias, seguida da dosagem de anticorpos na cobaia imunizada, na prole ao nascer e 15 dias após o nascimento. Outro grupo de animais previamente vacinado recebeu uma dose de reforço 30 dias antes do parto, medindo-se o nível de anticorpos na cobaia e na prole. Na segunda fase, em ensaio clínico, as gestantes humanas foram vacinadas com três injeções de anatoxina tetânica, com um intervalo de 30 dias, em qualquer período da gravidez, medindo-se, a seguir, a antitoxina tetânica. Nos recém-nascidos, os anticorpos foram medidos ao nascer e aos 15 dias de vida. RESULTADOS: O título de antitoxina no sangue da prole de cobaias vacinadas com anatoxina tetânica foi elevado ao nascimento e aos 15 dias de vida. A dose de reforço provocou elevação do título basal. Nas gestantes, a aplicação de três doses de toxóide antitetânico conferiu imunidade a 95% dos recém-nascidos estudados. Os recém-nascidos de mães vacinadas apresentaram títulos elevados de antitoxina que persistiram por mais de 15 dias de vida. CONCLUSÕES: A vacinação durante a gestação foi acompanhada de títulos protetores de antitoxina contra o tétano tanto nos filhotes de cobaias quanto nos recém-nascidos humanos.OBJECTIVE: To measure, in pregnant guinea pigs and women, the production of tetanus antitoxin, induced by vaccination with tetanus toxin, and to study the transmission of these antibodies to the offspring. METHODS: In an experimental design, pregnant guinea pigs were vaccinated with two doses of tetanus toxoid with a 15-day interval followed by determination of antibodies in the immunized guinea pig, in the offspring at birth

  16. 21 CFR 610.20 - Standard preparations.

    Science.gov (United States)

    2010-04-01

    ... and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS... potency all forms of the following: Antibodies Botulism Antitoxin, Type A. Botulism Antitoxin, Type B.... Perfringens Antitoxin. Antipertussis Serum. Antirabies Serum. Sordellii Antitoxin. Staphylococcus Antitoxin...

  17. Antitoxin activity of Mimosa pudica root extracts against Naja naja and Bangarus caerulus venoms

    Directory of Open Access Journals (Sweden)

    Subramani Meenatchisundaram

    2009-06-01

    Full Text Available Aqueous extract of dried roots of Mimosa pudica was tested for inhibitory activity on lethality, phospholipase activity, edema forming activity, fibrinolytic activity and hemorrhagic activity of Naja naja and Bangarus caerulus venoms. The aqueous extract displayed a significant inhibitory effect on the lethality, phospholipase activity, edema forming activity, fibrinolytic activity and hemorrhagic activity. About 0.14 mg and 0.16 mg of M. pudica extracts were able to completely neutralize the lethal activity of 2LD50 of Naja naja and Bangarus caerulus venoms respectively. The present finding suggests that aqueous extract of M. pudica root possesses compounds, which inhibit the activity of Naja naja and Bangarus caerulus venoms.

  18. Antitoxin activity of Mimosa pudica root extracts against Naja naja and Bangarus caerulus venoms

    Directory of Open Access Journals (Sweden)

    Subramani Meenatchisundaram, Selvin Priyagrace, Ramasamy Vijayaraghavan, Ambikapathi Velmurugan, Govindarajan Parameswari, Antonysamy Michael

    2009-12-01

    Full Text Available Aqueous extract of dried roots of Mimosa pudica was tested for inhibitory activity on lethality, phospholipase activity, edema forming activity, fibrinolytic activity and hemorrhagic activity of Naja naja and Bangarus caerulus venoms. The aqueous extract displayed a significant inhibitory effect on the lethality, phospholipase activity, edema forming activity, fibrinolytic activity and hemorrhagic activity. About 0.14 mg and 0.16 mg of M. pudica extracts were able to completely neutralize the lethal activity of 2LD50 of Naja naja and Bangarus caerulus venoms respectively. The present finding suggests that aqueous extract of M. pudica root possesses compounds, which inhibit the activity of Naja naja and Bangarus caerulus venoms.

  19. Monoclonal Antibody Combinations that Present Synergistic Neutralizing Activity: A Platform for Next-Generation Anti-Toxin Drugs.

    Science.gov (United States)

    Diamant, Eran; Torgeman, Amram; Ozeri, Eyal; Zichel, Ran

    2015-05-29

    Monoclonal antibodies (MAbs) are among the fastest-growing therapeutics and are being developed for a broad range of indications, including the neutralization of toxins, bacteria and viruses. Nevertheless, MAbs potency is still relatively low when compared to conventional polyclonal Ab preparations. Moreover, the efficacy of an individual neutralizing MAb may significantly be hampered by the potential absence or modification of its target epitope in a mutant or subtype of the infectious agent. These limitations of individual neutralizing MAbs can be overcome by using oligoclonal combinations of several MAbs with different specificities to the target antigen. Studies conducted in our lab and by others show that such combined MAb preparation may present substantial synergy in its potency over the calculated additive potency of its individual MAb components. Moreover, oligoclonal preparation is expected to be better suited to compensating for reduced efficacy due to epitope variation. In this review, the synergistic neutralization properties of combined oligoclonal Ab preparations are described. The effect of Ab affinity, autologous Fc fraction, and targeting a critical number of epitopes, as well as the unexpected contribution of non-neutralizing clones to the synergistic neutralizing effect are presented and discussed.

  20. Monoclonal Antibody Combinations that Present Synergistic Neutralizing Activity: A Platform for Next-Generation Anti-Toxin Drugs

    Directory of Open Access Journals (Sweden)

    Eran Diamant

    2015-05-01

    Full Text Available Monoclonal antibodies (MAbs are among the fastest-growing therapeutics and are being developed for a broad range of indications, including the neutralization of toxins, bacteria and viruses. Nevertheless, MAbs potency is still relatively low when compared to conventional polyclonal Ab preparations. Moreover, the efficacy of an individual neutralizing MAb may significantly be hampered by the potential absence or modification of its target epitope in a mutant or subtype of the infectious agent. These limitations of individual neutralizing MAbs can be overcome by using oligoclonal combinations of several MAbs with different specificities to the target antigen. Studies conducted in our lab and by others show that such combined MAb preparation may present substantial synergy in its potency over the calculated additive potency of its individual MAb components. Moreover, oligoclonal preparation is expected to be better suited to compensating for reduced efficacy due to epitope variation. In this review, the synergistic neutralization properties of combined oligoclonal Ab preparations are described. The effect of Ab affinity, autologous Fc fraction, and targeting a critical number of epitopes, as well as the unexpected contribution of non-neutralizing clones to the synergistic neutralizing effect are presented and discussed.

  1. Discovery of Peptide-Based Antitoxins against Neurotoxins from Green and Black Mamba (Dendroaspis Family)

    DEFF Research Database (Denmark)

    Jappe, Emma Christine; Munk, Andreas; Laustsen, Andreas Hougaard

    Globally, more than 5.5 million people are bitten by venomous snakes every year, leading to an estimated 125,000 deaths and 3 times as many amputations. The problem is most prevalent in Sub-Saharan Africa where affordability of antivenom is low, resulting in only 2% of snakebite victims receiving...... blood comprising venom-specific antibodies [4]. The incompatibility of these antivenoms with the human immune system canlead to serious adverse effects. A novel approach is needed in order to introduce safer, cheaper and more efficacious antivenoms that are compatible with the human immune system...

  2. Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

    Directory of Open Access Journals (Sweden)

    Goldman Ellen R

    2007-11-01

    Full Text Available Abstract Background Shark heavy chain antibody, also called new antigen receptor (NAR, consists of one single Variable domain (VH, containing only two complementarity-determining regions (CDRs. The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences. Results A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB, ricin, and botulinum toxin A (BoNT/A complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications. Conclusion We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins.

  3. [Paul Ehrlich and commercial serum production: on the control of diphtheria antitoxin in the laboratory and in industry].

    Science.gov (United States)

    Hardy, Anne I

    2006-01-01

    The state control of the anti-diphtheria serum is a prime example for the efficient cooperation between state authorities, industrials and scientists. The article describes firstly the dynamics of this development and places it in the context of already existing approaches to drugs control. Of special interest are the motives which led the different actors to support the state control of sera. Secondly the reciprocal influences between Ehrlich and the serum producers are analyzed. It is shown that practical problems inspired Ehrlich's theoretical work, which in return led to an improvement of measurement techniques.

  4. Fatal diphtheria myocarditis in a 3-year-old girl-related to late availability and administration of antitoxin?

    NARCIS (Netherlands)

    Van Damme, Karlijn; Peeters, Natasja; Jorens, Philippe G; Boiy, Tine; Deplancke, Marjan; Audiens, Hilde; Wojciechowski, Marek; De Dooy, Jozef; Te Wierik, Margreet; Vlieghe, Erika

    2017-01-01

    Sporadic cases of diphtheria are very rare throughout Europe. A 3-year-old incompletely vaccinated girl was admitted with pharyngotonsillitis caused by diphtheria. On day 9 of her illness, renal and cardiac failure with a third-degree AV-block occurred. Unfortunately, she died within 36 h of

  5. Plasmid vectors for Xylella fastidiosa utilizing a toxin-antitoxin system for plasmid stability in the absence of antibiotic selection

    Science.gov (United States)

    The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacte...

  6. The HigB/HigA Toxin/Antitoxin System of Pseudomonas aeruginosa Influences the Virulence Factors Pyochelin, Pyocyanin, and Biofilm Formation

    Science.gov (United States)

    2016-08-24

    resistance of uropathogenic Escherichia coli. PLoS Pathog. 8:e1002954. Ogura, T., and S. Hiraga. 1983. Mini- F plasmid genes that couple host cell division...chronic infections including those associated with cystic fibrosis (CF) (Moker et al. 2010), burn wound infections, bacterial keratitis, and urinary...Furthermore, the expression of the toxin genes are induced under stress conditions (Aizenman et al. 1996; Sat et al. 2001; Hazan et al. 2004), and

  7. Validierung der Wirksamkeitsprüfung für Clostridium tetani Impfstoffe ad usum veterinarium durch den direkten Nachweis von Tetanus-Antitoxin im Zieltier mittels ELISA

    OpenAIRE

    Roßkopf, Ute

    2007-01-01

    Die dargelegten Ergebnisse zur Validierung einer neuen Wirksamkeitsprüfung von Tetanus-Impfstoffen resultieren aus umfangreichen Feldstudien, durchgeführt mit neun unterschiedlichen Impfstoffen für das Pferd sowie acht Impfstoffen für das Schaf. Insgesamt wurden 102 Pferde und 82 Schafe entsprechend den Angaben in der Gebrauchsinformation immunisiert. Blutentnahmen fanden nach einem vorgegebenen Schema von bis zu zwei Jahren nach der ersten Impfung statt. Die Impfstoffe waren entweder monoval...

  8. Insight into the specific virulence related genes and toxin-antitoxin virulent pathogenicity islands in swine streptococcosis pathogen Streptococcus equi ssp. zooepidemicus strain ATCC35246

    OpenAIRE

    Geng, Jianing; Yi, Li; Xu, Bin; Jia, Ruoyu; Li, Yue; Meng, Qingshu; Fan, Hongjie; Hu, Songnian

    2013-01-01

    Background Streptococcus equi ssp. zooepidemicus (S. zooepidemicus) is an important pathogen causing swine streptococcosis in China. Pathogenicity islands (PAIs) of S. zooepidemicus have been transferred among bacteria through horizontal gene transfer (HGT) and play important roles in the adaptation and increased virulence of S. zooepidemicus. The present study used comparative genomics to examine the different pathogenicities of S. zooepidemicus. Results Genome of S. zooepidemicus ATCC35246 ...

  9. Possível interferência da antitoxina tetânica pré-existente, na vacinação antitetânica Possible interference of pre-existing tetanus antitoxin in antitetanus immunization

    Directory of Open Access Journals (Sweden)

    Rosalvo Guidolin

    1981-04-01

    Full Text Available Estudou-se qual a possível interferência no desenvolvimento da imunidade antitetânica ativa em cobaias e camundongos, filhos de fêmeas vacinadas contra o tétano em diferentes épocas durante o período da prenhês. Verificou-se que a vacinação das fêmeas, em gestação não interferiu, negativamente, no desenvolvimento da imunidade ativa dos animais filhos, quando submetidos à vacinação ao redor de 60 dias após o nascimento. A presença de baixos níveis de anticorpos circulantes, recebidos congenitamente, parece ter, em determinadas condições, estimulado a resposta imunitária quando, posteriormente, os animais filhos foram vacinados contra o tétano. Sugere-se que o complexo antígeno-anticorpo formado seja capaz de melhorar a resposta imunitária induzida pelo toxóide.Possibility of interference in the development of active antitetanic immunity was studied in the offspring of guinea pigs and mice whose mothers had received antitetanic vaccine during different periods of pregnancy. Vaccination of the females during pregnancy did not interfere negatively in the development of active immunity in their offspring when the latter were vaccinated about 60 days after birth. The presence of small amounts of circulating antibodies, congenitally received, seems, under certain conditions, to stimulate the immune response upon posterior vaccination against tetanus. This suggests that the acquired antigen-antibody complex heightens the immune response induced by the toxoid.

  10. HicA of Escherichia coli defines a novel family of translation-independent mRNA interferases in bacteria and archaea

    DEFF Research Database (Denmark)

    Jørgensen, Mikkel G; Pandey, Deo P; Jaskolska, Milena

    2009-01-01

    Toxin-antitoxin (TA) loci are common in free-living bacteria and archaea. TA loci encode a stable toxin that is neutralized by a metabolically unstable antitoxin. The antitoxin can be either a protein or an antisense RNA. So far, six different TA gene families, in which the antitoxins are protein...... a bacteriostatic rather than a bactericidal condition. Nutrient starvation induced strong hicAB transcription that depended on Lon protease. Mining of 218 prokaryotic genomes revealed that hicAB loci are abundant in bacteria and archaea.......Toxin-antitoxin (TA) loci are common in free-living bacteria and archaea. TA loci encode a stable toxin that is neutralized by a metabolically unstable antitoxin. The antitoxin can be either a protein or an antisense RNA. So far, six different TA gene families, in which the antitoxins are proteins....... Expression of HicB (145 aa) prevented HicA-mediated inhibition of cell growth. These results suggest that HicB neutralizes HicA and therefore functions as an antitoxin. As with other antitoxins (RelB and MazF), HicB could resuscitate cells inhibited by HicA, indicating that ectopic production of HicA induces...

  11. EFFECT OF TYPE B BOTULINAL TOXIN ON THE LEVEL OF PYRUVIC ACID IN TISSUES OF GUINEA PIGS,

    Science.gov (United States)

    CLOSTRIDIUM BOTULINUM, TOXINS AND ANTITOXINS), PYRUVIC ACID, TISSUES(BIOLOGY), CENTRAL NERVOUS SYSTEM, MUSCLES, BLOOD, TOXICITY, GUINEA PIGS , CARBON DIOXIDE, METABOLISM, RESPIRATION, MORPHOLOGY(BIOLOGY), USSR

  12. 9 CFR 113.109 - Clostridium Sordellii Bacterin-Toxoid.

    Science.gov (United States)

    2010-01-01

    ... prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released. (a... following words and terms shall mean: (i) International antitoxin unit. (I.U.) That quantity of antitoxin... distilled water; adjusting the pH to 7.2; autoclaving at 121 °C for 25 minutes; and storing at 4 °C until...

  13. 9 CFR 113.108 - Clostridium Novyi Bacterin-Toxoid.

    Science.gov (United States)

    2010-01-01

    ... serial found unsatisfactory by any prescribed test shall not be released. (a) Purity test. Final... words and terms shall mean: (i) International antitoxin unit. (I.U.) That quantity of Alpha Antitoxin... chloride in each 100 ml of distilled water; adjusting the pH to 7.2; autoclaving at 121 °C for 25 minutes...

  14. Bacillus globigii cell size is influenced by variants of the quorum sensing peptide extracellular death factor

    NARCIS (Netherlands)

    T. Sijbrandij (T.); W.E. Kaman (Wendy); A.J.M. Ligtenberg (A. J M); K. Nazmi (Kamran); E.C.I. Veerman (Enno); F.J. Bikker (Floris)

    2014-01-01

    textabstractToxin-antitoxin modules are necessary for the mode of action of several antibiotics. One of the most studied toxin-antitoxin modules is the quorum sensing - dependent MazEF system in Escherichia coli. The quorum sensing factor in this system is called the extracellular death factor

  15. Bacillus globigii cell size is influenced by variants of the quorum sensing peptide extracellular death factor

    NARCIS (Netherlands)

    Sijbrandij, T.; Kaman, W.E.; Ligtenberg, A.J.M.; Nazmi, K.; Veerman, E.C.I.; Bikker, F.J.

    2014-01-01

    Toxin-antitoxin modules are necessary for the mode of action of several antibiotics. One of the most studied toxin-antitoxin modules is the quorum sensing—dependent MazEF system in Escherichia coli. The quorum sensing factor in this system is called the extracellular death factor (EDF), a linear

  16. Bacillus globigii cell size is influenced by variants of the quorum sensing peptide extracellular death factor

    NARCIS (Netherlands)

    Sijbrandij, T.; Kaman, W.E.; Ligtenberg, A.J.M.; Nazmi, K.; Veerman, E.C.I.; Bikker, F.J.

    2014-01-01

    Toxin-antitoxin modules are necessary for the mode of action of several antibiotics. One of the most studied toxin-antitoxin modules is the quorum sensing - dependent MazEF system in Escherichia coli. The quorum sensing factor in this system is called the extracellular death factor (EDF), a linear

  17. Cluster of Botulism among dutch tourists in Turkey, june 2008

    NARCIS (Netherlands)

    Swaan, C.M.; Ouwerkerk, van M.; Roest, H.I.J.

    2010-01-01

    In June 2008, three Dutch tourists participating in a mini-cruise in Turkey needed urgent repatriation for antitoxin treatment because of symptoms of botulism. Because there was a shortage of antitoxin in the Netherlands, an emergency delivery was requested from the manufacturer in Germany. An

  18. Toxin inhibition in i>C. crescentus VapBC1 is mediated by a flexible pseudo-palindromic protein motif and modulated by DNA binding

    DEFF Research Database (Denmark)

    Bendtsen, Kirstine Louise; Xu, Kehan; Luckmann, Majbritt

    2017-01-01

    architectural rearrangement of conserved TA interactions in which C-terminal extended structures of the antitoxin VapB1 swap positions to interlock the complex in the DNA-bound state. We further show that a pseudo-palindromic protein sequence in the antitoxin is responsible for this interaction and required...

  19. Back to the roots

    DEFF Research Database (Denmark)

    Harms, Alexander; Gerdes, Kenn

    2016-01-01

    In this issue of Molecular Cell, Jankevicius et al. (2016) characterize the DarTG toxin-antitoxin module in which the DarT toxin ADP-ribosylates single-stranded DNA and the DarG antitoxin counteracts DarT by direct binding and by enzymatic removal of the ADP-ribosylation....

  20. PCR-based plasmid typing in Enterococcus faecium strains reveals widely distributed pRE25-, pRUM-, pIP501-and pHT beta-related replicons associated with glycopeptide resistance and stabilizing toxin-antitoxin systems

    DEFF Research Database (Denmark)

    Rosvoll, T.C.S.; Pedersen, T.; Sletvold, H.

    2010-01-01

    for about 60% of the total number of plasmids detected by S1 nuclease analyses, which revealed zero to seven plasmids (10 kb to > 200 kb) per isolate. Interestingly, strains belonging to the clinically important clonal complex 17 (CC17) yielded a significantly higher number of plasmids (3.1) and p...

  1. New Parvovirus Associated with Serum Hepatitis in Horses after Inoculation of Common Biological Product

    OpenAIRE

    Divers, Thomas J.; Tennant, Bud C.; Kumar, Arvind; McDonough, Sean; Cullen, John; Bhuva, Nishit; Jain, Komal; Chauhan, Lokendra Singh; Scheel, Troels Kasper Høyer; Lipkin, W. Ian; Laverack, Melissa; Trivedi, Sheetal; Srinivasa, Satyapramod; Beard, Laurie; Rice, Charles M.

    2018-01-01

    Equine serum hepatitis (i.e., Theiler’s disease) is a serious and often life-threatening disease of unknown etiology that affects horses. A horse in Nebraska, USA, with serum hepatitis died 65 days after treatment with equine-origin tetanus antitoxin. We identified an unknown parvovirus in serum and liver of the dead horse and in the administered antitoxin. The equine parvovirus-hepatitis (EqPV-H) shares

  2. The SOS Response is Permitted in Escherichia coli Strains Deficient in the Expression of the mazEF Pathway

    Science.gov (United States)

    2014-12-03

    KB, Edmeston JS (1972) Dominant mutations (lex) in Escherichia coli K-12 which affect radiation sensitivity and frequency of ultraviolet light -induced...antitoxin module mazEF or its downstream pathway is not functioning. Introduction The enteric bacterium E. coli, like most other bacteria , carries on...Amitai S (2005) mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria . J. of Cell Science 118: 4327–4332. 2

  3. Expression, Purification, and Functional Analysis of Novel RelE Operon from X. nematophila

    Directory of Open Access Journals (Sweden)

    Jitendra Singh Rathore

    2014-01-01

    Full Text Available Bacterial toxin-antitoxin (TA complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms. Escherichia coli RelB-RelE TA complex consists of a RelE toxin functionally counteracted by RelB antitoxin. In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin from Xenorhabdus nematophila possessing its own antitoxin designated as Xn-relEAT has been identified. Expression and purification of recombinant proteins under native conditions with GST and Ni-NTA chromatography prove the existence of novel TA module. The expression of recombinant Xn-relE under tightly regulated ara promoter in E. coli Top 10 cells confirms its toxic nature in endogenous toxicity assay. The neutralization activity in endogenous toxicity assay by Xn-relEAT antitoxin confirms its antidote nature when studying the whole TA operon under ara regulated promoter. This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

  4. Immunity to Diphtheria and Tetanus in Army Personnel and Adult Civilians in Mashhad, Iran.

    Science.gov (United States)

    Hosseini Shokouh, Seyyed Javad; Mohammadi, Babak; Rajabi, Jalil; Mohammadian Roshan, Ghasem

    2017-03-24

    This study aimed to investigate serologic immunity to diphtheria and tetanus in army personnel and a sample population of adult civilians in Mashhad, Iran. Army personnel (n = 180) and civilians (n = 83) who presented at Mashhad army hospital participated in this study. Diphtheria and tetanus antitoxin levels were determined by enzyme-linked immunosorbent assay. Approximately 77% and 94% of army personnel aged 18-34 years had at least basic protection against diphtheria (antitoxin level ≥0.1 IU/mL) and tetanus (antitoxin level >0.1 IU/mL), respectively. For civilians in this age group, the proportions were 76% for both diseases. Antitoxin levels waned with age. Thus, participants older than 50 years had lower immunity; this decrease in immunity was more pronounced for tetanus than for diphtheria in both army personnel and civilians. For both diseases, geometric mean antitoxin titers and the proportion of participants with at least basic protection were higher in subjects with a history of vaccination in the last 10 years (P diphtheria and tetanus. However, the large number of susceptible older adults (>50 years old) calls for improved booster vaccination protocols.

  5. AAU-Specific RNA Cleavage Mediated by MazF Toxin Endoribonuclease Conserved in Nitrosomonas europaea

    Directory of Open Access Journals (Sweden)

    Tatsuki Miyamoto

    2016-06-01

    Full Text Available Nitrosomonas europaea carries numerous toxin-antitoxin systems. However, despite the abundant representation in its chromosome, studies have not surveyed the underlying molecular functions in detail, and their biological roles remain enigmatic. In the present study, we found that a chromosomally-encoded MazF family member, predicted at the locus NE1181, is a functional toxin endoribonuclease, and constitutes a toxin-antitoxin system, together with its cognate antitoxin, MazE. Massive parallel sequencing provided strong evidence that this toxin endoribonuclease exhibits RNA cleavage activity, primarily against the AAU triplet. This sequence-specificity was supported by the results of fluorometric assays. Our results indicate that N. europaea alters the translation profile and regulates its growth using the MazF family of endoribonuclease under certain stressful conditions.

  6. Combined active-passive immunisation of horses against tetanus.

    Science.gov (United States)

    Liefman, C E

    1980-03-01

    The protection afforded by active, passive and combined active-passive methods of immunisation against tetanus was examined in previously unimmunised horses. Three groups of horses were injected; one with tetanus toxoid alone, one with tetanus antitoxin alone and one in which the tetanus toxoid and tetanus antitoxin were injected simultaneously. The protection afforded was determined by monitoring the levels of antitoxin achieved in the horses by each of these methods. The results obtained demonstrated the effectiveness of the combined active-passive method in affording rapid and prolonged protection and enabled the examination of some of the factors involved in active and in passive immunisation when used alone. The advantages obtained by the use of the combined active-passive method in protecting unimmunised horses suddenly placed at risk to infection are outlined.

  7. New Parvovirus Associated with Serum Hepatitis in Horses after Inoculation of Common Biological Product.

    Science.gov (United States)

    Divers, Thomas J; Tennant, Bud C; Kumar, Arvind; McDonough, Sean; Cullen, John; Bhuva, Nishit; Jain, Komal; Chauhan, Lokendra Singh; Scheel, Troels Kasper Høyer; Lipkin, W Ian; Laverack, Melissa; Trivedi, Sheetal; Srinivasa, Satyapramod; Beard, Laurie; Rice, Charles M; Burbelo, Peter D; Renshaw, Randall W; Dubovi, Edward; Kapoor, Amit

    2018-02-01

    Equine serum hepatitis (i.e., Theiler's disease) is a serious and often life-threatening disease of unknown etiology that affects horses. A horse in Nebraska, USA, with serum hepatitis died 65 days after treatment with equine-origin tetanus antitoxin. We identified an unknown parvovirus in serum and liver of the dead horse and in the administered antitoxin. The equine parvovirus-hepatitis (EqPV-H) shares horses using a tetanus antitoxin contaminated with EqPV-H. Viremia developed, the horses seroconverted, and acute hepatitis developed that was confirmed by clinical, biochemical, and histopathologic testing. We also determined that EqPV-H is an endemic infection because, in a cohort of 100 clinically normal adult horses, 13 were viremic and 15 were seropositive. We identified a new virus associated with equine serum hepatitis and confirmed its pathogenicity and transmissibility through contaminated biological products.

  8. Atypical tetanus in a completely immunized 14-year-old boy.

    Science.gov (United States)

    König, Kai; Ringe, Hannelore; Dorner, Brigitte G; Diers, Alexander; Uhlenberg, Birgit; Müller, Dominik; Varnholt, Verena; Gaedicke, Gerhard

    2007-11-01

    We report the uncommon clinical course of tetanus in a completely immunized 14-year-old boy. His initial symptoms, which included a flaccid paralysis, supported a diagnosis of botulism. Preliminary mouse-test results with combined botulinum antitoxins A, B, and E, obtained from tetanus-immunized horses, backed this diagnosis. The change in his clinical course from paralysis to rigor and the negative, more specific, botulinum mouse test with isolated botulinum antitoxins A, B, and E, obtained from nonvaccinated rabbits, disproved the diagnosis of botulism. Tetanus was suspected despite complete vaccination. The final results of a positive mouse test performed with isolated tetanus antitoxin confirmed the diagnosis. Adequate treatment was begun, and the boy recovered completely.

  9. A novel strain of Clostridium botulinum that produces type B and type H botulinum toxins.

    Science.gov (United States)

    Barash, Jason R; Arnon, Stephen S

    2014-01-15

    Clostridium botulinum strain IBCA10-7060, isolated from a patient with infant botulism, produced botulinum neurotoxin type B (BoNT/B) and another BoNT that, by use of the standard mouse bioassay, could not be neutralized by any of the Centers for Disease Control and Prevention-provided monovalent polyclonal botulinum antitoxins raised against BoNT types A-G.  The combining of antitoxins to neutralize the toxicity of known bivalent C. botulinum strains Ab, Ba, Af, and Bf also failed to neutralize the second BoNT. Analysis of culture filtrate by double immunodiffusion yielded a single line of immunoprecipitate with anti-A, anti-B, and anti-F botulinum antitoxins but not with anti-E antitoxin. A heptavalent F(ab')2 botulinum antitoxin A-G obtained from the US Army also did not neutralize the second BoNT. An antitoxin raised against IBCA10-7060 toxoid protected mice against BoNT/B (Okra) and against the second BoNT but did not protect mice against BoNT/A (Hall) or BoNT/F (Langeland). The second BoNT thus fulfilled classic criteria for being designated BoNT/H. IBCA10-7060 is the first C. botulinum type Bh strain to be identified. BoNT/H is the first new botulinum toxin type to be recognized in >40 years, and its recognition could not have been accomplished without the availability of the mouse bioassay.

  10. RNA decay by messenger RNA interferases

    DEFF Research Database (Denmark)

    Christensen-Dalsgaard, Mikkel; Overgaard, Martin; Winther, Kristoffer Skovbo

    2008-01-01

    Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mRNA...... cleaving enzymes such as RelE of Escherichia coli and the corresponding antitoxin RelB. In particular, we describe a set of plasmid vectors useful for the detailed analysis of cleavage sites in model mRNAs....

  11. ORIGINAL ARTICLE DIPHTHERIA MORTALITY IN NIGERIA: THE ...

    African Journals Online (AJOL)

    Dr Oboro VO

    whom eventually required tracheostomy. No child received the diphtheria antitoxin as this is not available in Nigeria. The public health unit investigated each case and reported the cases to the appropriate authorities. Family and close contacts were treated with erythromycin while unimmunized children were immunized.

  12. Discovery of Peptidic Anti-cobratoxins by Next Generation Phage Display

    DEFF Research Database (Denmark)

    Laustsen, Andreas Hougaard; Lynagh, Timothy; Kringelum, Jens Vindahl

    Antivenoms are still being produced by animal immunization protocols and are therefore associated with high immunogenicity for human recipients. Here we report the first step towards discovery of synthetic antitoxins that could be used for development of a fully synthetic antivenom against neurot...... neurotoxin from cobras (Naja genus)....

  13. New Parvovirus Associated with Serum Hepatitis in Horses after Inoculation of Common Biological Product

    DEFF Research Database (Denmark)

    Divers, Thomas J; Tennant, Bud C; Kumar, Arvind

    2018-01-01

    and liver of the dead horse and in the administered antitoxin. The equine parvovirus-hepatitis (EqPV-H) shares contaminated with EqPV-H. Viremia developed...... virus associated with equine serum hepatitis and confirmed its pathogenicity and transmissibility through contaminated biological products....

  14. Evaluation of diphtheria convalescent patients to serve as donors for the production of anti-diphtheria immunoglobulin preparations

    NARCIS (Netherlands)

    Bissumbhar, B.; Rakhmanova, A.G.; Berbers, G.; Iakolev, A.; Nosikova, E.; Melnick, O.; Ovtcharenko, E.; Rümke, H. C.; Ruitenberg, E.J.

    2004-01-01

    Aims: The study was conducted to evaluate the possibility of selecting convalescent diphtheria patients to serve in emergency situations as donors for the production of anti-diphtheria immunoglobulin. To select suitable donors, the criterion of an antitoxin titer ≥3.0 IU/ml was used. In addition,

  15. Impairment of antitoxic antitetanus immunity under the combined effect of radiation and heat

    International Nuclear Information System (INIS)

    Kurochkina, O.I.; Budagov, R.S.

    1990-01-01

    In experiments with mice a delay was noted in the development of secondary immune response to revaccination with a tetanic anatoxin after the combined effect of radiation and heat; the maximum antibody formation occured at later times. The low level of antitoxins within the first 10 days after the combined effect of radiation and heat correlated with the low tetanus resistance of animals

  16. Molecular biology. A Swiss army knife of immunity

    NARCIS (Netherlands)

    Brouns, S.J.J.

    2012-01-01

    Selfish genetic elements are more than a daily nuisance in the life of prokaryotes. Whereas viruses can multiply by reprogramming host cells, or integrate in the host genome as “stowaways,” conjugative plasmids (transferrable extrachromosomal DNA) make cells addicted to plasmid-encoded antitoxin

  17. Tetanus immunity in 50 Years of Age and Older Persons in Kashan

    Directory of Open Access Journals (Sweden)

    Reza Razaghi

    2011-10-01

    Full Text Available Ojectives: Tetanus is a neurologic disorder which occurs as sporadic cases in people with incomplete vaccination course.The most tetanus patients are people over 60 years of age.It is a important disease in elderly patients because of risk of severe forms and high rate of morbidity and mortality This study was conducted to evaluate tetanus immunity in 50 years of age and older people in kashan-Iran. Methods & Materials: This cross-sectional survey was conducted on 180 randomly selected adults, 50 years of age and older who visited Booali laboratory for check up examination in kashan -Iran in 2008.After taking consent form standard questionnaires consisting demographic data and history of vaccination were filled and 5cc blood taken from selected persons and tetanus toxoid-specific antibody were measured by ELISA method .Results were analyzed by SPSS and presented by descriptive statistics. Results: Sixty five per cent of adults 50 years of age and olderhad no protective levels of tetanus antitoxin (60 years, 6(7% had protective antitoxin levels IU/mL 0.1-1, and 5(5.8% had protective antitoxin levels  IU/mL≥1. Male gender and prior receipt of toxoid booster(s wereassociated with protective tetanus immunity. Tetanus antitoxin levels declined with age. Conclusion: It appears that most 50 years of age and older adults do not have protective levels of tetanus antitoxin because of inadequate vaccination coverage. There is a need to improve the immunity levels of this age group.

  18. Seroprevalence of tetanus toxoid antibody and booster vaccination efficacy in Japanese travelers.

    Science.gov (United States)

    Mizuno, Yasutaka; Yamamoto, Akihiko; Komiya, Takako; Takeshita, Nozomi; Takahashi, Motohide

    2014-01-01

    Tetanus can be prevented by vaccination, which is especially important for overseas travelers. However, despite booster vaccination every 10 years being recommended, most Japanese adults do not receive it in the absence of physical injury or overseas travel. We aimed to investigate the level of protective immunity against tetanus among Japanese travelers, which may provide valuable information for formulating booster vaccination recommendations. 113 Japanese travelers given tetanus toxoid were recruited. The collected samples included paired samples prior to and 3-5 weeks after receiving the booster vaccination. Travelers who did not return and those lacking sample collection at the second visit were excluded. Finally, 96 paired blood samples were collected. History of immunization against tetanus, including DPT and DT vaccines, was determined from interviews or immunization records. The pre-vaccination geometric mean titer for the 96 participants was 1.07 IU/mL; 76% had a protective antitoxin level (>0.1 IU/mL), and 50% had a long-term protective antitoxin level (>1.0 IU/mL). Most participants 50 years of age had protective immunity. Among the 23 participants without protective antitoxin levels (50 years of age. Although the tetanus antitoxin level decreases with age, booster vaccination helped to achieve an adequate protective antitoxin levels in Japanese travelers tetanus especially in those >50 years old need to obtain protective immunity against tetanus according to a basic immunization schedule to prevent tetanus in travelers and residents of Japan. Copyright © 2013 Japanese Society of Chemotherapy and the Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  19. Systemic antibody responses induced by a two-component Clostridium difficile toxoid vaccine protect against C. difficile-associated disease in hamsters.

    Science.gov (United States)

    Anosova, Natalie G; Brown, Anna M; Li, Lu; Liu, Nana; Cole, Leah E; Zhang, Jinrong; Mehta, Hersh; Kleanthous, Harry

    2013-09-01

    Clostridium difficile infection (CDI) has been identified as the leading cause of nosocomial diarrhoea and pseudomembranous colitis associated with antibiotic therapy. Recent epidemiological changes as well as increases in the number of outbreaks of strains associated with increased virulence and higher mortality rates underscore the importance of identifying alternatives to antibiotics to manage this important disease. Animal studies have clearly demonstrated the roles that toxins A and B play in gut inflammation as well as diarrhoea; therefore it is not surprising that serum anti-toxin A and B IgG are associated with protection against recurrent CDI. In humans, strong humoral toxin-specific immune responses elicited by natural C. difficile infection is associated with recovery and lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. The first generation of C. difficile vaccine that contained inactivated toxin A and B was found to be completely protective against death and diarrhoea in the hamster C. difficile challenge model. When tested in young healthy volunteers in Phase I clinical trials, this investigational vaccine was shown to be safe and immunogenic. Moreover, in a separate study this vaccine was able to prevent further relapses in three out of three patients who had previously suffered from chronic relapsing C. difficile-associated diarrhoea. Herein we examined the immunogenicity and protective activity of a next-generation Sanofi Pasteur two-component highly purified toxoid vaccine in a C. difficile hamster model. This model is widely recognized as a stringent and relevant choice for the evaluation of novel treatment strategies against C. difficile and was used in preclinical testing of the first-generation vaccine candidate. Intramuscular (i.m.) immunizations with increasing doses of this adjuvanted toxoid vaccine protected hamsters from mortality and disease symptoms in a dose-dependent manner. ELISA

  20. ¹H, ¹³C, and ¹⁵N backbone and side-chain chemical shift assignment of the toxin Doc in the unbound state.

    Science.gov (United States)

    De Gieter, Steven; Loris, Remy; van Nuland, Nico A J; Garcia-Pino, Abel

    2014-04-01

    Toxin-antitoxin (TA) modules in bacteria are involved in pathogenesis, antibiotic stress response, persister formation and programmed cell death. The toxin Doc, from the phd/doc module, blocks protein synthesis by targeting the translation machinery. Despite a large wealth of biophysical and biochemical data on the regulatory aspects of the operon transcription and role of Doc co-activator and co-repressor, little is still know on the molecular basis of Doc toxicity. Structural information about this toxin is only available for its inhibited state bound to the antitoxin Phd. Here we report the (1)H, (15)N and (13)C backbone and side chain chemical shift assignments of the toxin Doc from of bacteriophage P1 (the model protein from this family of TA modules) in its free state. The BMRB accession number is 18899.

  1. A New Selectable Marker System for Genetic Studies of Bacteria: Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Parsons, D; Tolmasky, M; Chain, P; Segelke, B W

    2011-03-18

    Genetic manipulations in bacteria currently rely on the introduction of antibiotic resistance genes into a bacterial strain; for those organisms that will be used for commercial or industrial applications, the genetic cassette encoding the antibiotic resistance is sometimes removed after selection. it is clear that if alternative technologies could obviate the need to introduce antibiotic resistance into bacteria, they would most certainly become a standard tool in molecular micriobiology for commercial, industrial as well as research applications. Here, they present the development of a novel genetic engineering technology based on toxin-antitoxin systems to modify bacterial genomes without the use of antibiotic resistance in the mutagenesis process. The primary goal is to develop antibiotic-free selection for genetically altered select agent pathogens. They are adapting the toxinc-antitoxin system to enable gene replacement in select agent pathogens since the NIH restrictions introducing antibiotic resistance into select agent pathogens have hindered research with select agent pathogens.

  2. Resonance assignments of a VapC family toxin from Clostridium thermocellum.

    Science.gov (United States)

    Wang, Chen; Xuan, Jinsong; Cui, Qiu; Feng, Yingang

    2016-10-01

    Toxin-antitoxin (TA) systems widely exist in bacterial plasmids, phages, and chromosomes and play important roles in growth persistence and host-pathogen interaction. Virulence associated protein BC (VapBC) family TAs are the most abundant TAs in bacteria and many pathogens contain a large number of vapBC loci in the genome which have been extensively studied. Clostridium thermocellum, a cellulolytic anaerobic gram-positive bacterium with promising applications in biofuel production, also contains a VapBC TA in the genome. Despite the structures of several VapBC family TAs have been determined, the toxin and anti-toxin components of C. thermocellum VapBC have very low sequence identity to the proteins in PDB. Therefore, the structure and functional mechanism of this TA is largely unknown. Here we reported the NMR resonance assignments of the VapC toxin from C. thermocellum as a basis for further structural and functional studies.

  3. Proteomic insight into the primycin fermentation process of Saccharomonospora azurea.

    Science.gov (United States)

    Valasek, Andrea; Kiss, Írisz Éva; Fodor, István; Kovács, Márk; Urbán, Péter; Jámbor, Éva; Fekete, Csaba; Kerepesi, Ildikó

    2016-12-01

    Saccharomonospora azurea SZMC 14600 is a member of the family Pseudonocardiaceae exclusively used for industrial scale production of primycin a large 36-membered non-polyene macrolide lactone antibiotic belonging to the polyketide class of natural products. Even though maximum antibiotic yield has been achieved by empirically optimized two-step fermentation process, little is known about the molecular components and mechanisms underlying the efficient antibiotic production. In order to identify differentially expressed proteins (DEPs) between the pre- and main-fermentation stages of primycin, comparative 2D-PAGE experiments were performed. In total, 98 DEP spots were reproducibly detected, out of which four spots were excised from gels, and identified through MALDI-TOF/TOF mass spectrometry. Peptide mass fingerprint analysis revealed peptide matches to HicB antitoxin for the HicAB toxin-antitoxin system (EHK86651), to a nucleoside diphosphate kinase regulator ((Ndk; EHK81899) and two other proteins with unknown function (EHK88946 and EHK86777).

  4. Quantitative estimation of diphtheria and tetanus toxoids. 4. Toxoids as international reference materials defining Lf-units for diphtheria and tetanus toxoids.

    Science.gov (United States)

    Lyng, J

    1990-01-01

    The Lf-unit, which is used in the control of diphtheria and tetanus toxoid production and in some countries also to follow immunization of horses for production of antitoxins, has hitherto been defined by means of antitoxin preparations. A diphtheria toxoid and a tetanus toxoid preparation, both freeze-dried, were examined in an international collaborative study for their suitability to serve as reference reagents in the flocculation tests and for defining the Lf-units. It was shown that flocculation tests using the reference toxoids are very reproducible and reliable and the WHO Expert Committee on Biological Standardization established: the toxoid called DIFT as the International Reference Reagent of Diphtheria Toxoid for Flocculation Test with a defined content of 900 Lf-units of diphtheria toxoid per ampoule; and the toxoid called TEFT as the International Reference Reagent of Tetanus Toxoid for Flocculation Test with a defined content of 1000 Lf-units of diphtheria toxoid per ampoule.

  5. Diphtheria outbreak with high mortality in northeastern Nigeria.

    Science.gov (United States)

    Besa, N C; Coldiron, M E; Bakri, A; Raji, A; Nsuami, M J; Rousseau, C; Hurtado, N; Porten, K

    2014-04-01

    SUMMARY A diphtheria outbreak occurred from February to November 2011 in the village of Kimba and its surrounding settlements, in Borno State, northeastern Nigeria. We conducted a retrospective outbreak investigation in Kimba village and the surrounding settlements to better describe the extent and clinical characteristics of this outbreak. Ninety-eight cases met the criteria of the case definition of diphtheria, 63 (64.3%) of whom were children aged diphtheria. None of the 98 cases received diphtheria antitoxin, penicillin, or erythromycin during their illness. The overall case-fatality ratio was 21.4%, and was highest in children aged 0-4 years (42.9%). Low rates of immunization, delayed clinical recognition of diphtheria and absence of treatment with antitoxin and appropriate antibiotics contributed to this epidemic and its severity.

  6. Translation-dependent mRNA cleavage by YhaV in Escherichia coli.

    Science.gov (United States)

    Choi, Wonho; Yamaguchi, Yoshihiro; Lee, Jae-Woo; Jang, Kyung-Min; Inouye, Masayori; Kim, Sung-Gun; Yoon, Min-Ho; Park, Jung-Ho

    2017-07-01

    Many bacteria have toxin-antitoxin (TA) systems, where toxin gene expression inhibits their own cell growth. mRNA is one of the well-known targets of the toxins in the type II toxin-antitoxin systems. Here, we examined the ribosome dependency of the endoribonuclease activity of YhaV, one of the toxins in type II TA systems, on mRNA in vitro and in vivo. A polysome profiling assay revealed that YhaV is bound to the 70S ribosomes and 50S ribosomal subunits. Moreover, we found that while YhaV cleaves ompF and lpp mRNAs in a translation-dependent manner, they did not cleave the 5' untranslated region in primer extension experiments. From these results, we conclude that YhaV is a ribosome-dependent toxin that cleaves mRNA in a translation-dependent manner. © 2017 Federation of European Biochemical Societies.

  7. A System for Discovering Bioengineering Threats by Knowledge Base Driven Mining of Toxin Data

    Science.gov (United States)

    2005-08-01

    warfare agents, (2) identifying and developing counter measures such as anti-toxins, vaccines, and inhibitors , and (3) developing a better understanding... inhibitors target abtoxin pore-former enzyme-catalyzed EC_number 3,.24.69 Botulinum toxin acts by inhibiting neurotransmitter release. It binds to...some results that do not yet have a thorough biochemical validation. First we present the result of comparing Thermolysin , Neprilysin and Botulinum

  8. Coxofemoral luxation in a border collie as a complication of a Clostridium tetani infection.

    Science.gov (United States)

    Goldhammer, M A; Chapman, P S; Grierson, J M

    2008-03-01

    A four-month-old male, entire, border collie was presented to the Queen Mother Hospital for Animals with a two day history of muscular spasms and "Risus sardonicus". Tetanus was diagnosed, and the dog was treated with tetanus antitoxin, antibiotics and supportive therapy. Coxofemoral luxation resulted as a complication of the tetanus and was successfully managed by performing a femoral head and neck excision. This is the first report of joint luxation associated with Clostridium tetani infection in a dog.

  9. Crystallization and preliminary crystallographic studies of the YafN–YafO complex from Escherichia coli

    International Nuclear Information System (INIS)

    Zhang, Fan; Xing, Li; Teng, Maikun; Li, Xu

    2012-01-01

    The ribosome-dependent mRNA interferase YafO from Escherichia coli belongs to a type II toxin–antitoxin (TA) system and its cognate antitoxin YafN neutralizes cell toxicity by forming a stable YafN–YafO complex. Here, the YafN–YafO complex has been expressed and crystallized. The ribosome-dependent mRNA interferase YafO from Escherichia coli belongs to a type II toxin–antitoxin (TA) system and its cognate antitoxin YafN neutralizes cell toxicity by forming a stable YafN–YafO complex. The YafN–YafO TA system is upregulated by the SOS response (a global response to DNA damage in which the cell cycle is arrested and mutagenesis is induced) and may then inhibit protein synthesis by endoribonuclease activity of YafO with the 50S ribosome subunit. Structural information on the YafN–YafO complex and related complexes would be helpful in order to understand the structural basis of the mechanism of mRNA recognition and cleavage, and the assembly of these complexes. Here, the YafN–YafO complex was expressed and crystallized. Crystals grown by the hanging-drop vapour-diffusion method diffracted to 3.50 Å resolution and belonged to the hexagonal space group P622, with unit-cell parameters a = 86.14, b = 86.14, c = 173.11 Å, α = β = 90, γ = 120°. Both Matthews coefficient analysis and the self-rotation function suggested the presence of one molecule per asymmetric unit in the crystal, with a solvent content of 65.69% (V M = 3.58 Å 3 Da −1 )

  10. Persistence of Vancomycin Resistance in Multiple Clones of Enterococcus faecium Isolated from Danish Broilers 15 Years after the Ban of Avoparcin

    DEFF Research Database (Denmark)

    Bortolaia, Valeria; Mander, Manuela; Jensen, Lars Bogø

    2015-01-01

    The occurrence and diversity of vancomycin-resistant Enterococcus faecium (VREF) were investigated in 100 Danish broiler flocks 15 years after the avoparcin ban. VREF occurred in 47 flocks at low fecal concentrations detectable only by selective enrichment. Vancomycin resistance was prevalently...... associated with a transferable nontypeable plasmid lineage occurring in multiple E. faecium clones. Coselection of sequence type 842 by tetracycline use only partly explained the persistence of vancomycin resistance in the absence of detectable plasmid coresistance and toxin-antitoxin systems....

  11. RESISTANCE OF GUINEA PIGS IMMUNIZED WITH BOTULINUM TOXOIDS TO AEROGENIC CHALLENGE WITH TOXIN

    Science.gov (United States)

    guinea pigs with botulinum toxoids afforded a high level of protection to challenge with botulinum toxins by the respiratory route. Resistance to challenge by this route was similar to resistance afforded to challenge by the parenteral and oral routes. The high survival percentages obtained in these studies made it impossible to establish any relationship between serum antitoxin titer and resistance to challenge. This report presents results of some studies to determine resistance afforded actively immunized guinea pigs to challenge with botulinum

  12. Orphan Toxin OrtT (YdcX) of Escherichia coli Reduces Growth during the Stringent Response

    Science.gov (United States)

    2015-01-29

    consists of two-components with the exception of the three-component system ω-ε-ζ of Streptococcus pyogenes [37]. Here, we demonstrate the importance of a...often found in cryptic prophage remnants such as PhyL in Bacillus anthracis [35] and LysA in Streptococcus gordonii [36] without the antitoxin genes...36. Ploeg, J.R.v.d. Characterization of Streptococcus gordonii prophage PH15: Complete genome sequence and functional analysis of phage-encoded

  13. Respiratory diphtheria in an asylum seeker from Afghanistan arriving to Finland via Sweden, December 2015.

    Science.gov (United States)

    Sane, Jussi; Sorvari, Tiina; Widerström, Micael; Kauma, Heikki; Kaukoniemi, Ulla; Tarkka, Eveliina; Puumalainen, Taneli; Kuusi, Markku; Salminen, Mika; Lyytikäinen, Outi

    2016-01-01

    In December 2015, an asylum seeker originating from Afghanistan was diagnosed with respiratory diphtheria in Finland. He arrived in Finland from Sweden where he had already been clinically suspected and tested for diphtheria. Corynebacterium diphtheriae was confirmed in Sweden and shown to be genotypically and phenotypically toxigenic. The event highlights the importance of early case detection, rapid communication within the country and internationally as well as preparedness plans of diphtheria antitoxin availability.

  14. The research progress and medicine application of the ScFv antibody

    International Nuclear Information System (INIS)

    Qin Lili; Zhang Chunming

    2005-01-01

    Since the scholar of England and Japan had found the diphtheria antitoxin, the research of the antibody has experienced three phases: polyclonal antibodies, monoclonal antibodies and genetically engineered antibodies. In recent years, far more attention has been paid to single-chain antibody by researchers owing to it's small molecular, strong ability of penetration, short half-lives in blood, high specificity to combine with the corresponding antibody, weak immunogenicity and possibility to be expressed in prokaryocyte. (authors)

  15. Identification of novel substrates of Shigella T3SA through analysis of its virulence plasmid-encoded secretome.

    Directory of Open Access Journals (Sweden)

    Laurie Pinaud

    Full Text Available Many human Gram-negative bacterial pathogens express a Type Three Secretion Apparatus (T3SA, including among the most notorious Shigella spp., Salmonella enterica, Yersinia enterocolitica and enteropathogenic Escherichia coli (EPEC. These bacteria express on their surface multiple copies of the T3SA that mediate the delivery into host cells of specific protein substrates critical to pathogenesis. Shigella spp. are Gram-negative bacterial pathogens responsible for human bacillary dysentery. The effector function of several Shigella T3SA substrates has largely been studied but their potential cellular targets are far from having been comprehensively delineated. In addition, it is likely that some T3SA substrates have escaped scrutiny as yet. Indeed, sequencing of the virulence plasmid of Shigella flexneri has revealed numerous open reading frames with unknown functions that could encode additional T3SA substrates. Taking advantage of label-free mass spectrometry detection of proteins secreted by a constitutively secreting strain of S. flexneri, we identified five novel substrates of the T3SA. We further confirmed their secretion through the T3SA and translocation into host cells using β-lactamase assays. The coding sequences of two of these novel T3SA substrates (Orf13 and Orf131a have a guanine-cytosine content comparable to those of T3SA components and effectors. The three other T3SA substrates identified (Orf48, Orf86 and Orf176 have significant homology with antitoxin moieties of type II Toxin-Antitoxin systems usually implicated in the maintenance of low copy plasmids. While Orf13 and Orf131a might constitute new virulence effectors contributing to S. flexneri pathogenicity, potential roles for the translocation into host cells of antitoxins or antitoxin-like proteins during Shigella infection are discussed.

  16. Clostridium tetani growth and toxin production in the intestines of germfree rats.

    OpenAIRE

    Wells, C L; Balish, E

    1983-01-01

    Germfree rats were challenged orally and intrarectally with spores of Clostridium tetani. Although C. tetani spores remained viable in the intestinal tract, they were unable to germinate. Germfree rats were then challenged orally with vegetative cells of C. tetani. Vegetative cells were able to colonize the intestinal tract, replicate, and produce toxin. Tetanus antitoxin, but no tetanus toxin, was detected in the sera of monoassociated rats.

  17. Clostridium tetani growth and toxin production in the intestines of germfree rats.

    Science.gov (United States)

    Wells, C L; Balish, E

    1983-01-01

    Germfree rats were challenged orally and intrarectally with spores of Clostridium tetani. Although C. tetani spores remained viable in the intestinal tract, they were unable to germinate. Germfree rats were then challenged orally with vegetative cells of C. tetani. Vegetative cells were able to colonize the intestinal tract, replicate, and produce toxin. Tetanus antitoxin, but no tetanus toxin, was detected in the sera of monoassociated rats. PMID:6347898

  18. Active immunisation of horses against tetanus including the booster dose and its application.

    Science.gov (United States)

    Liefman, C E

    1981-02-01

    Successful active immunisation of horses against tetanus is dependent on a number of factors of which the toxoid preparation used, its method of application and the ability of the individual horse to respond are fundamental. Two immunisation schedules using an aluminium-based toxoid preparation were examined and the protection determined by monitoring the level of antitoxin afforded by each schedule. The results obtained demonstrated that 2 doses of this toxoid are necessary to ensure 12 months protection in all horses. These results are discussed in relation to the factors involved in active immunisation against tetanus. Reference is also made to the occurrence of a transient phase of reduced levels of antitoxin following booster doses of toxoid in immunised horses during which it is considered these horses could become more susceptible to tetanus. The effect of a booster dose on immunised horses was examined and while there can be a reduction in the level of antitoxin in some immunised horses following this dose its effect is minimal, short-lived and for all practical purposes can be disregarded. The application of the booster dose in practice is also discussed.

  19. Seroprevalence of antibody against diphtheria among the population in Khon Kaen province, Thailand.

    Science.gov (United States)

    Bansiddhi, Hataichanok; Vuthitanachot, Viboonsuk; Vuthitanachot, Chanpim; Prachayangprecha, Slinporn; Theamboonlers, Apiradee; Poovorawan, Yong

    2015-03-01

    To assess diphtheria immunity in the northeastern region of Thailand, a seroepidemiological survey was undertaken in 2011 from 516 healthy individuals (age range 2-87 years) in Khon Kaen province. Diphtheria antitoxin levels were measured by enzyme-linked immunosorbent assay and titers of ≥0.1 IU/mL were considered to be protective antitoxin levels. Among the studied population, 94.8% have fully protective levels. The younger population (age range 2-19 years) has higher diphtheria immunity with seroprotection rates of 96.8% to 97.9%, compared with the adult population. The proportion of protective diphtheria antitoxin levels declines to 88.3% to 91.9% in the middle-aged group (20-50 years), and appeared to be higher again in the older age-group (50-70 years). To avoid epidemic spreading, promoting immunization booster programs will be helpful, especially among the adult population (20-50 years). Finally, this study may serve as a valuable guide in deciding exactly which age-groups should be targeted by such an effort. © 2012 APJPH.

  20. Humoral immunity 10 years after booster immunization with an adolescent and adult formulation combined tetanus, diphtheria, and 5-component acellular pertussis vaccine.

    Science.gov (United States)

    Tomovici, A; Barreto, L; Zickler, P; Meekison, W; Noya, F; Voloshen, T; Lavigne, P

    2012-03-30

    Persistence of antibodies after a single dose of Tdap vaccine (tetanus, diphtheria, and 5-component acellular pertussis vaccine) was evaluated in a follow-up study of adolescents (N=324) and adults (N=644) who had received Tdap in earlier clinical trials. Outcome measures were seroprotection (tetanus and diphtheria) or seropositivity (pertussis) and geometric mean concentrations. Humoral immune responses to all antigens were robust 1 month after initial immunization, decreased at subsequent measurements, but continued to exceed pre-immunization levels 1, 3, 5, and 10 years later. Protective levels of diphtheria and tetanus antitoxin persisted in 99.3% of adolescents 10 years after a booster dose of Tdap. Seropositivity to 1 or more pertussis antigens also persisted in most adolescents for 10 years. Although tetanus antitoxin responses were similar in adults to those observed in adolescents, diphtheria antitoxin titers were lower, reflecting the fact that a smaller proportion of adults had received diphtheria toxoid in the previous 10 years compared to adolescents. These data will contribute to the selection of the optimal interval for repeat doses of Tdap. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Cluster of botulism among Dutch tourists in Turkey, June 2008.

    Science.gov (United States)

    Swaan, C M; van Ouwerkerk, I M; Roest, H J

    2010-04-08

    In June 2008, three Dutch tourists participating in a mini-cruise in Turkey needed urgent repatriation for antitoxin treatment because of symptoms of botulism. Because there was a shortage of antitoxin in the Netherlands, an emergency delivery was requested from the manufacturer in Germany. An outbreak investigation was initiated into all nine cruise members, eight of whom developed symptoms. C. botulinum type B was isolated in stool culture from four of them. No other patients were notified locally. Food histories revealed locally purchased unprocessed black olives, consumed on board of the ship, as most likely source, but no left-overs were available for investigation. C. botulinum type D was detected in locally purchased canned peas, and whilst type D is not known to be a cause of human intoxication, its presence in a canned food product indicates an inadequate preserving process. With increasing tourism to areas where food-borne botulism is reported regularly special requests for botulism antitoxin may become necessary. Preparing an inventory of available reserve stock in Europe would appear to be a necessary and valuable undertaking.

  2. Comparative Genomic Analysis of Lactobacillus plantarum GB-LP1 Isolated from Traditional Korean Fermented Food.

    Science.gov (United States)

    Yu, Jihyun; Ahn, Sojin; Kim, Kwondo; Caetano-Anolles, Kelsey; Lee, Chanho; Kang, Jungsun; Cho, Kyungjin; Yoon, Sook Hee; Kang, Dae-Kyung; Kim, Heebal

    2017-08-28

    As probiotics play an important role in maintaining a healthy gut flora environment through antitoxin activity and inhibition of pathogen colonization, they have been of interest to the medical research community for quite some time now. Probiotic bacteria such as Lactobacillus plantarum , which can be found in fermented food, are of particular interest given their easy accessibility. We performed whole-genome sequencing and genomic analysis on a GB-LP1 strain of L. plantarum isolated from Korean traditional fermented food; this strain is well known for its functions in immune response, suppression of pathogen growth, and antitoxin effects. The complete genome sequence of GB-LP1 is a single chromosome of 3,040,388 bp with 2,899 predicted open reading frames. Genomic analysis of GB-LP1 revealed two CRISPR regions and genes showing accelerated evolution, which may have antibiotic and antitoxin functions. The aim of the present study was to predict strain specific-genomic characteristics and assess the potential of this new strain as lactic acid bacteria at the genomic level using in silico analysis. These results provide insight into the L. plantarum species as well as confirm the possibility of its utility as a candidate probiotic.

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

    Directory of Open Access Journals (Sweden)

    Chengxian Zhang

    Full Text Available 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.

  4. Functional characterization of replication and stability factors of an incompatibility group P-1 plasmid from Xylella fastidiosa.

    Science.gov (United States)

    Lee, Min Woo; Rogers, Elizabeth E; Stenger, Drake C

    2010-12-01

    Xylella fastidiosa strain riv11 harbors a 25-kbp plasmid (pXF-RIV11) belonging to the IncP-1 incompatibility group. Replication and stability factors of pXF-RIV11 were identified and used to construct plasmids able to replicate in X. fastidiosa and Escherichia coli. Replication in X. fastidiosa required a 1.4-kbp region from pXF-RIV11 containing a replication initiation gene (trfA) and the adjacent origin of DNA replication (oriV). Constructs containing trfA and oriV from pVEIS01, a related IncP-1 plasmid of the earthworm symbiont Verminephrobacter eiseniae, also were competent for replication in X. fastidiosa. Constructs derived from pXF-RIV11 but not pVEIS01 replicated in Agrobacterium tumefaciens, Xanthomonas campestris, and Pseudomonas syringae. Although plasmids bearing replication elements from pXF-RIV11 or pVEIS01 could be maintained in X. fastidiosa under antibiotic selection, removal of selection resulted in plasmid extinction after 3 weekly passages. Addition of a toxin-antitoxin addiction system (pemI/pemK) from pXF-RIV11 improved plasmid stability such that >80 to 90% of X. fastidiosa cells retained plasmid after 5 weekly passages in the absence of antibiotic selection. Expression of PemK in E. coli was toxic for cell growth, but toxicity was nullified by coexpression of PemI antitoxin. Deletion of N-terminal sequences of PemK containing the conserved motif RGD abolished toxicity. In vitro assays revealed a direct interaction of PemI with PemK, suggesting that antitoxin activity of PemI is mediated by toxin sequestration. IncP-1 plasmid replication and stability factors were added to an E. coli cloning vector to constitute a stable 6.0-kbp shuttle vector (pXF20-PEMIK) suitable for use in X. fastidiosa.

  5. Genomic analysis of Acidianus hospitalis W1 a host for studying crenarchaeal virus and plasmid life cycles

    DEFF Research Database (Denmark)

    You, X. Y.; Liu, Chao; Wang, S. Y.

    2011-01-01

    The Acidianus hospitalis W1 genome consists of a minimally sized chromosome of about 2.13 Mb and a conjugative plasmid pAH1 and it is a host for the model filamentous lipothrixvirus AFV1. The chromosome carries three putative replication origins in conserved genomic regions and two large regions...... where non-essential genes are clustered. Within these variable regions, a few orphan orfB and other elements of the IS200/607/605 family are concentrated with a novel class of MITE-like repeat elements. There are also 26 highly diverse vapBC antitoxin-toxin gene pairs proposed to facilitate maintenance...

  6. Clostridium oedematiens: observations on potency assaying.

    Science.gov (United States)

    Macheak, M E

    1976-01-01

    The United States has established a Standard Requirement for the potency testing of biological products containing Clostridium oedematiens belonging to two types: type B (Cl. novyi) and type D (Cl. haemolyticum). Guinea pig testing has provided widely varying results depending on the origin of the animals. The tests reported are intended to determine the efficacity of the vaccines (Cl. novyi and Cl. haemolyticum) depending on the animal tested: guinea pigs, sheep and bovines; they further establish a parallelism between the antitoxin titer and the immunity of the animal.

  7. Physico-Chemical-Managed Killing of Penicillin-Resistant Static and Growing Gram-Positive and Gram-Negative Vegetative Bacteria

    Science.gov (United States)

    Richmond, Robert Chaffee (Inventor); Schramm, Jr., Harry F. (Inventor); Defalco, Francis G. (Inventor); Farris, III, Alex F. (Inventor)

    2012-01-01

    Systems and methods for the use of compounds from the Hofmeister series coupled with specific pH and temperature to provide rapid physico-chemical-managed killing of penicillin-resistant static and growing Gram-positive and Gram-negative vegetative bacteria. The systems and methods represent the more general physico-chemical enhancement of susceptibility for a wide range of pathological macromolecular targets to clinical management by establishing the reactivity of those targets to topically applied drugs or anti-toxins.

  8. Clostridium sordellii necrotizing omphalitis: A case report and literature review

    Directory of Open Access Journals (Sweden)

    Eric J. Rellinger

    2016-07-01

    Full Text Available Omphalitis is an infrequent neonatal infection of the umbilicus. Necrotizing infections are a rare complication of omphalitis that requires prompt antibiotic therapy, surgical debridement, and supportive care. Here, we present a rare case of Clostridium sordellii necrotizing omphalitis that progressed to severe toxemia characterized by refractory hypotension, massive capillary leak, leukemoid reaction, and absence of fever. These clinical features are common in C. sordellii infections and harbor a poor prognosis with only one reported survivor (out of 12 of C. sordellii omphalitis reported in the literature. Early antibiotic and surgical intervention remain the mainstay of care as no early detection assays or antitoxins are commercially available.

  9. A retrospective study of 155 adult equids and 21 foals with tetanus from Western, Northern and Central Europe (2000-2014). Part 1

    DEFF Research Database (Denmark)

    van Galen, Gaby; Rijckaert, Joke; Claude, Saegerman

    2017-01-01

    ObjectiveTo describe clinical data of hospitalized adult equids and foals with tetanus.DesignMulticenter retrospective study (2000–2014).SettingTwenty Western, Northern, and Central European university teaching hospitals and private referral centers.AnimalsOne hundred fifty-five adult equids (>6...... months) and 21 foals (management-related data, clinical history, clinical examination and blood analysis on admission, complications, treatments, and outcomes were...... described and statistically compared between adults and foals. The described cases were often young horses. In 4 adult horses, tetanus developed despite appropriate vaccination and in 2 foals despite preventive tetanus antitoxin administration at birth. Castration, hoof abscesses, and wounds were the most...

  10. RelE-Mediated Dormancy Is Enhanced at High Cell Density in Escherichia coli

    OpenAIRE

    Tashiro, Yosuke; Kawata, Koji; Taniuchi, Asami; Kakinuma, Kenji; May, Thithiwat; Okabe, Satoshi

    2012-01-01

    Bacteria show remarkable adaptability under several stressful conditions by shifting themselves into a dormant state. Less is known, however, about the mechanism underlying the cell transition to dormancy. Here, we report that the transition to dormant states is mediated by one of the major toxin-antitoxin systems, RelEB, in a cell density-dependent manner in Escherichia coli K-12 MG1655. We constructed a strain, IKA121, which expresses the toxin RelE in the presence of rhamnose and lacks chr...

  11. [Development of autoimmune reactions in horses serving to produce antitetanus serum].

    Science.gov (United States)

    Georgadze, I A; Nozadze, Z M

    1986-03-01

    The hyperimmunization of horses with large doses of tetanus toxoid is accompanied by an increase in the levels of both specific antitoxic antibodies and autoantibodies to the tissue antigens of the liver, the spleen, the heart. The reverse relationship between the level of autoantibodies and the titer of antitoxin has been established. The authors suggest that the synthesis of autoantibodies is stimulated by the presence of antigen-antibody immune complexes in the circulating blood, as well as by the action of exo- and endopolyclonal stimulators.

  12. Dynamic changes of horse serum T-globulin immunization with snake venoms, tetanus and diphtheria toxoids.

    Science.gov (United States)

    Lee, H F; Lee, J D; Lee, Y C

    1979-12-01

    In course of immunizing horses with snake venoms, tetanus and diphtheria toxoids, a new serum component, T-globulin, was formed and migrated between the beta- and gamma-globulins. The T-globulin content was parallel with the antibody titre after the middle course of immunization. There were many components in snake antivenin and T-globulin was composed of most of those components. The components of diphtheria T-globulin were the same as those of crude antitoxin and tetanus T-globulin except one precipitin.

  13. Biotechnological Trends in Spider and Scorpion Antivenom Development

    DEFF Research Database (Denmark)

    Laustsen, Andreas Hougaard; Solà, Mireia; Jappe, Emma Christine

    2016-01-01

    Spiders and scorpions are notorious for their fearful dispositions and their ability to inject venom into prey and predators, causing symptoms such as necrosis, paralysis, and excruciating pain. Information on venom composition and the toxins present in these species is growing due to an interest...... at an increasingly faster pace. In this review, the current knowledge of spider and scorpion venoms is presented, followed by a discussion of all published biotechnological efforts within development of spider and scorpion antitoxins based on small molecules, antibodies and fragments thereof, and next generation...

  14. Diphtheria in a 7-year-old child in north-eastern Nigeria - management in a resource-poor setting.

    Science.gov (United States)

    Goni, Baba Waru; Gofama, Mustapha M; Lawan, Gana M; Haruna, Yusuph; Bukar, Bakki; Musa, Kida I

    2014-01-01

    We report a case of a 7-year-old unimmunized child who presented with a 2 week history of nasal quality speech, hoarseness of the voice, regurgitation of feeds, and unstable gait. He had a previous history of fever, severe sore throat and bloody nasal discharge. A throat swab was negative for Corynebacterium diphtheria; however, he had received antibiotics at a primary care clinic prior to presentation. A clinical diagnosis of diphtheria with neurologic complication was made and the child was started on oral erythromycin, nasogastric tube feeding and daily physiotherapy, following which he improved. We did not prescribe diphtheria anti-toxin because of its unavailability.

  15. Diphtheria, pertussis, and tetanus: evidence-based management of pediatric patients in the emergency department

    Science.gov (United States)

    Zibners, Lara

    2017-02-01

    Diphtheria, pertussis, and tetanus are potentially deadly bacterial infections that are largely preventable through vaccination, though they remain in the population. This issue reviews the epidemiology, pathophysiology, diagnosis, and current recommended emergency management of these conditions. Disease-specific medications, as well as treatment of the secondary complications, are examined in light of the best current evidence. Resources include obtaining diphtheria antitoxin from the United States Centers for Disease Control and Prevention and best-practice recommendations with regard to testing, involvement of government health agencies, isolation of the patient, and identification and treatment of close contacts. Most importantly, issues regarding vaccination and prevention are highlighted.

  16. Experimental investigations on the immunity development in irradiated mice after local and parenteral immunization with tetanus-toxoid

    International Nuclear Information System (INIS)

    Baljer, G.; Schaller, M.; Schick, P.; Sailer, J.; Messerschmidt, O.; Mayr, A.

    1977-01-01

    Using the 'tetanus-mouse' model the differences in immunity development between animals exposed to irradiation (300 R and 400 R) and animals not exposed to irradiation after nasal and subcutaneous vaccination was investigated. Immunization was carried out 1, 3, 6 and 10 days before and after exposure to irradiation. Efficacy of immunization was tested by challenge with 10 x LD 50 tetanus-toxin and by antitoxin determination with L+-method. The degree of the immune response was dependent on 1) the irradiation dose, 2) the interval between active immunization and the ensuing or preceding irradiation, 3) the mode of vaccination (local or parenteral) and 4) the vaccination dose. (orig.) [de

  17. A micro-enzyme-linked immunosorbent assay (ELISA) and radioimmunosorbent technique (RIST) for the detection of immunity to clinical tetanus

    International Nuclear Information System (INIS)

    Layton, G.T.

    1980-01-01

    Enzyme-linked immunosorbent assay (ELISA), and radioimmunosorbent assay (RIST) techniques for the detection of tetanus toxin antibodies are described. Both methods proved to be highly sensitive, and allowed the measurement of 5 x 10 -3 units/ml tetanus antitoxin in human serum or plasma, sensitivity and reproducibility comparing well with other techniques previously described, and being superior to haemagglutination and latex agglutination tests. Results of the two methods correlated well, and reflected the immunization histories obtained. Micro ELISA and micro RIST would seem to be suitable for the detection of immunity, or non-immunity to clinical tetanus. (author)

  18. Complete sequence of the IncA/C1 plasmid pCf587 carrying blaPER-2 from Citrobacter freundii.

    Science.gov (United States)

    Ruggiero, Melina; Girlich, Delphine; Dabos, Laura; Power, Pablo; Naas, Thierry; Gutkind, Gabriel

    2018-02-20

    The bla PER-2 harboring plasmid pCf587 (191,541 bp) belongs to lineage IncA/C 1 and is closely related to pRA1. It contains a large resistance island including the bla PER-2 gene between two copies of IS Kox2 -like elements, the toxin-antitoxin module pemK-pemI, several other resistance genes inserted within a Tn 2 transposon, a Tn 21 -like structure, and a class 1 integron. pCf587 belongs into ST 13, a new pMLST. Copyright © 2018 American Society for Microbiology.

  19. [An atypical case of necrotizing staphylococcal pneumonia].

    Science.gov (United States)

    Bertreau, E; Grard, S; Baudry, T; Freymond, N

    2017-11-01

    Some strains of Staphylococcus aureus produce a toxin known as Panton-Valentine leukocidin. These strains notably cause a necrotizing pneumonia which is associated with a high mortality. A 70-year-old woman presented with sub-acute onset dyspnea, low-grade fever, and hemoptysis after a trip to Dubai and New Zealand. Computed tomography showed bilateral necrotizing pneumonia, suggesting the diagnosis of pneumonia caused by S. aureus producing Panton-Valentine toxin. It was confirmed by microbiological investigation. The rapid initiation of adequate antimicrobial therapy including an effective antitoxin was essential for successful treatment, without the need for ventilatory support. Necrotizing pneumonia caused by S. aureus producing Panton-Valentine leukocidin usually occurs in young subjects without comorbidities. Typical symptoms are a combination of hypoxemia, high fever, hemoptysis, leukopenia, and a rapidly worsening condition. Panton-Valentine leukocidin should not be discarded if not all the symptoms are typical. Antibiotic therapy including an antitoxin drug such as linezolid or clindamycin should be initiated promptly. Copyright © 2017 SPLF. Published by Elsevier Masson SAS. All rights reserved.

  20. Doc toxin is a kinase that inactivates elongation factor Tu.

    Science.gov (United States)

    Cruz, Jonathan W; Rothenbacher, Francesca P; Maehigashi, Tatsuya; Lane, William S; Dunham, Christine M; Woychik, Nancy A

    2014-03-14

    The Doc toxin from bacteriophage P1 (of the phd-doc toxin-antitoxin system) has served as a model for the family of Doc toxins, many of which are harbored in the genomes of pathogens. We have shown previously that the mode of action of this toxin is distinct from the majority derived from toxin-antitoxin systems: it does not cleave RNA; in fact P1 Doc expression leads to mRNA stabilization. However, the molecular triggers that lead to translation arrest are not understood. The presence of a Fic domain, albeit slightly altered in length and at the catalytic site, provided a clue to the mechanism of P1 Doc action, as most proteins with this conserved domain inactivate GTPases through addition of an adenylyl group (also referred to as AMPylation). We demonstrated that P1 Doc added a single phosphate group to the essential translation elongation factor and GTPase, elongation factor (EF)-Tu. The phosphorylation site was at a highly conserved threonine, Thr-382, which was blocked when EF-Tu was treated with the antibiotic kirromycin. Therefore, we have established that Fic domain proteins can function as kinases. This distinct enzymatic activity exhibited by P1 Doc also solves the mystery of the degenerate Fic motif unique to the Doc family of toxins. Moreover, we have established that all characterized Fic domain proteins, even those that phosphorylate, target pivotal GTPases for inactivation through a post-translational modification at a single functionally critical acceptor site.

  1. Doc Toxin Is a Kinase That Inactivates Elongation Factor Tu*

    Science.gov (United States)

    Cruz, Jonathan W.; Rothenbacher, Francesca P.; Maehigashi, Tatsuya; Lane, William S.; Dunham, Christine M.; Woychik, Nancy A.

    2014-01-01

    The Doc toxin from bacteriophage P1 (of the phd-doc toxin-antitoxin system) has served as a model for the family of Doc toxins, many of which are harbored in the genomes of pathogens. We have shown previously that the mode of action of this toxin is distinct from the majority derived from toxin-antitoxin systems: it does not cleave RNA; in fact P1 Doc expression leads to mRNA stabilization. However, the molecular triggers that lead to translation arrest are not understood. The presence of a Fic domain, albeit slightly altered in length and at the catalytic site, provided a clue to the mechanism of P1 Doc action, as most proteins with this conserved domain inactivate GTPases through addition of an adenylyl group (also referred to as AMPylation). We demonstrated that P1 Doc added a single phosphate group to the essential translation elongation factor and GTPase, elongation factor (EF)-Tu. The phosphorylation site was at a highly conserved threonine, Thr-382, which was blocked when EF-Tu was treated with the antibiotic kirromycin. Therefore, we have established that Fic domain proteins can function as kinases. This distinct enzymatic activity exhibited by P1 Doc also solves the mystery of the degenerate Fic motif unique to the Doc family of toxins. Moreover, we have established that all characterized Fic domain proteins, even those that phosphorylate, target pivotal GTPases for inactivation through a post-translational modification at a single functionally critical acceptor site. PMID:24448800

  2. Treatments for Pulmonary Ricin Intoxication: Current Aspects and Future Prospects

    Directory of Open Access Journals (Sweden)

    Yoav Gal

    2017-10-01

    Full Text Available Ricin, a plant-derived toxin originating from the seeds of Ricinus communis (castor beans, is one of the most lethal toxins known, particularly if inhaled. Ricin is considered a potential biological threat agent due to its high availability and ease of production. The clinical manifestation of pulmonary ricin intoxication in animal models is closely related to acute respiratory distress syndrome (ARDS, which involves pulmonary proinflammatory cytokine upregulation, massive neutrophil infiltration and severe edema. Currently, the only post-exposure measure that is effective against pulmonary ricinosis at clinically relevant time-points following intoxication in pre-clinical studies is passive immunization with anti-ricin neutralizing antibodies. The efficacy of this antitoxin treatment depends on antibody affinity and the time of treatment initiation within a limited therapeutic time window. Small-molecule compounds that interfere directly with the toxin or inhibit its intracellular trafficking may also be beneficial against ricinosis. Another approach relies on the co-administration of antitoxin antibodies with immunomodulatory drugs, thereby neutralizing the toxin while attenuating lung injury. Immunomodulators and other pharmacological-based treatment options should be tailored according to the particular pathogenesis pathways of pulmonary ricinosis. This review focuses on the current treatment options for pulmonary ricin intoxication using anti-ricin antibodies, disease-modifying countermeasures, anti-ricin small molecules and their various combinations.

  3. Modeling new immunoregulatory therapeutics as antimicrobial alternatives for treating Clostridium difficile infection.

    Science.gov (United States)

    Leber, Andrew; Hontecillas, Raquel; Abedi, Vida; Tubau-Juni, Nuria; Zoccoli-Rodriguez, Victoria; Stewart, Caroline; Bassaganya-Riera, Josep

    2017-05-01

    The current treatment paradigm in Clostridium difficile infection is the administration of antibiotics contributing to the high rates of recurrent infections. Recent alternative strategies, such as fecal microbiome transplantation and anti-toxin antibodies, have shown similar efficacy in the treatment of C. difficile associated disease (CDAD). However, barriers exist for either treatment or other novel treatments to displace antibiotics as the standard of care. To aid in the comparison of these and future treatments in CDAD, we developed an in silico pipeline to predict clinical efficacy with nonclinical results. The pipeline combines an ordinary differential equation (ODE)-based model, describing the immunological and microbial interactions in the gastrointestinal (GI) mucosa, with machine learning algorithms to translate simulated output quantities (i.e. time of clearance, quantity of commensal bacteria, T cell ratios) into clinical predictions based on prior preclinical, translational and clinical trial data. As a use case, we compare the efficacy of lanthionine synthetase C-like 2 (LANCL2), a novel immunoregulatory target with promising efficacy in inflammatory bowel disease (IBD), activation with antibiotics, fecal microbiome transplantation and anti-toxin antibodies in the treatment of CDAD. We further validate the potential of LANCL2 pathway activation, in a mouse model of C. difficile infection in which it displays an ability to decrease weight loss and inflammatory cell types while protecting against mortality. The computational pipeline can serve as an important resource in the development of new treatment modalities. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Toxin ζ Reversible Induces Dormancy and Reduces the UDP-N-Acetylglucosamine Pool as One of the Protective Responses to Cope with Stress

    Directory of Open Access Journals (Sweden)

    Mariangela Tabone

    2014-09-01

    Full Text Available Toxins of the ζ/PezT family, found in the genome of major human pathogens, phosphorylate the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG leading to unreactive UNAG-3P. Transient over-expression of a PezT variant impairs cell wall biosynthesis and triggers autolysis in Escherichia coli. Conversely, physiological levels of ζ reversibly induce dormancy produce a sub-fraction of membrane-compromised cells, and a minor subpopulation of Bacillus subtilis cells become tolerant of toxin action. We report here that purified ζ is a strong UNAG-dependent ATPase, being GTP a lower competitor. In vitro, ζ toxin phosphorylates a fraction of UNAG. In vivo, ζ-mediated inactivation of UNAG by phosphorylation does not deplete the active UNAG pool, because expression of the toxin enhances the efficacy of genuine cell wall inhibitors (fosfomycin, vancomycin or ampicillin. Transient ζ expression together with fosfomycin treatment halt cell proliferation, but ε2 antitoxin expression facilitates the exit of ζ-induced dormancy, suggesting that there is sufficient UNAG for growth. We propose that ζ induces diverse cellular responses to cope with stress, being the reduction of the UNAG pool one among them. If the action of ζ is not inhibited, e.g., by de novo ε2 antitoxin synthesis, the toxin markedly enhances the efficacy of antimicrobial treatment without massive autolysis in Firmicutes.

  5. RelE-mediated dormancy is enhanced at high cell density in Escherichia coli.

    Science.gov (United States)

    Tashiro, Yosuke; Kawata, Koji; Taniuchi, Asami; Kakinuma, Kenji; May, Thithiwat; Okabe, Satoshi

    2012-03-01

    Bacteria show remarkable adaptability under several stressful conditions by shifting themselves into a dormant state. Less is known, however, about the mechanism underlying the cell transition to dormancy. Here, we report that the transition to dormant states is mediated by one of the major toxin-antitoxin systems, RelEB, in a cell density-dependent manner in Escherichia coli K-12 MG1655. We constructed a strain, IKA121, which expresses the toxin RelE in the presence of rhamnose and lacks chromosomal relBE and rhaBAD. With this strain, we demonstrated that RelE-mediated dormancy is enhanced at high cell densities compared to that at low cell densities. The initiation of expression of the antitoxin RelB from a plasmid, pCA24N, reversed RelE-mediated dormancy in bacterial cultures. The activation of RelE increased the appearance of persister cells against β-lactams, quinolones, and aminoglycosides, and more persister cells appeared at high cell densities than at low cell densities. Further analysis indicated that amino acid starvation and an uncharacterized extracellular heat-labile substance promote RelE-mediated dormancy. This is a first report on the induction of RelE-mediated dormancy by high cell density. This work establishes a population-based dormancy mechanism to help explain E. coli survival in stressful environments.

  6. HigB of Pseudomonas aeruginosa enhances killing of phagocytes by up-regulating the type III secretion system in ciprofloxacin induced persister cells

    Directory of Open Access Journals (Sweden)

    Mei Li

    2016-10-01

    Full Text Available Bacterial persister cells are dormant and highly tolerant to lethal antibiotics, which are believed to be the major cause of recurring and chronic infections. Activation of toxins of bacterial toxin-antitoxin systems inhibits bacterial growth and plays an important role in persister formation. However, little is known about the overall gene expression profile upon toxin activation. More importantly, how the dormant bacterial persisters evade host immune clearance remains poorly understood. Here we demonstrate that a Pseudomonas aeruginosa toxin-antitoxin system HigB-HigA is required for the ciprofloxacin induced persister formation. Transcriptome analysis of a higA::Tn mutant revealed up regulation of type III secretion systems (T3SS genes. Overexpression of HigB increased the expression of T3SS genes as well as bacterial cytotoxicity. We further demonstrate that wild type bacteria that survived ciprofloxacin treatment contain higher levels of T3SS proteins and display increased cytotoxicity to macrophage compared to vegetative bacterial cells. These results suggest that P. aeruginosa accumulates T3SS proteins during persister formation, which can protect the persister cells from host clearance by efficiently killing host immune cells.

  7. HigB of Pseudomonas aeruginosa Enhances Killing of Phagocytes by Up-Regulating the Type III Secretion System in Ciprofloxacin Induced Persister Cells

    Science.gov (United States)

    Li, Mei; Long, Yuqing; Liu, Ying; Liu, Yang; Chen, Ronghao; Shi, Jing; Zhang, Lu; Jin, Yongxin; Yang, Liang; Bai, Fang; Jin, Shouguang; Cheng, Zhihui; Wu, Weihui

    2016-01-01

    Bacterial persister cells are dormant and highly tolerant to lethal antibiotics, which are believed to be the major cause of recurring and chronic infections. Activation of toxins of bacterial toxin-antitoxin systems inhibits bacterial growth and plays an important role in persister formation. However, little is known about the overall gene expression profile upon toxin activation. More importantly, how the dormant bacterial persisters evade host immune clearance remains poorly understood. Here we demonstrate that a Pseudomonas aeruginosa toxin-antitoxin system HigB-HigA is required for the ciprofloxacin induced persister formation. Transcriptome analysis of a higA::Tn mutant revealed up regulation of type III secretion systems (T3SS) genes. Overexpression of HigB increased the expression of T3SS genes as well as bacterial cytotoxicity. We further demonstrate that wild type bacteria that survived ciprofloxacin treatment contain higher levels of T3SS proteins and display increased cytotoxicity to macrophage compared to vegetative bacterial cells. These results suggest that P. aeruginosa accumulates T3SS proteins during persister formation, which can protect the persister cells from host clearance by efficiently killing host immune cells. PMID:27790409

  8. A Chimeric protein of CFA/I, CS6 subunits and LTB/STa toxoid protects immunized mice against enterotoxigenic Escherichia coli.

    Science.gov (United States)

    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.

  9. Tetanus in the horse: a review of 20 cases (1970 to 1990).

    Science.gov (United States)

    Green, S L; Little, C B; Baird, J D; Tremblay, R R; Smith-Maxie, L L

    1994-01-01

    The case records of 20 horses with tetanus referred to the Ontario Veterinary College-Veterinary Teaching Hospital between 1970 and 1990 were reviewed. The fatality rate was 75%. There was a strong association with previous vaccination and survival (P = .03). Most of the animals had been injured an average of 9 days (range 2 to 21 days) prior to development of clinical signs. Hyperesthesia and prolapse of the third eyelid were the most common clinical signs. Treatment regimens varied during hospitalization; however, all horses received parenteral penicillin, tranquilizers, tetanus toxoid, and antitoxin. Five of the nonsurviving animals were given intrathecal tetanus antitoxin. One animal had seizures as a complication of intrathecal treatment. The prognosis was best for horses that (1) had been vaccinated prior to the injury, (2) responded to the phenothiazine tranquilizers, and (3) did not rapidly (over 24 to 48 hours) become recumbent. Considering the species susceptibility, potential for contaminated wounds, and the increased survival of vaccinated horses, yearly revaccination is recommended.

  10. TETANUS—Prophylaxis and Treatment of the Disease

    Science.gov (United States)

    Ross, Donald E.; Kraut, J. J.

    1959-01-01

    Cleansing and debridement is paramount in dealing with tetanus-prone wounds (severe crushing injuries, piercing wounds, blisters and burns are outstanding examples, particularly if contaminated with dirt, grass or other debris). Prophylaxis then is relatively easy in persons who have been actively immunized by toxoid injections. For them, a “booster” injection is indicated. Use of antitoxin, however, is hazardous, whether for prophylaxis or for treatment of the disease. Since it may in itself cause severe disease, including anaphylactic reaction and serum sickness, decision to use it must be weighed against the possibility of the development of tetanus in each case. To prepare for use of it, careful history should be taken, with particular reference to sensitivity to horse dander. Dermal tests, and perhaps ophthalmic tests, for sensitivity to the serum should be carried out. Even the tests may be hazardous and precautions should be taken accordingly. If it is decided that the use of antitoxin is necessary even though the patient is sensitive to the material, desensitization must be carried out promptly, with adequate preparation for severe reaction. There is experimental evidence that antibiotics of the tetracycline group, given soon after injury, may have prophylactic effect against tetanus. PMID:13651954

  11. Tetanus: prophylaxis and treatment of the disease.

    Science.gov (United States)

    ROSS, D E; KRAUT, J J

    1959-05-01

    Cleansing and debridement is paramount in dealing with tetanus-prone wounds (severe crushing injuries, piercing wounds, blisters and burns are outstanding examples, particularly if contaminated with dirt, grass or other debris). Prophylaxis then is relatively easy in persons who have been actively immunized by toxoid injections. For them, a "booster" injection is indicated. Use of antitoxin, however, is hazardous, whether for prophylaxis or for treatment of the disease. Since it may in itself cause severe disease, including anaphylactic reaction and serum sickness, decision to use it must be weighed against the possibility of the development of tetanus in each case. To prepare for use of it, careful history should be taken, with particular reference to sensitivity to horse dander. Dermal tests, and perhaps ophthalmic tests, for sensitivity to the serum should be carried out. Even the tests may be hazardous and precautions should be taken accordingly. If it is decided that the use of antitoxin is necessary even though the patient is sensitive to the material, desensitization must be carried out promptly, with adequate preparation for severe reaction. There is experimental evidence that antibiotics of the tetracycline group, given soon after injury, may have prophylactic effect against tetanus.

  12. Isolation of bacteriophages and their application to control Pseudomonas aeruginosa in planktonic and biofilm models.

    Science.gov (United States)

    Kwiatek, Magdalena; Parasion, Sylwia; Rutyna, Paweł; Mizak, Lidia; Gryko, Romuald; Niemcewicz, Marcin; Olender, Alina; Łobocka, Małgorzata

    2017-04-01

    Pseudomonas aeruginosa is frequently identified as a cause of diverse infections and chronic diseases. It forms biofilms and has natural resistance to several antibiotics. Strains of this pathogen resistant to new-generation beta-lactams have emerged. Due to the difficulties associated with treating chronic P. aeruginosa infections, bacteriophages are amongst the alternative therapeutic options being actively researched. Two obligatorily lytic P. aeruginosa phages, vB_PaeM_MAG1 (MAG1) and vB_PaeP_MAG4 (MAG4), have been isolated and characterized. These phages belong to the PAK_P1likevirus genus of the Myoviridae family and the LIT1virus genus of the Podoviridae family, respectively. They adsorb quickly to their hosts (∼90% in 5 min), have a short latent period (15 min), and are stable during storage. Each individual phage propagated in approximately 50% of P. aeruginosa strains tested, which increased to 72.9% when phages were combined into a cocktail. While MAG4 reduced biofilm more effectively after a short time of treatment, MAG1 was more effective after a longer time and selected less for phage-resistant clones. A MAG1-encoded homolog of YefM antitoxin of the bacterial toxin-antitoxin system may contribute to the superiority of MAG1 over MAG4. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  13. Complete genome sequence of a commensal bacterium, Hafnia alvei CBA7124, isolated from human feces.

    Science.gov (United States)

    Song, Hye Seon; Kim, Joon Yong; Kim, Yeon Bee; Jeong, Myeong Seon; Kang, Jisu; Rhee, Jin-Kyu; Kwon, Joseph; Kim, Ju Suk; Choi, Jong-Soon; Choi, Hak-Jong; Nam, Young-Do; Roh, Seong Woon

    2017-01-01

    Members of the genus Hafnia have been isolated from the feces of mammals, birds, reptiles, and fish, as well as from soil, water, sewage, and foods. Hafnia alvei is an opportunistic pathogen that has been implicated in intestinal and extraintestinal infections in humans. However, its pathogenicity is still unclear. In this study, we isolated H. alvei from human feces and performed sequencing as well as comparative genomic analysis to better understand its pathogenicity. The genome of H. alvei CBA7124 comprised a single circular chromosome with 4,585,298 bp and a GC content of 48.8%. The genome contained 25 rRNA genes (9 5S rRNA genes, 8 16S rRNA genes, and 8 23S rRNA genes), 88 tRNA genes, and 4043 protein-coding genes. Using comparative genomic analysis, the genome of this strain was found to have 72 strain-specific singletons. The genome also contained genes for antibiotic and antimicrobial resistance, as well as toxin-antitoxin systems. We revealed the complete genome sequence of the opportunistic gut pathogen, H. alvei CBA7124. We also performed comparative genomic analysis of the sequences in the genome of H. alvei CBA7124, and found that it contained strain-specific singletons, antibiotic resistance genes, and toxin-antitoxin systems. These results could improve our understanding of the pathogenicity and the mechanism behind the antibiotic resistance of H. alvei strains.

  14. Difteria: situação imunitária de uma população infantil urbana de São Paulo, SP, Brasil Diphtheria: immunity in an infant population in the city of S. Paulo, SP, Brazil

    Directory of Open Access Journals (Sweden)

    Hideyo Iizuka

    1980-12-01

    Full Text Available A verificação do teor de anticorpos antidiftéricos provenientes de 130 crianças de 7 a 10 anos de idade, do município de São Paulo, Brasil, revelou 31, 14 e 5% de indivíduos susceptíveis nas idades de 7, 8 e 9 anos, respectivamente. Todas as crianças de 10 anos de idade apresentaram proteção contra a difteria, revelando teor de antitoxina circulante em níveis superiores a 0,01 UI/ml. O teor médio de antitoxina diftérica encontrada variou de 0,0385 a 0,1315 UI/ml de soro, na população examinada.An ascertainment of the level diphtheria antibodies in 130 children, 7 to 10 years old, in the city of S. Paulo (Brazil, revealed susceptibility in 31% of the 7-year-olds, 14% in the eight-year-olds, and 5% in the nine-year-olds. All ten-year-olds had protective circulating antitoxin at levels superior to 0.01 IU/ml. Analysis of the results thus showed that susceptibility varies inversely to age. In the population examined, the mean diphtheric antitoxin content oscillated between 0.0385 and 0.1315 IU/ml of serum.

  15. One in five mortality in non-menstrual toxic shock syndrome versus no mortality in menstrual cases in a balanced French series of 55 cases.

    Science.gov (United States)

    Descloux, E; Perpoint, T; Ferry, T; Lina, G; Bes, M; Vandenesch, F; Mohammedi, I; Etienne, J

    2008-01-01

    Staphylococcus aureus superantigenic toxins are responsible for menstrual and non-menstrual toxic shock syndrome (TSS). We compared the clinical and biological characteristics of 21 cases of menstrual TSS (MTSS) with 34 cases of non-menstrual TSS (NMTSS) diagnosed in France from December 2003 to June 2006. All 55 S. aureus isolates had been spontaneously referred to the French National Staphylococcal Reference Center, where they were screened for superantigenic toxin gene sequences. Most of the patients had previously been in good health. The most striking differences between MTSS and NMTSS were the higher frequency in NMTSS of neurological disorders (p=0.028), of S. aureus isolation by blood culture (50% versus 0% in MTSS), and the higher mortality rate in NMTSS (22% versus 0% in MTSS). The tst and sea genes were less frequent in isolates causing NMTSS than in those causing MTSS (p<0.001 and 0.051, respectively). Higher mortality was significantly associated with the presence of the sed gene (p=0.041), but when considering NMTSS survivors and non-survivors, no clinical or bacteriological factors predictive of vital outcome were identified. Specific antitoxinic therapy was rarely prescribed, and never in fatal cases. Higher mortality was observed in NMTSS than in MTSS, and no definite factors could explain the higher severity of NMTSS. NMTSS would require more aggressive therapy, comprising systematic rapid wound debridement, antistaphylococcal agents, including an antitoxin antibiotics, and intravenous immunoglobulin.

  16. Production, biophysical characterization and crystallization of Pseudomonas putida GraA and its complexes with GraT and the graTA operator.

    Science.gov (United States)

    Talavera, Ariel; Tamman, Hedvig; Ainelo, Andres; Hadǽi, San; Garcia-Pino, Abel; Hõrak, Rita; Konijnenberg, Albert; Loris, Remy

    2017-08-01

    The graTA operon from Pseudomonas putida encodes a toxin-antitoxin module with an unusually moderate toxin. Here, the production, SAXS analysis and crystallization of the antitoxin GraA, the GraTA complex and the complex of GraA with a 33 bp operator fragment are reported. GraA forms a homodimer in solution and crystallizes in space group P2 1 , with unit-cell parameters a = 66.9, b = 48.9, c = 62.7 Å, β = 92.6°. The crystals are likely to contain two GraA dimers in the asymmetric unit and diffract to 1.9 Å resolution. The GraTA complex forms a heterotetramer in solution. Crystals of the GraTA complex diffracted to 2.2 Å resolution and are most likely to contain a single heterotetrameric GraTA complex in the asymmetric unit. They belong to space group P4 1 or P4 3 , with unit-cell parameters a = b = 56.0, c = 128.2 Å. The GraA-operator complex consists of a 33 bp operator region that binds two GraA dimers. It crystallizes in space group P3 1 or P3 2 , with unit-cell parameters a = b = 105.6, c = 149.9 Å. These crystals diffract to 3.8 Å resolution.

  17. Nanoparticle-detained toxins for safe and effective vaccination

    Science.gov (United States)

    Hu, Che-Ming J.; Fang, Ronnie H.; Luk, Brian T.; Zhang, Liangfang

    2013-12-01

    Toxoid vaccines--vaccines based on inactivated bacterial toxins--are routinely used to promote antitoxin immunity for the treatment and prevention of bacterial infections. Following chemical or heat denaturation, inactivated toxins can be administered to mount toxin-specific immune responses. However, retaining faithful antigenic presentation while removing toxin virulence remains a major challenge and presents a trade-off between efficacy and safety in toxoid development. Here, we show a nanoparticle-based toxin-detainment strategy that safely delivers non-disrupted pore-forming toxins for immune processing. Using erythrocyte membrane-coated nanoparticles and staphylococcal α-haemolysin, we demonstrate effective virulence neutralization via spontaneous particle entrapment. Compared with vaccination with heat-denatured toxin, mice vaccinated with the nanoparticle-detained toxin showed superior protective immunity against toxin-mediated adverse effects. We find that the non-disruptive detoxification approach benefited the immunogenicity and efficacy of toxoid vaccines. We anticipate that this study will open new possibilities in the preparation of antitoxin vaccines against the many virulence factors that threaten public health.

  18. Global occurrence and heterogeneity of the Roseobacter-clade species Ruegeria mobilis

    DEFF Research Database (Denmark)

    Sonnenschein, Eva; Nielsen, Kristian Fog; D'Alvise, Paul

    2017-01-01

    environment. We isolated 42 TDA-producing R. mobilis strains during a global marine research cruise. While highly similar on the 16S ribosomal RNA gene level (99–100% identity), the strains separated into four sub-clusters in a multilocus sequence analysis. They were further differentiated to the strain level...... by average nucleotide identity using pairwise genome comparison. The four sub-clusters could not be associated with a specific environmental niche, however, correlated with the pattern of sub-typing using co-isolated phages, the number of prophages in the genomes and the distribution in ocean provinces....... Major genomic differences within the sub-clusters include prophages and toxin-antitoxin systems. In general, the genome of R. mobilis revealed adaptation to a particle-associated life style and querying TARA ocean data confirmed that R. mobilis is more abundant in the particle-associated fraction than...

  19. Generalised tetanus in a 2-week-old foal: use of physiotherapy to aid recovery.

    Science.gov (United States)

    Mykkänen, A K; Hyytiäinen, H K; McGowan, C M

    2011-11-01

    A 2-week-old Estonian Draft foal presented with signs of severe generalised tetanus, recumbency and inability to drink. The suspected source of infection was the umbilicus. Medical treatment was administered, including tetanus antitoxin, antimicrobial therapy and phenobarbital to control tetanic spasms. In addition, an intensive physiotherapy program was carried out during the recovery period. Techniques designed for syndromes involving upper motor neuron spasticity in humans were applied. Exercises aimed at weight-bearing and mobility were executed with the help of a walking-frame. The foal made a complete recovery. To our knowledge, this is the first report of the use of physiotherapy in the treatment of tetanus in horses. © 2011 The Authors. Australian Veterinary Journal © 2011 Australian Veterinary Association.

  20. Biological and molecular properties of yellow venom of the Amazonian coral snake Micrurus surinamensis.

    Science.gov (United States)

    Oliveira, Fabiana da Rocha; Noronha, Maria das Dores Nogueira; Lozano, Jorge Luis Lopez

    2017-01-01

    The coral snake Micrurus surinamensis, which is widely distributed throughout Amazonia, has a neurotoxic venom. It is important to characterize the biological and molecular properties of this venom in order to develop effective antitoxins. Toxins from the venom of M. surinamensis were analyzed by two-dimensional polyacrylamide gel electrophoresis and their neurotoxic effects in vivo were evaluated. Most proteins in the venom had masses < 14kDa, low phospholipase A2 activity, and no proteolytic activity. The toxins inhibited the coagulation cascade. The venom had neurotoxic effects in mice, with a median lethal dose upon intravenous administration of 700 µg/kg. Immunogenic studies revealed abundant cross-reactivity of antielapidic serum with 14kDa toxins and limited cross-reactivity with toxins < 10kDa. These results indicate that antielapidic serum against M. surinamensis venom has weak potency (0.35mg/ml) in mice.

  1. Immunity to tetanus and diphtheria in rural Africa

    DEFF Research Database (Denmark)

    Kurtzhals, J A; Kjeldsen, K; Hey, A S

    1997-01-01

    To assess the effect of the Expanded Program on Immunization (EPI) in rural Africa, blood samples were collected in two Kenyan sublocations. Serum antibodies against tetanus toxoid were measured in 155 individuals 1-70 years of age. Titers greater than the protective level of 0.01 IU/ml were found...... in 47% of the population. Protection was significantly higher in children born after the launching of the EPI (68%) and in women who had been at childbearing age since then (69%). Significantly lower protection was demonstrated in other age and sex-groups. The level of protection in children was equal...... in the two populations, whereas protection in fertile women was significantly lower in the population living a long distance from a health center. Diphtheria anti-toxin was measured in the samples from one sublocation, and 70 of 84 individuals (83%) had antibody levels greater than the protective level...

  2. High Persister Mutants in Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Heather L Torrey

    Full Text Available Mycobacterium tuberculosis forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in M. tuberculosis is not well understood. In this study, we selected for high persister (hip mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the hip mutants obtained in vitro with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical hip isolates exhibited greater ex vivo survival than the low persister isolates. Our data suggest that M. tuberculosis persister formation involves multiple pathways, and hip mutants may contribute to the recalcitrance of the infection.

  3. Transcriptome changes in STSV2-infected Sulfolobus islandicus REY15A undergoing continuous CRISPR spacer acquisition

    DEFF Research Database (Denmark)

    León-Sobrino, Carlos; Kot, Witold P; Garrett, Roger A

    2016-01-01

    A transcriptome study was performed on Sulfolobus islandicus REY15A actively undergoing CRISPR spacer acquisition from the crenarchaeal monocaudavirus STSV2 in rich and basal media over a 6 day period. Spacer acquisition preceded strong host growth retardation, altered transcriptional activity...... of four different CRISPR-Cas modules and changes in viral copy numbers, and with significant differences in the two media. Transcript levels of proteins involved in the cell cycle were reduced, while those of DNA replication, DNA repair, transcriptional regulation, and some antitoxin-toxin pairs...... and transposases were unchanged or enhanced. Antisense RNAs were implicated in the transcriptional regulation of adaptation and interference modules of the type I-A CRISPR-Cas system and evidence was found for the occurrence of functional coordination between the single CRISPR-Cas adaptation module...

  4. Diphtheria, pertussis, and tetanus: evidence-based management of pediatric patients in the emergency department [digest].

    Science.gov (United States)

    Zibners, Lara; Chaudhari, Pradip

    2017-02-22

    Diphtheria, pertussis, and tetanus are potentially deadly bacterial infections that are largely preventable through vaccination, though they remain in the population. This issue reviews the epidemiology, pathophysiology, diagnosis, and current recommended emergency management of these conditions. Disease-specific medications, as well as treatment of the secondary complications, are examined in light of the best current evidence. Resources include obtaining diphtheria antitoxin from the United States Centers for Disease Control and Prevention and best-practice recommendations with regard to testing, involvement of government health agencies, isolation of the patient, and identification and treatment of close contacts. Most importantly, issues regarding vaccination and prevention are highlighted. [Points & Pearls is a digest of Pediatric Emergency Medicine Practice].

  5. [Study of the receptor for black widow spider neurotoxin. I. Characteristics of membrane-bound and solubilized receptors from the bovine brain].

    Science.gov (United States)

    Petrenko, A G; Shamotienko, O G; Surkova, I N; Kovalenko, V A; Grishin, E V

    1990-02-01

    Iodine-125 labelled alpha-latrotoxin from the venom of Central Asia black widow spider Latrodectus mactans tredecimguttatus binds specifically to the bovine brain membrane receptor producing a stable slowly dissociating complex with Kd = 1.6 x 10(-10) M and Bmax = 0.5 pmol/mg protein. Treatment of the complex with alkaline high-salt buffer induces reversible dissociation of the bound toxin. The antitoxin polyclonal antibody does not increase the dissociation rate of the bound toxin. Wheat germ lectin as well as concanavalin A inhibit the toxin binding to the membrane receptor. The receptor is solubilized with ionic and non-ionic detergents, and methods of latrotoxin binding assay are developed. The solubilized receptor is shown to retain high affinity to toxin, its binding activity being stable but critically dependent on the presence of calcium ions. Chromatographic properties of the receptor suggest its glycoprotein nature.

  6. High prevalence of subclass-specific binding and neutralizing antibodies against Clostridium difficile toxins in adult cystic fibrosis sera: possible mode of immunoprotection against symptomatic C. difficile infection

    Directory of Open Access Journals (Sweden)

    Monaghan TM

    2017-07-01

    Full Text Available Tanya M Monaghan,1 Ola H Negm,2,3 Brendon MacKenzie,4 Mohamed R Hamed,2,3 Clifford C Shone,5 David P Humphreys,4 K Ravi Acharya,6 Mark H Wilcox7 1Nottingham Digestive Diseases Centre, NIHR Nottingham Digestive Diseases Biomedical Research Unit, School of Medicine, University of Nottingham, Nottingham, 2Breast Surgery Group, Division of Medical Sciences and Graduate Entry Medicine, School of Medicine, Queen’s Medical Centre, University of Nottingham, Nottingham, UK; 3Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt; 4Antibody Biology, UCB-New Medicines, UCB Celltech, Slough, UK; 5Toxins Group, National Infection Service, Public Health England, Salisbury, UK; 6Department of Biology and Biochemistry, University of Bath, Bath, UK; 7Leeds Institute of Biomedical and Clinical Sciences, University of Leeds, Leeds, UK Objectives: Despite multiple risk factors and a high rate of colonization for Clostridium difficile, the occurrence of C. difficile infection in patients with cystic fibrosis is rare. The aim of this study was to compare the prevalence of binding C. difficile toxin-specific immunoglobulin (IgA, IgG and anti-toxin neutralizing antibodies in the sera of adults with cystic fibrosis, symptomatic C. difficile infection (without cystic fibrosis and healthy controls. Methods: Subclass-specific IgA and IgG responses to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087, toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309 and precursor form of B fragment of binary toxin, pCDTb, were determined by protein microarray. Neutralizing antibodies to C. difficile toxins A and B were evaluated using a Caco-2 cell-based neutralization assay. Results: Serum IgA anti-toxin A and B levels and neutralizing antibodies against toxin A were significantly higher in adult cystic fibrosis patients (n=16 compared with healthy controls (n=17 and

  7. Autoproteolytic Activation of Bacterial Toxins

    Directory of Open Access Journals (Sweden)

    Aimee Shen

    2010-05-01

    Full Text Available Protease domains within toxins typically act as the primary effector domain within target cells. By contrast, the primary function of the cysteine protease domain (CPD in Multifunctional Autoprocessing RTX-like (MARTX and Clostridium sp. glucosylating toxin families is to proteolytically cleave the toxin and release its cognate effector domains. The CPD becomes activated upon binding to the eukaryotic-specific small molecule, inositol hexakisphosphate (InsP6, which is found abundantly in the eukaryotic cytosol. This property allows the CPD to spatially and temporally regulate toxin activation, making it a prime candidate for developing anti-toxin therapeutics. In this review, we summarize recent findings related to defining the regulation of toxin function by the CPD and the development of inhibitors to prevent CPD-mediated activation of bacterial toxins.

  8. Role of Homologous Fc Fragment in the Potency and Efficacy of Anti‐Botulinum Antibody Preparations

    Directory of Open Access Journals (Sweden)

    Amram Torgeman

    2017-05-01

    Full Text Available The only approved treatment for botulism relies on passive immunity which is mostly based on antibody preparations collected from hyper‐immune horses. The IgG Fc fragment is commonly removed from these heterologous preparations to reduce the incidence of hyper‐sensitivity reactions. New‐generation therapies entering the pipeline are based on a combination of humanized monoclonal antibodies (MAbs, which exhibit improved safety and pharmacokinetics. In the current study, a systematic and quantitative approach was applied to measure the direct contribution of homologous Fc to the potency of monoclonal and polyclonal antitoxin preparations in mice. Homologous Fc increased the potency of three individual anti‐botulinum toxin MAbs by up to one order of magnitude. Moreover, Fc fragment removal almost completely abolished the synergistic potency obtained from a combined preparation of these three MAbs. The MAb mixture neutralized a 400‐mouse median lethal dose (MsLD50 of botulinum toxin, whereas the F(ab′2 combination failed to neutralize 10 MsLD50 of botulinum toxin. Notably, increased avidity did not compensate for this phenomenon, as a polyclonal, hyper‐immune, homologous preparation lost 90% of its potency as well upon Fc removal. Finally, the addition of homologous Fc arms to a heterologous pharmaceutical anti‐botulinum toxin polyclonal horse F(ab′2 preparation improved its efficacy when administered to intoxicated symptomatic mice. Our study extends the aspects by which switching from animal‐based to human‐based antitoxins will improve not only the safety but also the potency and efficacy of passive immunity against toxins.

  9. Immunogenicity of a low-dose diphtheria, tetanus and acellular pertussis combination vaccine with either inactivated or oral polio vaccine compared to standard-dose diphtheria, tetanus, acellular pertussis when used as a pre-school booster in UK children: A 5-year follow-up of a randomised controlled study.

    Science.gov (United States)

    John, T; Voysey, M; Yu, L M; McCarthy, N; Baudin, M; Richard, P; Fiquet, A; Kitchin, N; Pollard, A J

    2015-08-26

    This serological follow up study assessed the kinetics of antibody response in children who previously participated in a single centre, open-label, randomised controlled trial of low-dose compared to standard-dose diphtheria booster preschool vaccinations in the United Kingdom (UK). Children had previously been randomised to receive one of three combination vaccines: either a combined adsorbed tetanus, low-dose diphtheria, 5-component acellular pertussis and inactivated polio vaccine (IPV) (Tdap-IPV, Repevax(®); Sanofi Pasteur MSD); a combined adsorbed tetanus, low-dose diphtheria and 5-component acellular pertussis vaccine (Tdap, Covaxis(®); Sanofi Pasteur MSD) given concomitantly with oral polio vaccine (OPV); or a combined adsorbed standard-dose diphtheria, tetanus, 2-component acellular pertussis and IPV (DTap-IPV, Tetravac(®); Sanofi Pasteur MSD). Blood samples for the follow-up study were taken at 1, 3 and 5 years after participation in the original trial (median, 5.07 years of age at year 1), and antibody persistence to each vaccine antigen measured against defined serological thresholds of protection. All participants had evidence of immunity to diphtheria with antitoxin concentrations greater than 0.01IU/mL five years after booster vaccination and 75%, 67% and 79% of children who received Tdap-IPV, Tdap+OPV and DTap-IPV, respectively, had protective antitoxin levels greater than 0.1IU/mL. Long lasting protective immune responses to tetanus and polio antigens were also observed in all groups, though polio responses were lower in the sera of those who received OPV. Low-dose diphtheria vaccines provided comparable protection to the standard-dose vaccine and are suitable for use for pre-school booster vaccination. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Development of a flow cytometric bead immunoassay and its assessment as a possible aid to potency evaluation of enterotoxaemia vaccines

    Directory of Open Access Journals (Sweden)

    Angela Buys

    2014-03-01

    Full Text Available Enterotoxaemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens type D. The only practical means of controlling the occurrence of enterotoxaemia is to immunise animals by vaccination. The vaccine is prepared by deriving a toxoid from the bacterial culture filtrate and the potency of the vaccine is tested with the in vivo mouse neutralisation test (MNT. Due to ethical, economic and technical reasons, alternative in vitro assays are needed. In this study an indirect cytometric bead immunoassay (I-CBA was developed for use in vaccine potency testing and the results were compared with those obtained using an indirect enzyme-linked immunosorbent assay (I-ELISA and the MNT. Sera were collected from guinea pigs immunised with three different production batches of enterotoxaemia vaccine and the levels of anti-epsilon toxin antibodies were determined. Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels determined by both the I-ELISA and indirect cytometric bead immunoassay (I-CBA tests were higher than those of the MNT assay. In contrast to the MNT, all of the serum samples were identified as having antitoxin levels above the required minimum (not less than 5 U/mL. These results indicate that the respective in vitro tests in their current formats are not yet suitable alternatives to the in vivo MNT. The growing demand for a more humane, cost-effective and efficient method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus for further optimisation and standardisation of the I-CBA assay but further analytical research is required.

  11. Passive antibodies derived from intramuscularly immunized toxoid fusion 3xSTaN12S-dmLT protect against STa+ enterotoxigenic Escherichia coli (ETEC) diarrhea in a pig model.

    Science.gov (United States)

    Nandre, Rahul M; Duan, Qiangde; Wang, Yin; Zhang, Weiping

    2017-01-23

    Enterotoxigenic Escherichia coli (ETEC) strains are among the most common causes of children's diarrhea and travelers' diarrhea. Developing effective vaccines against ETEC associated diarrhea becomes a top priority. ETEC heat-labile toxin (LT) and heat-stable toxin (STa) toxoid fusion 3xSTa N12S -dmLT was demonstrated recently to induce neutralizing antitoxin antibodies in intraperitoneally or subcutaneously immunized mice. However, whether antibodies derived from this toxoid fusion are protective against ETEC diarrhea has not been examined. In this study, we intramuscularly immunized pregnant gilts with toxoid fusion 3xSTa N12S -dmLT, challenged suckling piglets with a STa-positive ETEC strain, and assessed protective efficacy of passive acquire antitoxin antibodies against ETEC diarrhea. Data showed all three immunized gilts developed anti-STa IgG and IgA antibodies, and piglets born to the immunized dams acquired anti-STa and anti-LT antibodies. When challenged with a STa+ ETEC strain, none of the piglets born to the immunized dams developed watery diarrhea, with 20 piglets remained normal and the other 8 piglets developed mild diarrhea indicated with stained butt. In contrast, the control dams and born piglets had no anti-STa or anti-LT antibodies detected, and 26 out 32 piglets developed watery diarrhea after challenge of the STa+ ETEC strain. These results indicated that passive acquired anti-STa antibodies are protective against ETEC diarrhea, and suggested potential application of toxoid fusion 3xSTa N12S -dmLT in ETEC vaccine development. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Treatment of hyperphosphatemia, secondary hyperparathyroidism and hypocalcemia with calcium carbonate favorably modulates vaccination response in uremic rats.

    Science.gov (United States)

    Hannula, P M; Pörsti, I H; Saha, H H T; Kalliovalkama, J; Jolma, P M; Kööbi, P; Olander, R M; Antonen, J A

    2004-06-01

    Immune dysfunction is characteristic of renal failure, leading to suboptimal antibody generation and increased susceptibility to infections. We tested whether the treatment of uremic phosphate retention by increased calcium carbonate intake will beneficially influence vaccination response in 5/6-nephrectomized rats. The nephrectomized (uremic) and sham-operated (control) rats were either fed 0.3% calcium diet (NTX and Sham groups, respectively) or 3% high-calcium diet (Ca-NTX and Ca-Sham groups). All rats were immunized with tetanus toxoid 6 weeks after the operations, and antitoxin levels were measured 7 weeks later. Plasma creatinine was significantly elevated after the nephrectomy: the values (mean +/- SD) in the NTX (n = 16), Ca-NTX (n = 11), Sham (n = 14) and Ca-Sham (n = 8) groups were 97 +/- 14, 93 +/- 17, 66 +/- 7, and 69 +/- 8 micromol/l, respectively. The NTX group developed phosphate retention and secondary hyperparathyroidism, which were completely prevented by the high calcium diet. The mean tetanus antitoxin concentrations of the groups were: NTX 0.25 +/- 0.32; Ca-NTX 0.45 +/- 0.44; Sham 0.58 +/- 0.24 and Ca-Sham 0.64 +/- 0.25 IU/ml (log of geometric mean concentration). The antibody response in the NTX group was significantly lower, i.e. 43% of that in the Sham group (p = 0.003), while the response in the Ca-NTX group was not different from that in the Sham group. The tetanus response of all the uremic rats inversely correlated with the plasma levels of phosphate (r = 0.447, p = 0.02), parathormone (r = -0.409, p = 0.03) and creatinine (r = 0.578, p = 0.002). We conclude that renal failure impairs vaccination response in rats, the impairment of which can be favorably modulated by phosphate-binding and PTH-suppressing high-calcium diet.

  13. Efficacy demonstration of tetanus vaccines by double antigen ELISA.

    Science.gov (United States)

    Rosskopf, U; Noeske, K; Werner, E

    2005-09-01

    This paper describes a double antigen ELISA (DAE) for rapid, specific and reliable assessment of the antitetanus immune status of horses and sheep. Compared with the indirect ELISA, the double antigen ELISA has the advantage of species-independent testing of sera. Thanks to its test design, it is more specific since the detected antibodies are forced to bind tetanus toxoid twice. In addition, it is very sensitive to tetanus antibodies, enabling the detection of low antibody titres, in range which is relevant for the assessment of the protective status (tetanus toxin neutralising antibodies). The detection limit of the DAE for tetanus antibodies is in the order of 10(-4) EU/ml. A comparison of in vitro results of individual sera with in vivo titres showed that horse sera with titres of 0.04 and 0.05 EU/ml in the DAE showed titres of > 0.05 IU and 0.034 IU/ml respectively during in vivo testing thus indicating good agreement. For tested sheep sera which were rated > 0.05 IU/ml in vivo, the corresponding titre in the DAE was 0.24 EU/ml. Clear tetanus antitoxin establishment of protective ELISA limits requires further comparative examination of sera with low titres (tetanus vaccines ad us. vet. As a consequence, the toxin neutralisation test (still being the standard method of choice for quantifying tetanus toxin neutralising antitoxin titres) could be replaced, since it requires too great a number of animals per test and involves considerable suffering for the animals. The test described here reduces the use of mice and guinea pigs within vaccine efficacy testing. In addition, it involves less exposure of the laboratory personnel to toxin.

  14. Retrospective evaluation of 155 adult equids and 21 foals with tetanus in Western, Northern, and Central Europe (2000-2014). Part 1: Description of history and clinical evolution.

    Science.gov (United States)

    van Galen, Gaby; Saegerman, Claude; Rijckaert, Joke; Amory, Helene; Armengou, Lara; Bezdekova, Barbora; Durie, Inge; Findshøj Delany, Rikke; Fouché, Nathalie; Haley, Laura; Hewetson, Michael; van den Hoven, Rene; Kendall, Anna; Malalana, Fernando; Muller Cavalleri, Jessika; Picavet, Tresemiek; Roscher, Katja; Verwilghen, Denis; Wehrli Eser, Meret; Westermann, Cornélie; Mair, Tim

    2017-11-01

    To describe clinical data of hospitalized adult equids and foals with tetanus. Multicenter retrospective study (2000-2014). Twenty Western, Northern, and Central European university teaching hospitals and private referral centers. One hundred fifty-five adult equids (>6 months) and 21 foals (tetanus. None. Information on geographic, annual and seasonal data, demographic- and management-related data, clinical history, clinical examination and blood analysis on admission, complications, treatments, and outcomes were described and statistically compared between adults and foals. The described cases were often young horses. In 4 adult horses, tetanus developed despite appropriate vaccination and in 2 foals despite preventive tetanus antitoxin administration at birth. Castration, hoof abscesses, and wounds were the most common entry sites for adults; umbilical cord infections and wounds for foals. Stiffness was the commonest observed initial clinical sign. Blood analyses frequently revealed an inflammatory response, hemoconcentration, muscle damage, azotemia, negative energy balance, liver damage, and electrolyte and acid base disturbances. Common complications or clinical signs developing during hospitalization included dysphagia, dyspnea, recumbency, hyperthermia, seizures, hyperlipemia, gastrointestinal impactions, dysuria, and laryngeal spasms. Cases were supported with wound debridement, antimicrobial treatment, tetanus antitoxin, muscle spasm and seizure control, analgesia, anti-inflammatory drugs, fluid therapy, and nutritional support. Mortality rates were 68.4% in adult horses and 66.7% in foals. Foals differed from adult horses with respect to months of occurrence, signalment, management-related data, potential causative events, clinical signs on admission, blood analysis, complications, and severity grades. This is the first study that rigorously describes a large population of equids affected by tetanus. The information provided is potentially useful to

  15. Defense Islands in Bacterial and Archaeal Genomes and Prediction of Novel Defense Systems ▿†‡

    Science.gov (United States)

    Makarova, Kira S.; Wolf, Yuri I.; Snir, Sagi; Koonin, Eugene V.

    2011-01-01

    The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic “sinks” that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands. PMID:21908672

  16. Defense islands in bacterial and archaeal genomes and prediction of novel defense systems.

    Science.gov (United States)

    Makarova, Kira S; Wolf, Yuri I; Snir, Sagi; Koonin, Eugene V

    2011-11-01

    The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic "sinks" that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands.

  17. Long-Term Protection against Diphtheria in the Netherlands after 50 Years of Vaccination: Results from a Seroepidemiological Study.

    Science.gov (United States)

    Swart, E M; van Gageldonk, P G M; de Melker, H E; van der Klis, F R; Berbers, G A M; Mollema, L

    2016-01-01

    To evaluate the National Immunisation Programme (NIP) a population-based cross-sectional seroepidemiological study was performed in the Netherlands. We assessed diphtheria antitoxin levels in the general Dutch population and in low vaccination coverage (LVC) areas where a relatively high proportion of orthodox Protestants live who decline vaccination based on religious grounds. Results were compared with a nationwide seroepidemiological study performed 11 years earlier. In 2006/2007 a national serum bank was established. Blood samples were tested for diphtheria antitoxin IgG concentrations using a multiplex immunoassay for 6383 participants from the national sample (NS) and 1518 participants from LVC municipalities. A cut-off above 0.01 international units per ml (IU/ml) was used as minimum protective level. In the NS 91% of the population had antibody levels above 0.01 IU/ml compared to 88% in the 1995/1996 serosurvey (pdiphtheria vaccination in the NIP and 46% (vs. 37% in the 1995/1996 serosurvey, p = 0.11) of orthodox Protestants living in LVC areas had antibody levels above 0.01 IU/ml. Linear regression analysis among fully immunized individuals (six vaccinations) without evidence of revaccination indicated a continuous decline in antibodies in both serosurveys, but geometric mean antibodies remained well above 0.01 IU/ml in all age groups. The NIP provides long-term protection against diphtheria, although antibody levels decline after vaccination. As a result of natural waning immunity, a substantial proportion of individuals born before introduction of diphtheria vaccination in the NIP lack adequate levels of diphtheria antibodies. Susceptibility due to lack of vaccination is highest among strictly orthodox Protestants. The potential risk of spread of diphtheria within the geographically clustered orthodox Protestant community after introduction in the Netherlands has not disappeared, despite national long-term high vaccination coverage.

  18. Sequencing and phylogenetic analysis of neurotoxin gene from an environmental isolate of Clostridium sp.: comparison with other clostridial neurotoxins.

    Science.gov (United States)

    Dixit, Aparna; Alam, Syed Imteyaz; Singh, Lokendra

    2006-07-01

    A Clostridium sp. isolated from intestine of decaying fish exhibited 99% sequence identity with C. tetani at 16S rRNA level. It produced a neurotoxin that was neutralized by botulinum antitoxin (A+B+E) as well as tetanus antitoxin. The gene fragments for light chain, C-terminal and N-terminal regions of the heavy chain of the toxin were amplified using three reported primer sets for tetanus neurotoxin (TeNT). The neurotoxin gene fragments were cloned in Escherichia coli and sequenced. The sequences obtained exhibited approximately 98, 99 and 98% sequence identity with reported gene sequences of TeNT/LC, TeNT/HC and TeNT/HN, respectively. The phylogenetic interrelationship between the neurotoxin gene of Clostridium sp. with previously reported gene sequences of Clostridium botulinum A to G and C. tetani was examined by analysis of differences in the nucleotide sequences. Six amino acids were substituted at four different positions in the light chain of neurotoxin from the isolate when compared with the reported closest sequence of TeNT. Of these, four were located in the beta15 motif at a solvent inaccessible, buried region of the protein molecule. One of these substitutions were on the solvent accessible surface residue of alpha1 motif, previously shown to have strong sequence conservation. A substitution of two amino acids observed in N-terminal region of heavy chain were buried residues, located in the beta21 and beta37 motifs showing variability in other related sequences. The C-terminal region responsible for binding to receptor was conserved, showing no changes in the amino acid sequence.

  19. Purification and crystallization of Vibrio fischeri CcdB and its complexes with fragments of gyrase and CcdA

    Energy Technology Data Exchange (ETDEWEB)

    De Jonge, Natalie, E-mail: ndejonge@vub.ac.be; Buts, Lieven; Vangelooven, Joris [Department of Molecular and Cellular Interactions, VIB, Pleinlaan 2, 1050 Brussels (Belgium); Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels (Belgium); Mine, Natacha; Van Melderen, Laurence [Laboratoire de Génétique des Procaryotes, Institut de Biologie et de Médecine, Université Libre de Bruxelles, Gosselies (Belgium); Wyns, Lode; Loris, Remy [Department of Molecular and Cellular Interactions, VIB, Pleinlaan 2, 1050 Brussels (Belgium); Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels (Belgium)

    2007-04-01

    A CcdB homologue from V. fischeri was overexpressed in E. coli and purified. The free protein was crystallized, as were its complexes with fragments of E. coli and V. fischeri gyrase and with the F-plasmid CcdA C-terminal domain. The ccd toxin–antitoxin module from the Escherichia coli F plasmid has a homologue on the Vibrio fischeri integron. The homologue of the toxin (CcdB{sub Vfi}) was crystallized in two different crystal forms. The first form belongs to space group I23 or I2{sub 1}3, with unit-cell parameter a = 84.5 Å, and diffracts to 1.5 Å resolution. The second crystal form belongs to space group C2, with unit-cell parameters a = 58.5, b = 43.6, c = 37.5 Å, β = 110.0°, and diffracts to 1.7 Å resolution. The complex of CcdB{sub Vfi} with the GyrA14{sub Vfi} fragment of V. fischeri gyrase crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.5, b = 94.6, c = 58.1 Å, and diffracts to 2.2 Å resolution. The corresponding mixed complex with E. coli GyrA14{sub Ec} crystallizes in space group C2, with unit-cell parameters a = 130.1, b = 90.8, c = 58.1 Å, β = 102.6°, and diffracts to 1.95 Å. Finally, a complex between CcdB{sub Vfi} and part of the F-plasmid antitoxin CcdA{sub F} crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.9, b = 62.6, c = 82.0 Å, and diffracts to 1.9 Å resolution.

  20. ACTIVE IMMUNIZATION OF GUINEA PIGS WITH THE VIRUS OF EQUINE ENCEPHALOMYELITIS

    Science.gov (United States)

    Cox, Herald R.; Olitsky, Peter K.

    1936-01-01

    not be the direct antigenic stimulus but that some substance on which it acts and which becomes liberated from infected cells may be the factor responsible. While this subject awaits the results of further study, we believe that formalin inactivates the infective agent in virus suspensions and preserves the antigenic component therein, whatever its nature may be. It would be of interest if this phenomenon of prevention of antigenic capacity by proper amounts of immune serum might apply to such materials which by their very nature do not multiply in the body of the host, e.g., toxin and antitoxin. Theobald Smith (8) and later Park (9) demonstrated that in mixtures of diphtheria toxin-antitoxin, when smaller amounts of immune serum (antitoxin) are used, the toxicity of the mixture is retained and immunity results; if the serum is increased, toxicity is reduced and immunity occurs irregularly, and if more serum is added, no toxicity nor immunity results. This is supported by the experiments of Hartley (10) on washed precipitates from underneutralized, neutral, and overneutralized mixtures of antitoxin and toxin: those derived from underneutralized material are toxic and powerfully antigenic; those from neutralized, atoxic and of good antigenic action, and from overneutralized, atoxic and of low antigenicity. Hartley states, moreover, that the precipitate reactions of toxicity and antigenicity bear a close relationship to the nature of the mixture from which they are produced. There is, therefore, a connection between the preventive reactions of the serum on the two forms of virus and of antitoxin on toxin in respect to toxicity and antigenicity. Furthermore, the toxin is rendered atoxic with retention of immunizing capacity by formalin: the production of toxoid or anatoxin (Glenny and Hopkins (11), Ramon (12))—again a condition related to the effects following formolization of the virus. It has, however, been stated that "in an immunizing mixture prepared with modified

  1. Antibodies derived from a toxoid MEFA (multiepitope fusion antigen) show neutralizing activities against heat-labile toxin (LT), heat-stable toxins (STa, STb), and Shiga toxin 2e (Stx2e) of porcine enterotoxigenic Escherichia coli (ETEC).

    Science.gov (United States)

    Rausch, Dana; Ruan, Xiaosai; Nandre, Rahul; Duan, Qiangde; Hashish, Emad; Casey, Thomas A; Zhang, Weiping

    2017-04-01

    Enterotoxigenic Escherichia coli (ETEC) strains are the main cause of diarrhea in pigs. Pig diarrhea especially post-weaning diarrhea remains one of the most important swine diseases. ETEC bacterial fimbriae including K88, F18, 987P, K99 and F41 promote bacterial attachment to intestinal epithelial cells and facilitate ETEC colonization in pig small intestine. ETEC enterotoxins including heat-labile toxin (LT) and heat-stable toxins type Ia (porcine-type STa) and type II (STb) stimulate fluid hyper-secretion, leading to watery diarrhea. Blocking bacteria colonization and/or neutralizing enterotoxicity of ETEC toxins are considered effective prevention against ETEC diarrhea. In this study, we applied the MEFA (multiepitope fusion antigen) strategy to create toxoid MEFAs that carried antigenic elements of ETEC toxins, and examined for broad antitoxin immunogenicity in a murine model. By embedding STa toxoid STa P12F (NTFYCCELCCNFACAGCY), a STb epitope (KKDLCEHY), and an epitope of Stx2e A subunit (QSYVSSLN) into the A1 peptide of a monomeric LT toxoid (LT R192G ), two toxoid MEFAs, 'LT R192G -STb-Stx2e-STa P12F ' and 'LT R192G -STb-Stx2e-3xSTa P12F ' which carried three copies of STa P12F , were constructed. Mice intraperitoneally immunized with each toxoid MEFA developed IgG antibodies to all four toxins. Induced antibodies showed in vitro neutralizing activities against LT, STa, STb and Stx2e toxins. Moreover, suckling piglets born by a gilt immunized with 'LT R192G -STb-Stx2e-3xSTa P12F ' were protected when challenged with ETEC strains, whereas piglets born by a control gilt developed diarrhea. Results from this study showed that the toxoid MEFA induced broadly antitoxin antibodies, and suggested potential application of the toxoid MEFA for developing a broad-spectrum vaccine against ETEC diarrhea in pigs. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Contribution of the Chromosomal ccdAB Operon to Bacterial Drug Tolerance.

    Science.gov (United States)

    Gupta, Kritika; Tripathi, Arti; Sahu, Alishan; Varadarajan, Raghavan

    2017-10-01

    One of the first identified and best-studied toxin-antitoxin (TA) systems in Escherichia coli is the F-plasmid-based CcdAB system. This system is involved in plasmid maintenance through postsegregational killing. More recently, ccdAB homologs have been found on the chromosome, including in pathogenic strains of E. coli and other bacteria. However, the functional role of chromosomal ccdAB genes, if any, has remained unclear. We show that both the native ccd operon of the E. coli O157 strain ( ccd O157 ) and the ccd operon from the F plasmid ( ccd F ), when inserted on the E. coli chromosome, lead to protection from cell death under multiple antibiotic stress conditions through formation of persisters, with the O157 operon showing higher protection. While the plasmid-encoded CcdB toxin is a potent gyrase inhibitor and leads to bacterial cell death even under fully repressed conditions, the chromosomally encoded toxin leads to growth inhibition, except at high expression levels, where some cell death is seen. This was further confirmed by transiently activating the chromosomal ccd operon through overexpression of an active-site inactive mutant of F-plasmid-encoded CcdB. Both the ccd F and ccd O157 operons may share common mechanisms for activation under stress conditions, eventually leading to multidrug-tolerant persister cells. This study clearly demonstrates an important role for chromosomal ccd systems in bacterial persistence. IMPORTANCE A large number of free-living and pathogenic bacteria are known to harbor multiple toxin-antitoxin systems, on plasmids as well as on chromosomes. The F-plasmid CcdAB system has been extensively studied and is known to be involved in plasmid maintenance. However, little is known about the function of its chromosomal counterpart, found in several pathogenic E. coli strains. We show that the native chromosomal ccd operon of the E. coli O157 strain is involved in drug tolerance and confers protection from cell death under multiple

  3. Comparative metagenomic analysis of plasmid encoded functions in the human gut microbiome

    Directory of Open Access Journals (Sweden)

    Marchesi Julian R

    2010-01-01

    Full Text Available Abstract Background Little is known regarding the pool of mobile genetic elements associated with the human gut microbiome. In this study we employed the culture independent TRACA system to isolate novel plasmids from the human gut microbiota, and a comparative metagenomic analysis to investigate the distribution and relative abundance of functions encoded by these plasmids in the human gut microbiome. Results Novel plasmids were acquired from the human gut microbiome, and homologous nucleotide sequences with high identity (>90% to two plasmids (pTRACA10 and pTRACA22 were identified in the multiple human gut microbiomes analysed here. However, no homologous nucleotide sequences to these plasmids were identified in the murine gut or environmental metagenomes. Functions encoded by the plasmids pTRACA10 and pTRACA22 were found to be more prevalent in the human gut microbiome when compared to microbial communities from other environments. Among the most prevalent functions identified was a putative RelBE toxin-antitoxin (TA addiction module, and subsequent analysis revealed that this was most closely related to putative TA modules from gut associated bacteria belonging to the Firmicutes. A broad phylogenetic distribution of RelE toxin genes was observed in gut associated bacterial species (Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria, but no RelE homologues were identified in gut associated archaeal species. We also provide indirect evidence for the horizontal transfer of these genes between bacterial species belonging to disparate phylogenetic divisions, namely Gram negative Proteobacteria and Gram positive species from the Firmicutes division. Conclusions The application of a culture independent system to capture novel plasmids from the human gut mobile metagenome, coupled with subsequent comparative metagenomic analysis, highlighted the unexpected prevalence of plasmid encoded functions in the gut microbial ecosystem. In

  4. Enterotoxigenic Escherichia coli Adhesin-Toxoid Multiepitope Fusion Antigen CFA/I/II/IV-3xSTaN12S-mnLTG192G/L211A-Derived Antibodies Inhibit Adherence of Seven Adhesins, Neutralize Enterotoxicity of LT and STa Toxins, and Protect Piglets against Diarrhea.

    Science.gov (United States)

    Nandre, Rahul; Ruan, Xiaosai; Lu, Ti; Duan, Qiangde; Sack, David; Zhang, Weiping

    2018-03-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's diarrhea and travelers' diarrhea. Vaccines inducing antibodies to broadly inhibit bacterial adherence and to neutralize toxin enterotoxicity are expected to be effective against ETEC-associated diarrhea. 6×His-tagged adhesin-toxoid fusion proteins were shown to induce neutralizing antibodies to several adhesins and LT and STa toxins (X. Ruan, D. A. Sack, W. Zhang, PLoS One 10:e0121623, 2015, https://doi.org/10.1371/journal.pone.0121623). However, antibodies derived from His-tagged CFA/I/II/IV-2xSTa A14Q -dmLT or CFA/I/II/IV-2xSTa N12S -dmLT protein were less effective in neutralizing STa enterotoxicity and were not evaluated in vivo for efficacy against ETEC diarrhea. Additionally, His-tagged proteins are considered less desirable for human vaccines. In this study, we produced a tagless adhesin-toxoid MEFA (multiepitope fusion antigen) protein, enhanced anti-STa immunogenicity by including a third copy of STa toxoid STa N12S , and examined antigen immunogenicity in a murine model. Moreover, we immunized pregnant pigs with the tagless adhesin-toxoid MEFA protein and evaluated passive antibody protection against STa + or LT + ETEC infection in a pig challenge model. Results showed that tagless adhesin-toxoid MEFA CFA/I/II/IV-3xSTa N12S -mnLT R192G/L211A induced broad antiadhesin and antitoxin antibody responses in the intraperitoneally immunized mice and the intramuscularly immunized pigs. Mouse and pig serum antibodies significantly inhibited adherence of seven colonization factor antigen (CFA) adhesins (CFA/I and CS1 to CS6) and effectively neutralized both toxins. More importantly, suckling piglets born to the immunized mothers acquired antibodies and were protected against STa + ETEC and LT + ETEC diarrhea. These results indicated that tagless CFA/I/II/IV-3xSTa N12S -mnLT R192G/L211A induced broadly protective antiadhesin and antitoxin antibodies and demonstrate that this adhesin

  5. Replacement of the in vivo neutralisation test for efficacy demonstration of tetanus vaccines ad us. vet.

    Science.gov (United States)

    Rosskopf, Ute; Noeske, Kerstin; Werner, Esther

    2005-01-01

    The bacterium Clostridium (C.) tetani is an ubiquitous pathogen. This anaerobic, gram-positive bacterium can form spores and can be found in the whole environment. It enters the body via injuries of the skin and wounds where it releases the neurotoxin "tetanospasmin" (= tetanus toxin). The animals most susceptible to tetanus infection are horses and sheep. Only active immunisation by tetanus vaccine provides effective protection against tetanus intoxication. The marketing authorisation requirements stipulate that efficacy of tetanus vaccines ad us. vet. must be demonstrated in all target animal species via determination of neutralising tetanus serum antitoxin concentrations. The standard method used for this purpose is still the toxin neutralisation test (TNT), as it quantifies the tetanus toxin-neutralising effect of tetanus serum antibodies in vivo. In this test, tetanus toxin is added to dilutions of serum from vaccinated horse and sheep. The serum dilutions are then administered to mice or guinea pigs, which are observed for toxic symptoms. Against the background of animal protection, the goal of one project of the Paul-Ehrlich-Institut (Bundesministerium fuer Bildung und Forschung (Federal Ministry for Education and Research), 0312636) was to establish an alternative to the toxin neutralisation test, enabling the testing of efficacy of tetanus vaccines with serological in vitro methods. For this purpose, a so-called double antigen ELISA (DAE) was established which enables the testing of sera of different species in one assay. In addition, the sera were tested in an indirect ELISA for horses and sheep separately. Altogether, ten groups of horses and eight groups of sheep were immunised with ten animals per group each. The tetanus vaccines comprised almost all products authorised for the German market at the start of the project. 564 horse sera and 257 sheep sera were tested using the two ELISA methods. Some sera were also tested in vivo. The kinetics of

  6. Unearthing Bacillus endophytes from desert plants that enhance growth of Arabidopsis thaliana under abiotic stress conditions

    KAUST Repository

    Bokhari, Ameerah M

    2018-04-01

    Here, we embarked a bioprospecting project that focuses on the isolation and characterization of plant root endophytes, collected from the Thar Desert. A total of 381 endophytes were isolated and based on their 16S rRNA gene sequences, genus Bacillus (58 strains) was identified as the major taxon and only endophytes from this genus were isolated from all plant types. Of the 58 Bacillus strains, only 16 strains were selected for screening of plant growth promotion traits such as P and Zn solubilization, indole-3-acetic acid and siderophore production, and antimicrobial activity. Based on the presence of specific plant growth promotion traits 10 strains were shortlisted for further in vitro screening with A. thaliana; to confirm that these bacteria can confer resilience to plants under salt stress conditions. B. circulans (PK3-15 and PK3-109), B. cereus (PK6-15) B. subtilis (PK3-9) and B. licheniformis (PK5-26) displayed the ability to increased the fresh weight of A. thaliana under salt stress conditions by more than 50 % compared to the uninoculated control. An interesting observation was that B. circulans (PK3-109) (shown to produce IAA exopolysaccharide) and B. circulans (PK3-138) (shown to produce IAA) in vitro results were substantially different as B. circulans (PK3-138) decreased the total fresh weight of A. thaliana by 47 %, whilst B. circulans (PK3-109) was one of the best performing strains. Thus, the genomes of these two strains were sequences to unravel the molecular versatility of B. circulans strains, specifically with respect to their interaction with plants. Most of the genome of these strains is identical but the most interesting feature was the presence of 1/ the DegS–DegU two-component system that is known to mediate the salt stress response and DegU also represses toxin wapA similar to antitoxin wapI, and 2/ YxiG, a gene in the unique orthogroup of PK3-109 was found to be linked to WapI. Thus, PK3-138 substantially decreasing the total fresh

  7. Live virus-free or die: coupling of antivirus immunity and programmed suicide or dormancy in prokaryotes

    Directory of Open Access Journals (Sweden)

    Makarova Kira S

    2012-11-01

    Full Text Available Abstract Background The virus-host arms race is a major theater for evolutionary innovation. Archaea and bacteria have evolved diverse, elaborate antivirus defense systems that function on two general principles: i immune systems that discriminate self DNA from nonself DNA and specifically destroy the foreign, in particular viral, genomes, whereas the host genome is protected, or ii programmed cell suicide or dormancy induced by infection. Presentation of the hypothesis Almost all genomic loci encoding immunity systems such as CRISPR-Cas, restriction-modification and DNA phosphorothioation also encompass suicide genes, in particular those encoding known and predicted toxin nucleases, which do not appear to be directly involved in immunity. In contrast, the immunity systems do not appear to encode antitoxins found in typical toxin-antitoxin systems. This raises the possibility that components of the immunity system themselves act as reversible inhibitors of the associated toxin proteins or domains as has been demonstrated for the Escherichia coli anticodon nuclease PrrC that interacts with the PrrI restriction-modification system. We hypothesize that coupling of diverse immunity and suicide/dormancy systems in prokaryotes evolved under selective pressure to provide robustness to the antivirus response. We further propose that the involvement of suicide/dormancy systems in the coupled antivirus response could take two distinct forms: 1 induction of a dormancy-like state in the infected cell to ‘buy time’ for activation of adaptive immunity; 2 suicide or dormancy as the final recourse to prevent viral spread triggered by the failure of immunity. Testing the hypothesis This hypothesis entails many experimentally testable predictions. Specifically, we predict that Cas2 protein present in all cas operons is a mRNA-cleaving nuclease (interferase that might be activated at an early stage of virus infection to enable incorporation of virus

  8. Long-Term Protection against Diphtheria in the Netherlands after 50 Years of Vaccination: Results from a Seroepidemiological Study.

    Directory of Open Access Journals (Sweden)

    E M Swart

    Full Text Available To evaluate the National Immunisation Programme (NIP a population-based cross-sectional seroepidemiological study was performed in the Netherlands. We assessed diphtheria antitoxin levels in the general Dutch population and in low vaccination coverage (LVC areas where a relatively high proportion of orthodox Protestants live who decline vaccination based on religious grounds. Results were compared with a nationwide seroepidemiological study performed 11 years earlier.In 2006/2007 a national serum bank was established. Blood samples were tested for diphtheria antitoxin IgG concentrations using a multiplex immunoassay for 6383 participants from the national sample (NS and 1518 participants from LVC municipalities. A cut-off above 0.01 international units per ml (IU/ml was used as minimum protective level.In the NS 91% of the population had antibody levels above 0.01 IU/ml compared to 88% in the 1995/1996 serosurvey (p<0.05. On average, 82% (vs. 78% in the 1995/1996 serosurvey, p<0.05 of individuals from the NS born before introduction of diphtheria vaccination in the NIP and 46% (vs. 37% in the 1995/1996 serosurvey, p = 0.11 of orthodox Protestants living in LVC areas had antibody levels above 0.01 IU/ml. Linear regression analysis among fully immunized individuals (six vaccinations without evidence of revaccination indicated a continuous decline in antibodies in both serosurveys, but geometric mean antibodies remained well above 0.01 IU/ml in all age groups.The NIP provides long-term protection against diphtheria, although antibody levels decline after vaccination. As a result of natural waning immunity, a substantial proportion of individuals born before introduction of diphtheria vaccination in the NIP lack adequate levels of diphtheria antibodies. Susceptibility due to lack of vaccination is highest among strictly orthodox Protestants. The potential risk of spread of diphtheria within the geographically clustered orthodox Protestant community

  9. Foodborne Botulism, I Only Had Nacho Cheese: A Case Report.

    Science.gov (United States)

    Choudhry, Saad A; Anwar, Muhammad Jahanzaib; Afzal, Muhammad; Shah, Abdul

    2017-09-08

    A 32-year-old female presented to the emergency department with complaints of diplopia, followed by dyspnea, chest tightness, congestion, and dysphagia. The patient was resuscitated and initial investigations were done. Within a few hours of the admission, she started developing signs of respiratory failure and was intubated and placed on the mechanical ventilator. The patient denied any ingestion of exotic food, shellfish, raw meat, or raw fish. The patient also denied traveling to any exotic place or recent camping trips. The edrophonium tensilon test and lumbar puncture came out to be negative. The botulinum toxin test was positive, the patient was started on botulinum antitoxin, and the rest of symptomatic treatment was continued. The Centre for Disease Control (CDC) tracked the events related to the presentation and found she had eaten nacho cheese from a gas station the day before the appearance of the symptoms. A total of 10 cases were associated with this source within days and one death was reported.

  10. A Family Outbreak of Foodborne Botulism Following Consumption of Home-Canned Doogh in Hamadan, West of Iran

    Directory of Open Access Journals (Sweden)

    Hashemi

    2015-02-01

    Full Text Available Background Food-borne botulism is one of the potentially fatal forms of food poisoning, usually caused by ingestion of home-canned vegetables, fruits and fish products. Objectives The aim of this study was to report an outbreak of botulism due to homemade doogh in Hamadan, Iran. Patients and Methods During an outbreak, 10 members of a family referred to the hospital because of food poisoning. All patients had a history of consumption of doogh, a traditional drink. After careful physical examination, all of them were hospitalized. Botulism was suspected in all patients except for the first patient. Results The first patient was a 76-year-old man who died after 12 hours of admission due to respiratory distress. Nine subsequent patients were diagnosed as botulism with the following symptoms: diplopia (90%, dizziness (70%, nausea and vomiting (80%, ptosis (60%, symmetric weakness of extremities (60%, dysarthria (30%, chest discomfort (30%, mydriasis (20%, dysphasia (20% and dry mouth (20%. All of the nine patients received botulinum antitoxin and improved during 5-15 days of hospitalization. Conclusions Immediate diagnosis based on careful history and physical examination are essential for management of botulism. People should be notified about proper food handling and preparation of traditional homemade foods.

  11. Inhibitory effect of Thymus vulgaris and Origanum vulgare essential oils on virulence factors of phytopathogenic Pseudomonas syringae strains.

    Science.gov (United States)

    Carezzano, M E; Sotelo, J P; Primo, E; Reinoso, E B; Paletti Rovey, M F; Demo, M S; Giordano, W F; Oliva, M de Las M

    2017-07-01

    Pseudomonas syringae is a phytopathogenic bacterium that causes lesions in leaves during the colonisation process. The damage is associated with production of many virulence factors, such as biofilm and phytotoxins. The essential oils of Thymus vulgaris (thyme) and Origanum vulgare (oregano) have been demonstrated to inhibit P. syringae. The aim of this study was to investigate the effects of T. vulgaris and O. vulgare essential oils on production of virulence factors of phytopathogenic P. syringae strains, including anti-biofilm and anti-toxins activities. The broth microdilution method was used for determination of MIC and biofilm inhibition assays. Coronatine, syringomycin and tabtoxin were pheno- and genotypically evaluated. Both oils showed good inhibitory activity against P. syringae, with MIC values from 1.43 to 11.5 mg·ml -1 for thyme and 5.8 to 11.6 mg·ml -1 for oregano. Biofilm formation, production of coronatine, syringomycin and tabtoxin were inhibited by thyme and oregano essential oil in most strains. The results presented here are promising, demonstrating the bactericidal activity and reduction of virulence factor production after treatment with thyme and oregano oil, providing insight into how they exert their antibacterial activity. These natural products could be considered in the future for the control of diseases caused by P. syringae. © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.

  12. Persistence and resistance as complementary bacterial adaptations to antibiotics.

    Science.gov (United States)

    Vogwill, T; Comfort, A C; Furió, V; MacLean, R C

    2016-06-01

    Bacterial persistence represents a simple of phenotypic heterogeneity, whereby a proportion of cells in an isogenic bacterial population can survive exposure to lethal stresses such as antibiotics. In contrast, genetically based antibiotic resistance allows for continued growth in the presence of antibiotics. It is unclear, however, whether resistance and persistence are complementary or alternative evolutionary adaptations to antibiotics. Here, we investigate the co-evolution of resistance and persistence across the genus Pseudomonas using comparative methods that correct for phylogenetic nonindependence. We find that strains of Pseudomonas vary extensively in both their intrinsic resistance to antibiotics (ciprofloxacin and rifampicin) and persistence following exposure to these antibiotics. Crucially, we find that persistence correlates positively to antibiotic resistance across strains. However, we find that different genes control resistance and persistence implying that they are independent traits. Specifically, we find that the number of type II toxin-antitoxin systems (TAs) in the genome of a strain is correlated to persistence, but not resistance. Our study shows that persistence and antibiotic resistance are complementary, but independent, evolutionary adaptations to stress and it highlights the key role played by TAs in the evolution of persistence. © 2016 The Authors. Journal of Evolutionary Biology published by John Wiley & Sons Ltd on behalf of European Society for Evolutionary Biology.

  13. Crystal structure of the toxin Msmeg_6760, the structural homolog of Mycobacterium tuberculosis Rv2035, a novel type II toxin involved in the hypoxic response

    Energy Technology Data Exchange (ETDEWEB)

    Bajaj, R. Alexandra; Arbing, Mark A.; Shin, Annie; Cascio, Duilio; Miallau, Linda (UCLA)

    2016-11-19

    The structure of Msmeg_6760, a protein of unknown function, has been determined. Biochemical and bioinformatics analyses determined that Msmeg_6760 interacts with a protein encoded in the same operon, Msmeg_6762, and predicted that the operon is a toxin–antitoxin (TA) system. Structural comparison of Msmeg_6760 with proteins of known function suggests that Msmeg_6760 binds a hydrophobic ligand in a buried cavity lined by large hydrophobic residues. Access to this cavity could be controlled by a gate–latch mechanism. The function of the Msmeg_6760 toxin is unknown, but structure-based predictions revealed that Msmeg_6760 and Msmeg_6762 are homologous to Rv2034 and Rv2035, a predicted novel TA system involved inMycobacterium tuberculosislatency during macrophage infection. The Msmeg_6760 toxin fold has not been previously described for bacterial toxins and its unique structural features suggest that toxin activation is likely to be mediated by a novel mechanism.

  14. How World War 1 changed global attitudes to war and infectious diseases.

    Science.gov (United States)

    Shanks, G Dennis

    2014-11-08

    World War 1 was a key transition point towards scientific medicine. Medical officers incorporated Louis Pasteur's discoveries into their understanding of microorganisms as the cause of infectious diseases, which were therefore susceptible to rational control and treatment measures even in the pre-antibiotic era. Typhoid vaccination led to the successful evasion of the disastrous epidemics of previous wars. The incidence of tetanus was probably decreased by giving millions of doses of horse antitoxin to wounded soldiers. Quinine treated but could not control malaria; its use required mass compulsion. Tuberculosis was not a great military problem during World War 1, although mortality in civilian populations increased substantially. Treatment of sexually transmitted infections remained a matter of aversive conditioning, with invasive antiseptics used in the absence of antibiotics. Pandemic influenza in 1918-19 killed more people than died during the entire war, showing how much remained beyond the capability of the scientists and doctors who fought infectious diseases during World War 1. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Polyclonal Antibody Therapies for Clostridium difficile Infection

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    Michael R. Simon

    2014-10-01

    Full Text Available Clostridium difficile infection has emerged as a growing worldwide health problem. The colitis of Clostridium difficile infection results from the synergistic action of C. difficile secreted toxins A and B upon the colon mucosa. A human monoclonal IgG anti-toxin has demonstrated the ability in combination therapy to reduce mortality in C. difficile challenged hamsters. This antibody is currently in a clinical trial for the treatment of human Clostridium difficile infection. More than one group of investigators has considered using polyclonal bovine colostral antibodies to toxins A and B as an oral passive immunization. A significant proportion of the healthy human population possesses polyclonal antibodies to the Clostridium difficile toxins. We have demonstrated that polyclonal IgA derived from the pooled plasma of healthy donors possesses specificity to toxins A and B and can neutralize these toxins in a cell-based assay. This suggests that secretory IgA prepared from such pooled plasma IgA may be able to be used as an oral treatment for Clostridium difficile infection.

  16. The role of the hok/sok locus in bacterial response to stressful growth conditions.

    Science.gov (United States)

    Chukwudi, Chinwe U; Good, Liam

    2015-02-01

    The hok/sok locus is renowned for its plasmid stabilization effect via post-segregational killing of plasmid-free daughter cells. However, the function(s) of the chromosome-encoded loci, which are more abundant in pathogenic strains of a broad range of enteric bacteria, are yet to be understood. Also, the frequent occurrence of this toxin/antitoxin addiction system in multi-drug resistance plasmids suggests additional roles. In this study, the effects of the hok/sok locus on the growth of bacteria in stressful growth-limiting conditions such as high temperature and antibiotic burden were investigated using hok/sok plasmids. The results showed that the hok/sok locus prolonged the lag phase of host cell cultures, thereby enabling the cells to adapt, respond to the stress and eventually thrive in these growth-limiting conditions by increasing the growth rate at exponential phase. The hok/sok locus also enhanced the survival and growth of cells in low cell density cultures irrespective of unfavourable growth conditions, and may complement existing or defective SOS mechanism. In addition to the plasmid stabilization function, these effects would enhance the ability of pathogenic bacteria to establish infections and propagate the antibiotic resistance elements carried on these plasmids, thereby contributing to the virulence of such bacteria. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Persister formation in Staphylococcus aureus is associated with ATP depletion

    Energy Technology Data Exchange (ETDEWEB)

    Conlon, Brian P.; Rowe, Sarah E.; Gandt, Autumn Brown; Nuxoll, Austin S.; Donegan, Niles P.; Zalis, Eliza A.; Clair, Geremy; Adkins, Joshua N.; Cheung, Ambrose L.; Lewis, Kim

    2016-04-18

    Persisters are dormant phenotypic variants of bacterial cells that are tolerant to killing by antibiotics1. Persisters are associated with chronic bacterial infection and antibiotic treatment failure. In Escherichia coli, toxin/antitoxin (TA) modules are responsible for persister formation. The mechanism of persister formation in Gram positive bacteria is unknown. Staphylococcus aureus is a major human pathogen, responsible for a variety of chronic and relapsing infections such as osteomyelitis, endocarditis and infections of implanted devices. Deleting TA modules in S. aureus did not affect the level of persisters. Here we show that S. aureus persisters are produced due to a stochastic entrance to stationary phase accompanied by a drop in intracellular ATP. Cells expressing stationary state markers are present throughout the growth phase, increasing in frequency with cell density. Cell sorting revealed that expression of stationary markers was associated with a 100-1000 fold increased likelihood of survival to antibiotic challenge. We find that the antibiotic tolerance of these cells is due to a drop in intracellular ATP. The ATP level of the cell is predictive of bactericidal antibiotic efficacy and explains bacterial tolerance to antibiotic treatment.

  18. Time-resolved, single-cell analysis of induced and programmed cell death via non-invasive propidium iodide and counterstain perfusion.

    Science.gov (United States)

    Krämer, Christina E M; Wiechert, Wolfgang; Kohlheyer, Dietrich

    2016-09-01

    Conventional propidium iodide (PI) staining requires the execution of multiple steps prior to analysis, potentially affecting assay results as well as cell vitality. In this study, this multistep analysis method has been transformed into a single-step, non-toxic, real-time method via live-cell imaging during perfusion with 0.1 μM PI inside a microfluidic cultivation device. Dynamic PI staining was an effective live/dead analytical tool and demonstrated consistent results for single-cell death initiated by direct or indirect triggers. Application of this method for the first time revealed the apparent antibiotic tolerance of wild-type Corynebacterium glutamicum cells, as indicated by the conversion of violet fluorogenic calcein acetoxymethyl ester (CvAM). Additional implementation of this method provided insight into the induced cell lysis of Escherichia coli cells expressing a lytic toxin-antitoxin module, providing evidence for non-lytic cell death and cell resistance to toxin production. Finally, our dynamic PI staining method distinguished necrotic-like and apoptotic-like cell death phenotypes in Saccharomyces cerevisiae among predisposed descendants of nutrient-deprived ancestor cells using PO-PRO-1 or green fluorogenic calcein acetoxymethyl ester (CgAM) as counterstains. The combination of single-cell cultivation, fluorescent time-lapse imaging, and PI perfusion facilitates spatiotemporally resolved observations that deliver new insights into the dynamics of cellular behaviour.

  19. Clinical evaluation strategies for a live attenuated tetravalent dengue vaccine.

    Science.gov (United States)

    Precioso, Alexander Roberto; Palacios, Ricardo; Thomé, Beatriz; Mondini, Gabriella; Braga, Patrícia; Kalil, Jorge

    2015-12-10

    Butantan Institute is a public Brazilian biomedical research-manufacturer center affiliated to the São Paulo State Secretary of Health. Currently, Butantan is one of the main public producers of vaccines, antivenoms, and antitoxins in Latin America. The partnership between Butantan and the National Institutes of Health (NIH) of the United Sates has been one of the longest and most successful partnerships in the development and manufacturing of new vaccines. Recently, Butantan Institute has developed and manufactured a lyophilized tetravalent live attenuated dengue vaccine with the four dengue viruses attenuated and licensed from the Laboratory of Infectious Diseases at The National Institutes of Allergy and Infectious Diseases (LID/NIAID/NIH). The objective of this paper is to describe the clinical evaluation strategies of a live attenuated tetravalent dengue vaccine (Butantan-DV) developed and manufactured by Butantan Institute. These clinical strategies will be used to evaluate the Butantan-DV Phase III trial to support the Butantan-DV licensure for protection against any symptomatic dengue caused by any serotype in people aged 2 to 59 years. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. RelB and RelE of Escherichia coli Form a Tight Complex That Represses Transcription via The Ribbon-Helix-Helix Motif in RelB

    DEFF Research Database (Denmark)

    Overgaard, Martin; Borch, Jonas; Gerdes, Kenn

    2009-01-01

    RelB, the Ribbon-Helix-Helix (RHH) repressor encoded by the relBE toxin-antitoxin locus of Escherichia coli, forms a tight complex with RelE and thereby counteracts the mRNA cleavage activity of RelE. In addition, RelB dimers repress the strong relBE promoter and this repression by RelB is enhanced...... by RelE - that is - RelE functions as a transcriptional co-repressor. RelB is a Lon protease substrate and Lon is required both for activation of relBE transcription and for activation of the mRNA cleavage activity of RelE. Here we characterize the molecular interactions important for transcriptional...... motif recognizes four 6 bp repeats within the bipartite binding site. The spacing between each half-site was found to be essential for cooperative interactions between adjacently bound RelB dimers stabilized by the co-repressor RelE. Kinetic and stoichiometric measurements of the interaction between Rel...

  1. Investigations into an Outbreak of Botulism Caused by Clostridium botulinum Type C/D in Laying Hens.

    Science.gov (United States)

    Skarin, Hanna; Lindgren, Ylva; Jansson, Désirée S

    2015-06-01

    This case report describes a recent botulism outbreak in commercial laying hens with a history of increased mortality and flaccid paralysis. Routine diagnostic gross examination and microscopy from seven hens were inconclusive, but botulinum neurotoxin (BoNT) in peripheral blood was neutralized with both type C and type D antitoxins in the mouse bioassay. During a farm visit, 10 additional hens from a 34-wk-old flock on the farm were selected for clinical examination and further sampling. Nine hens were observed in sternal recumbency, with flaccid paralysis of the neck, drooping wings and tail, inability to escape, and bilateral ptosis, and one hen showed nonspecific clinical signs. Samples from cecum and liver were collected, and the gene coding for BoNT was detected by PCR in all 10 cecal samples and in four of the liver samples. Clostridium botulinum mosaic type C/D was isolated from 5 out of 10 hens from either cecum or liver, and the isolates were subjected to pulsed-field gel electrophoresis subtyping. All five isolates produced the same banding pattern, which was identical or showed >90% similarity to isolates from three different outbreaks on broiler farms in Sweden and Denmark during the 2007-10 period. However, they were clearly distinguishable from the predominantly reported pulsotype associated with avian botulism outbreaks in Europe. The authors are unaware of any previous report of C. botulinum mosaic type C/D isolates from laying hens.

  2. Pan-genome analysis of Clostridium botulinum reveals unique targets for drug development.

    Science.gov (United States)

    Bhardwaj, Tulika; Somvanshi, Pallavi

    2017-08-05

    Clostridium botulinum, a formidable pathogen is responsible for the emerging cause of food poisoning cases on the global canvas. The endemicity of bacterium Clostridium botulinum is reflected by the sudden hospital outbreaks and increased resistance towards multiple drugs. Therefore, a combined approach of in-silico comparative genomic analysis with statistical analysis was applied to overcome the limitation of bench-top technologies. Owing to the paucity of genomic data available by the advent of third generation sequencing technologies, several 'omics' technologies were applied to understand the underlying evolutionary pattern and lifestyle of the bacterial pathogen using phylogenomics. The calculation of pan-genome, core genome and singletons provides view of genetic repertoire of the bacterial pathogen lineage at the successive level, orthology shared and specific gene subsets. In addition, assessment of pathogenomic potential, resistome, toxin/antitoxin family in successive pathogenic strains of Clostridium botulinum aids in revealing more specific targets for drug design and development. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Bioterrorism in Canada: An Economic Assessment of Prevention and Postattack Response

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    Ronald St John

    2001-01-01

    Full Text Available The present paper calculates the human and economic consequences of a bioterrorist attack on Canadian soil using aerosolized Bacillus anthracis and Clostridium botulinum. The study assumed that 100,000 people in a Canadian suburban neighbourhood were exposed over a 2 h period to an infectious dose of one of the agents. Using an epidemic curve based on the epidemiology and management of anthrax and botulinum poisoning, the costs of intervention and treatment after an attack were compared with the costs of preparedness before a bioterrorist attack. The results show that an investment in planning and preparedness to manage the consequences of an attack can reduce morbidity, mortality and economic costs. The sooner that an intervention program is instituted, the more significant are the health and economic benefits. The greatest benefits were realized when postattack intervention was initiated before day 3 after the event. The economic impact of a bioterrorist attack in Canada could range from $6.4 billion/100,000 exposed to B anthracis to $8.6 billion/100,000 exposed in an attack using C botulinum. Without the benefit of an effective consequence management program, predicted deaths totalled 32,875 from anthrax and 30,000 from botulinum toxin. Rapid implementation of a postattack prophylaxis program that includes the stockpiling of antibiotics, vaccines and antitoxins; training of first responders in the diagnosis, handling and treatment of pathogens; and the general enhancement of Canada's response capability would reduce both human and economic losses.

  4. The Mechanisms of Virulence Regulation by Small Noncoding RNAs in Low GC Gram-Positive Pathogens

    Directory of Open Access Journals (Sweden)

    Stephanie Pitman

    2015-12-01

    Full Text Available The discovery of small noncoding regulatory RNAs (sRNAs in bacteria has grown tremendously recently, giving new insights into gene regulation. The implementation of computational analysis and RNA sequencing has provided new tools to discover and analyze potential sRNAs. Small regulatory RNAs that act by base-pairing to target mRNAs have been found to be ubiquitous and are the most abundant class of post-transcriptional regulators in bacteria. The majority of sRNA studies has been limited to E. coli and other gram-negative bacteria. However, examples of sRNAs in gram-positive bacteria are still plentiful although the detailed gene regulation mechanisms behind them are not as well understood. Strict virulence control is critical for a pathogen’s survival and many sRNAs have been found to be involved in that process. This review outlines the targets and currently known mechanisms of trans-acting sRNAs involved in virulence regulation in various gram-positive pathogens. In addition, their shared characteristics such as CU interaction motifs, the role of Hfq, and involvement in two-component regulators, riboswitches, quorum sensing, or toxin/antitoxin systems are described.

  5. Mapping of genotype-phenotype diversity among clinical isolates of mycobacterium tuberculosis by sequence-based transcriptional profiling.

    Science.gov (United States)

    Rose, Graham; Cortes, Teresa; Comas, Iñaki; Coscolla, Mireia; Gagneux, Sebastien; Young, Douglas B

    2013-01-01

    Genome sequencing has identified an extensive repertoire of single nucleotide polymorphisms among clinical isolates of Mycobacterium tuberculosis, but the extent to which these differences influence phenotypic properties of the bacteria remains to be elucidated. To determine whether these polymorphisms give rise to phenotypic diversity, we have integrated genome data sets with RNA sequencing to assess their impact on the comparative transcriptome profiles of strains belonging to M. tuberculosis Lineages 1 and 2. We observed clear correlations between genotype and transcriptional phenotype. These arose by three mechanisms. First, lineage-specific changes in amino acid sequence of transcriptional regulators were associated with alterations in their ability to control gene expression. Second, changes in nucleotide sequence were associated with alteration of promoter activity and generation of novel transcriptional start sites in intergenic regions and within coding sequences. We show that in some cases this mechanism is expected to generate functionally active truncated proteins involved in innate immune recognition. Finally, genes showing lineage-specific patterns of differential expression not linked directly to primary mutations were characterized by a striking overrepresentation of toxin-antitoxin pairs. Taken together, these findings advance our understanding of mycobacterial evolution, contribute to a systems level understanding of this important human pathogen, and more broadly demonstrate the application of state-of-the-art techniques to provide novel insight into mechanisms by which intergenic and silent mutations contribute to diversity.

  6. The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu.

    Science.gov (United States)

    Castro-Roa, Daniel; Garcia-Pino, Abel; De Gieter, Steven; van Nuland, Nico A J; Loris, Remy; Zenkin, Nikolay

    2013-12-01

    Fic proteins are ubiquitous in all of the domains of life and have critical roles in multiple cellular processes through AMPylation of (transfer of AMP to) target proteins. Doc from the doc-phd toxin-antitoxin module is a member of the Fic family and inhibits bacterial translation by an unknown mechanism. Here we show that, in contrast to having AMPylating activity, Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu, rendering EF-Tu unable to bind aminoacylated tRNAs. We provide evidence that EF-Tu phosphorylation diverged from AMPylation by antiparallel binding of the NTP relative to the catalytic residues of the conserved Fic catalytic core of Doc. The results bring insights into the mechanism and role of phosphorylation of EF-Tu in bacterial physiology as well as represent an example of the catalytic plasticity of enzymes and a mechanism for the evolution of new enzymatic activities.

  7. Natural Inhibitors of Snake Venom Metalloendopeptidases: History and Current Challenges

    Directory of Open Access Journals (Sweden)

    Viviane A. Bastos

    2016-08-01

    Full Text Available The research on natural snake venom metalloendopeptidase inhibitors (SVMPIs began in the 18th century with the pioneering work of Fontana on the resistance that vipers exhibited to their own venom. During the past 40 years, SVMPIs have been isolated mainly from the sera of resistant animals, and characterized to different extents. They are acidic oligomeric glycoproteins that remain biologically active over a wide range of pH and temperature values. Based on primary structure determination, mammalian plasmatic SVMPIs are classified as members of the immunoglobulin (Ig supergene protein family, while the one isolated from muscle belongs to the ficolin/opsonin P35 family. On the other hand, SVMPIs from snake plasma have been placed in the cystatin superfamily. These natural antitoxins constitute the first line of defense against snake venoms, inhibiting the catalytic activities of snake venom metalloendopeptidases through the establishment of high-affinity, non-covalent interactions. This review presents a historical account of the field of natural resistance, summarizing its main discoveries and current challenges, which are mostly related to the limitations that preclude three-dimensional structural determinations of these inhibitors using “gold-standard” methods; perspectives on how to circumvent such limitations are presented. Potential applications of these SVMPIs in medicine are also highlighted.

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

    Science.gov (United States)

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

    2014-08-01

    To obtain a recombinant enterotoxigenic Escherichia coli (ETEC) fusion enterotoxin protein LTB-STa-STb (Bab) that can express the immunogenicity of the haptens STa and STb and induce their corresponding neutralizing antibodies. The three important ETEC enterotoxin genes coding LTB, STa and STb were PCR-amplified, and the amplified products were fused to construct the trivalent enterotoxin expression vector pET30-Bab. SDS-PAGE and Western blot were used to verify the expression of the fusion protein Bab by E. coli BL21 carrying plasmid pET30-Bab. Laying hens immunized with Bab developed high egg yolk immunoglobulin (IgY) titres specific to LTB, STa and STb, and all were significantly higher than those in the control group (P protein containing three important ETEC enterotoxins may serve as an effective and convenient polyvalent toxoid that can be used to produce multiple antitoxin IgYs to prevent colibacillosis caused by ETEC with various fimbriae in young animals. © 2014 The Society for Applied Microbiology.

  9. Novel fusion protein protects against adherence and toxicity of enterohemorrhagic Escherichia coli O157:H7 in mice.

    Science.gov (United States)

    Gao, Xiang; Cai, Kun; Li, Tao; Wang, Qin; Hou, Xiaojun; Tian, Renmao; Liu, Hao; Tu, Wei; Xiao, Le; Fang, Lihua; Luo, Sen; Liu, Yuenan; Wang, Hui

    2011-09-02

    Infection with Escherichia coli (E. coli) O157:H7 may develop into bloody diarrhea, or hemorrhagic uremic syndrome (HUS), which usually causes kidney failure or even death. Considered as the pathogenesis mechanism of E. coli O157:H7 infection, attachment or adhesion that is directly mediated by intimin is the first step of E. coli O157:H7 interaction with its host, and all these serious sequelae are mainly due to Shiga toxins (Stxs) released by E. coli O157:H7. In this study, a novel SSI fusion protein that contains the critical toxin-antigens Stx2B and Stx1B, and the critical adhesion-antigen fragment Int281 was constructed. The protein induced complete immune protection, with both anti-toxin and anti-adhesion effects. The dominant increase in IgG1 and the high level of Th2-typical cytokine (IL-4 and IL-10) expression showed that SSI significantly induced Th2-mediated humoral immune response. In the mouse model, the SSI fusion protein not only elicited neutralizing antibodies against both Stx1 and Stx2 toxins, but also induced a high level of anti-adhesion antibodies. The SSI-immunized mice did not show any pathologic changes. SSI provides evident protection with two-time immunization against a highly lethal dose of E. coli O157:H7. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. An Integrative Genomic Island Affects the Adaptations of Piezophilic Hyperthermophilic Archaeon Pyrococcus yayanosii to High Temperature and High Hydrostatic Pressure

    Directory of Open Access Journals (Sweden)

    Zhen Li

    2016-11-01

    Full Text Available Deep-sea hydrothermal vent environments are characterized by high hydrostatic pressure and sharp temperature and chemical gradients. Horizontal gene transfer is thought to play an important role in the microbial adaptation to such an extreme environment. In this study, a 21.4-kb DNA fragment was identified as a genomic island, designated PYG1, in the genomic sequence of the piezophilic hyperthermophile Pyrococcus yayanosii. According to the sequence alignment and functional annotation, the genes in PYG1 could tentatively be divided into five modules, with functions related to mobility, DNA repair, metabolic processes and the toxin-antitoxin system. Integrase can mediate the site-specific integration and excision of PYG1 in the chromosome of P. yayanosii A1. Gene replacement of PYG1 with a SimR cassette was successful. The growth of the mutant strain ∆PYG1 was compared with its parent strain P. yayanosii A2 under various stress conditions, including different pH, salinity, temperature and hydrostatic pressure. The ∆PYG1 mutant strain showed reduced growth when grown at 100 °C, while the biomass of ∆PYG1 increased significantly when cultured at 80 MPa. Differential expression of the genes in module Ⅲ of PYG1 was observed under different temperature and pressure conditions. This study demonstrates the first example of an archaeal integrative genomic island that could affect the adaptation of the hyperthermophilic piezophile P. yayanosii to high temperature and high hydrostatic pressure.

  11. Culex pipiens crossing type diversity is governed by an amplified and polymorphic operon of Wolbachia.

    Science.gov (United States)

    Bonneau, Manon; Atyame, Celestine; Beji, Marwa; Justy, Fabienne; Cohen-Gonsaud, Martin; Sicard, Mathieu; Weill, Mylène

    2018-01-22

    Culex pipiens mosquitoes are infected with Wolbachia (wPip) that cause an important diversity of cytoplasmic incompatibilities (CIs). Functional transgenic studies have implicated the cidA-cidB operon from wPip and its homolog in wMel in CI between infected Drosophila males and uninfected females. However, the genetic basis of the CI diversity induced by different Wolbachia strains was unknown. We show here that the remarkable diversity of CI in the C. pipiens complex is due to the presence, in all tested wPip genomes, of several copies of the cidA-cidB operon, which undergoes diversification through recombination events. In 183 isofemale lines of C. pipiens collected worldwide, specific variations of the cidA-cidB gene repertoires are found to match crossing types. The diversification of cidA-cidB is consistent with the hypothesis of a toxin-antitoxin system in which the gene cidB co-diversifies with the gene cidA, particularly in putative domains of reciprocal interactions.

  12. Quantification and detoxification of aflatoxin in food items

    International Nuclear Information System (INIS)

    Nisa, A.U.; Hina, S.; Ejaz, N.

    2013-01-01

    The present study was conducted to quantify and detoxify the antitoxins in food items. For this purpose, total 30 samples of food were collected. The samples were quantified using thin layer chromatography (TLC) for the presence of aflatoxin level in food items. Out of them aflatoxins were not found in 10 samples. Remaining 20 aflatoxins +ve samples were treated with various chemical solutions i.e. 0.1% HCl, 0.3%HCl, 0.5% HCI, 10% citric acid, 30% citric acid, 50% calcium hydroxide, 0.2 and 0.3% NaOCl, 96% ethanol and 99% acetone for detoxification. The aflatoxins were reduced to 55.1%, 90.9%, 28.08% and 80.0% in Super Sella rice, Super Basmati rice, Brown rice and White rice, respectively. The aflatoxin level was reduced in maize grain, damaged wheat, peanut, figs and dates upto 31.3 %, 64.3 %, 63.6%, 42.7% and 19.8%, respectively. Aflatoxins were detoxified in cereals Dal Chana, Dal Mash, Dal Masoor, turmeric (Haldi) and Nigela seeds (Kalwangi) upto 70.5%, 83.0%, 46.2%, 82.09% and 36.9%, respectively. Reduction of aflatoxins was carried out 39.7 %,7.l % 39.5% 82.0% and 62.0% in red chilli, makhana, corn flakes, desert (Kheer Mix) and pistachio. The significant results (p = 0.042) of detoxification of aflatoxins in food items were obtained from present study. (author)

  13. An update on antibody-based immunotherapies for Clostridium difficile infection

    Directory of Open Access Journals (Sweden)

    Hussack G

    2016-08-01

    Full Text Available Greg Hussack,1 Jamshid Tanha1–3 1Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, 2School of Environmental Sciences, University of Guelph, Guelph, 3Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada Abstract: Clostridium difficile continues to be one of the most prevalent hospital-acquired bacterial infections in the developed world, despite the recent introduction of a novel and effective antibiotic agent (fidaxomicin. Alternative approaches under investigation to combat the anaerobic Gram-positive bacteria include fecal transplantation therapy, vaccines, and antibody-based immunotherapies. In this review, we catalog the recent advances in antibody-based approaches under development and in the clinic for the treatment of C. difficile infection. By and large, inhibitory antibodies that recognize the primary C. difficile virulence factors, toxin A and toxin B, are the most popular passive immunotherapies under investigation. We provide a detailed summary of the toxin epitopes recognized by various antitoxin antibodies and discuss general trends on toxin inhibition efficacy. In addition, antibodies to other C. difficile targets, such as surface-layer proteins, binary toxin, motility factors, and adherence and colonization factors, are introduced in this review. Keywords: antibody, Clostridium difficile, immunotherapy, toxin

  14. Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium Synechocystis sp. PCC 6803

    Directory of Open Access Journals (Sweden)

    Helena Čelešnik

    2016-04-01

    Full Text Available In recent years, photosynthetic autotrophic cyanobacteria have attracted interest for biotechnological applications for sustainable production of valuable metabolites. Although biosafety issues can have a great impact on public acceptance of cyanobacterial biotechnology, biosafety of genetically modified cyanobacteria has remained largely unexplored. We set out to incorporate biocontainment systems in the model cyanobacterium Synechocystis sp. PCC 6803. Plasmid-encoded safeguards were constructed using the nonspecific nuclease NucA from Anabaena combined with different metal-ion inducible promoters. In this manner, conditional lethality was dependent on intracellular DNA degradation for regulated autokilling as well as preclusion of horizontal gene transfer. In cells carrying the suicide switch comprising the nucA gene fused to a variant of the copM promoter, efficient inducible autokilling was elicited. Parallel to nuclease-based safeguards, cyanobacterial toxin/antitoxin (TA modules were examined in biosafety switches. Rewiring of Synechocystis TA pairs ssr1114/slr0664 and slr6101/slr6100 for conditional lethality using metal-ion responsive promoters resulted in reduced growth, rather than cell killing, suggesting cells could cope with elevated toxin levels. Overall, promoter properties and translation efficiency influenced the efficacy of biocontainment systems. Several metal-ion promoters were tested in the context of safeguards, and selected promoters, including a nrsB variant, were characterized by beta-galactosidase reporter assay.

  15. Analysing the Structural Effect of Point Mutations of Cytotoxic Necrotizing Factor 1 (CNF1 on Lu/BCAM Adhesion Glycoprotein Association

    Directory of Open Access Journals (Sweden)

    Alexandre G. de Brevern

    2018-03-01

    Full Text Available Cytotoxic Necrotizing Factor 1 (CNF1 was identified in 1983 as a protein toxin produced by certain pathogenic strains of Escherichia coli. Since then, numerous studies have investigated its particularities. For instance, it is associated with the single chain AB-toxin family, and can be divided into different functional and structural domains, e.g., catalytic and transmembrane domain and interaction sites. A few years ago, the identification of the Lutheran (Lu adhesion glycoprotein/basal cell adhesion molecule (BCAM as a cellular receptor for CNF1 provided new insights into the adhesion process of CNF1. Very recently, the Ig-like domain 2 of Lu/BCAM was confirmed as the main interaction site using protein-protein interaction and competition studies with various different mutants. Here, I present in silico approaches that precisely explain the impact of these mutations, leading to a better explanation of these experimental studies. These results can be used in the development of future antitoxin strategies.

  16. Toxigenic Corynebacterium ulcerans isolated from a hunting dog and its diphtheria toxin antibody titer.

    Science.gov (United States)

    Katsukawa, Chihiro; Komiya, Takako; Umeda, Kaoru; Goto, Minami; Yanai, Tokuma; Takahashi, Motohide; Yamamoto, Akihiko; Iwaki, Masaaki

    2016-03-01

    Toxigenic Corynebacterium ulcerans is a zoonotic pathogen that produces diphtheria toxin and causes a diphtheria-like illness in humans. The organism is known to infect and circulate among dogs, which can then transmit it to humans. Furthermore, previous studies have found that C. ulcerans is carried by wild animals, including game animals. In the present study, we tested hunting and companion dogs for the presence of toxigenic C. ulcerans and succeeded in isolating the bacterium from a hunting dog. Moreover, several hunting dogs had serum diphtheria antitoxin titers that were higher than the titers required for protection in humans, suggesting a history of exposure to toxigenic Corynebacterium strains. Notably, ribotyping, pulsed-field gel electrophoresis and tox gene sequencing demonstrated that the isolate from the hunting dog clustered with previously characterized C. ulcerans strains isolated from wild animals, as opposed to groups of isolates from humans and companion dogs. Interestingly, the wild animal cluster also contains an isolate from an outdoor breeding dog, which could have formed a bridge between isolates from wild animals and those from companion dogs. The results presented herein provide insight into the mechanism by which the zoonotic pathogen C. ulcerans circulates among wild animals, hunting and companion dogs, and humans. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

  17. Molecular Structure of Endotoxins from Gram-negative Marine Bacteria: An Update

    Directory of Open Access Journals (Sweden)

    Antonio Molinaro

    2007-09-01

    Full Text Available Marine bacteria are microrganisms that have adapted, through millions of years, to survival in environments often characterized by one or more extreme physical or chemical parameters, namely pressure, temperature and salinity. The main interest in the research on marine bacteria is due to their ability to produce several biologically active molecules, such as antibiotics, toxins and antitoxins, antitumor and antimicrobial agents. Nonetheless, lipopolysaccharides (LPSs, or their portions, from Gram-negative marine bacteria, have often shown low virulence, and represent potential candidates in the development of drugs to prevent septic shock. Besides, the molecular architecture of such molecules is related to the possibility of thriving in marine habitats, shielding the cell from the disrupting action of natural stress factors. Over the last few years, the depiction of a variety of structures of lipids A, core oligosaccharides and O-specific polysaccharides from LPSs of marine microrganisms has been given. In particular, here we will examine the most recently encountered structures for bacteria belonging to the genera Shewanella, Pseudoalteromonas and Alteromonas, of the γ-Proteobacteria phylum, and to the genera Flavobacterium, Cellulophaga, Arenibacter and Chryseobacterium, of the Cytophaga- Flavobacterium-Bacteroides phylum. Particular attention will be paid to the chemical features expressed by these structures (characteristic monosaccharides, non-glycidic appendages, phosphate groups, to the typifying traits of LPSs from marine bacteria and to the possible correlation existing between such features and the adaptation, over years, of bacteria to marine environments.

  18. Genome sequence and comparative genomic analysis of a clinically important strain CD11-4 ofJanibacter melonisisolated from celiac disease patient.

    Science.gov (United States)

    Chander, Atul Munish; Kochhar, Rakesh; Dhawan, Devinder Kumar; Bhadada, Sanjay Kumar; Mayilraj, Shanmugam

    2018-01-01

    Janibacter melonis and other member of this genus are known to cause bacteremia and serious clinical comorbidities, but there is no study reporting about pathogenicity attributes of J. melonis. Janibacter terrae is known to cause lethal infection. Reporting the genome of J. melonis CD11-4 and comparative genomics with other members of genus has provided some novel insights that can enable us to understand the mechanisms responsible for its pathogenicity in humans. Comparative genomic analysis by Rapid Annotation Server and Technology revealed the presence of similar virulence determinant genes in both J. terrae NBRC 107853 T and J. melonis CD11-4. Like J. terrae NBRC 107853 T , J. melonis CD11-4 contained two genes responsible for resistance against β-lactam class of antibiotics and two genes for resistance against fluoroquinolones. Interestingly, J. melonis CD11-4 contained a unique gene coding for multidrug resistance efflux pumps unlike all other members of this genus. It also contained two genes involved in Toxin-antitoxin Systems that were absent in J. terrae NBRC 107853 T but were present in some other members of genus. Genome annotations of J. melonis CD11-4 revealed that it contained similar or more virulence repertoire like J. terrae NBRC 107853 T . Like other gut pathogens, J. melonis possesses key virulence determinant genes for antibiotic resistance, invasion, adhesion, biofilm formation, iron acquisition and to cope with stress response, thereby indicating that strain J. melonis CD11-4 could be a gut pathogen.

  19. The genome sequence of Rickettsia felis identifies the first putative conjugative plasmid in an obligate intracellular parasite.

    Directory of Open Access Journals (Sweden)

    2005-08-01

    Full Text Available We sequenced the genome of Rickettsia felis, a flea-associated obligate intracellular alpha-proteobacterium causing spotted fever in humans. Besides a circular chromosome of 1,485,148 bp, R. felis exhibits the first putative conjugative plasmid identified among obligate intracellular bacteria. This plasmid is found in a short (39,263 bp and a long (62,829 bp form. R. felis contrasts with previously sequenced Rickettsia in terms of many other features, including a number of transposases, several chromosomal toxin-antitoxin genes, many more spoT genes, and a very large number of ankyrin- and tetratricopeptide-motif-containing genes. Host-invasion-related genes for patatin and RickA were found. Several phenotypes predicted from genome analysis were experimentally tested: conjugative pili and mating were observed, as well as beta-lactamase activity, actin-polymerization-driven mobility, and hemolytic properties. Our study demonstrates that complete genome sequencing is the fastest approach to reveal phenotypic characters of recently cultured obligate intracellular bacteria.

  20. The genome sequence of Rickettsia felis identifies the first putative conjugative plasmid in an obligate intracellular parasite.

    Science.gov (United States)

    Ogata, Hiroyuki; Renesto, Patricia; Audic, Stéphane; Robert, Catherine; Blanc, Guillaume; Fournier, Pierre-Edouard; Parinello, Hugues; Claverie, Jean-Michel; Raoult, Didier

    2005-08-01

    We sequenced the genome of Rickettsia felis, a flea-associated obligate intracellular alpha-proteobacterium causing spotted fever in humans. Besides a circular chromosome of 1,485,148 bp, R. felis exhibits the first putative conjugative plasmid identified among obligate intracellular bacteria. This plasmid is found in a short (39,263 bp) and a long (62,829 bp) form. R. felis contrasts with previously sequenced Rickettsia in terms of many other features, including a number of transposases, several chromosomal toxin-antitoxin genes, many more spoT genes, and a very large number of ankyrin- and tetratricopeptide-motif-containing genes. Host-invasion-related genes for patatin and RickA were found. Several phenotypes predicted from genome analysis were experimentally tested: conjugative pili and mating were observed, as well as beta-lactamase activity, actin-polymerization-driven mobility, and hemolytic properties. Our study demonstrates that complete genome sequencing is the fastest approach to reveal phenotypic characters of recently cultured obligate intracellular bacteria.

  1. The Escherichia coli replication inhibitor CspD is subject to growth-regulated degradation by the Lon protease.

    Science.gov (United States)

    Langklotz, Sina; Narberhaus, Franz

    2011-06-01

    Post-translational proteolysis-dependent regulation of critical cellular processes is a common feature in bacteria. The Escherichia coli Lon protease is involved in the control of the SOS response, acid tolerance and nutritional deprivation. Moreover, Lon plays a role in the regulation of toxin-antitoxin (TA) systems and thereby is linked to persister cell induction. Persister cells represent a small subpopulation that has reversibly switched to a dormant and non-dividing state without genomic alterations. Formation of persister cells permits viability upon nutritional depletion and severe environmental stresses. CspD is a replication inhibitor, which is induced in stationary phase or upon carbon starvation and increases the production of persister cells. It has remained unknown how CspD activity is counteracted when growth is resumed. Here we report that CspD is subject to proteolysis by the Lon protease both in vivo and in vitro. Turnover of CspD by Lon is strictly adjusted to the growth rate and growth phase of E. coli, reflecting the necessity to control CspD levels according to the physiological conditions. © 2011 Blackwell Publishing Ltd.

  2. Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Tobias Dörr

    2010-02-01

    Full Text Available Bacteria induce stress responses that protect the cell from lethal factors such as DNA-damaging agents. Bacterial populations also form persisters, dormant cells that are highly tolerant to antibiotics and play an important role in recalcitrance of biofilm infections. Stress response and dormancy appear to represent alternative strategies of cell survival. The mechanism of persister formation is unknown, but isolated persisters show increased levels of toxin/antitoxin (TA transcripts. We have found previously that one or more components of the SOS response induce persister formation after exposure to a DNA-damaging antibiotic. The SOS response induces several TA genes in Escherichia coli. Here, we show that a knockout of a particular SOS-TA locus, tisAB/istR, had a sharply decreased level of persisters tolerant to ciprofloxacin, an antibiotic that causes DNA damage. Step-wise administration of ciprofloxacin induced persister formation in a tisAB-dependent manner, and cells producing TisB toxin were tolerant to multiple antibiotics. TisB is a membrane peptide that was shown to decrease proton motive force and ATP levels, consistent with its role in forming dormant cells. These results suggest that a DNA damage-induced toxin controls production of multidrug tolerant cells and thus provide a model of persister formation.

  3. Tetanus in the equine species: a retrospective study of 31 cases.

    Science.gov (United States)

    van Galen, G; Delguste, C; Sandersen, C; Verwilghen, D; Grulke, S; Amory, H

    2008-06-15

    Few studies exist about factors affecting the outcome of horses with tetanus. 31 equids (30 horses and 1 donkey) with a clinical diagnosis of tetanus admitted to the Equine Clinic of the University of Liege between 1991 and 2006. The cases were divided into two groups according to the outcome (survivors and non-survivors). The clinical data of survivors and non-survivors were compared using an ANOVA (continuous data) or a Fisher's test (discrete data). The survival rate was 32%. Most animals were 5 years or younger, and none had been appropriately vaccinated. The non-survivors were significantly younger than the survivors. The development of dyspnoea, recumbency, and the combination of dysphagia, dyspnoea, and recumbency was observed significantly more in the non-survivors than in the survivors. The timing of tetanus antitoxin administration (either immediately after the onset of suggestive signs or after a delay) was not different between the two groups. The time between the occurrence of a wound and the first signs ranged from 2 days to 2 months and was not significantly different between groups. All non-survivors died within 8 days of the first signs. This study suggests that young animals are affected more often and more severely by tetanus than older animals. Dyspnoea, recumbency, and the combination of dysphagia, dyspnoea, and recumbency can be considered as indicators of a poor prognosis in equids suffering from tetanus.

  4. An imaging flow cytometry method to assess ricin trafficking in A549 human lung epithelial cells.

    Science.gov (United States)

    Jenner, Dominic; Chong, Damien; Walker, Nicola; Green, A Christopher

    2018-02-01

    The endocytosis and trafficking of ricin in mammalian cells is an important area of research for those producing ricin anti-toxins and other ricin therapeutics. Ricin trafficking is usually observed by fluorescence microscopy techniques. This gives good resolution and leads to a detailed understanding of the internal movement of ricin within cells. However, microscopy techniques are often hampered by complex analysis and quantification techniques, and the inability to look at ricin trafficking in large populations of cells. In these studies we have directly labelled ricin and assessed if its trafficking can be observed using Imaging Flow Cytometry (IFC) both to the cytoplasmic region of cells and specifically to the Golgi apparatus. Using IDEAS® data analysis software the specific fluorescence location of the ricin within the cells was analysed. Then, using cytoplasmic masking techniques to quantify the number of cells with endocytosed cytoplasmic ricin or cells with Golgi-associated ricin, kinetic endocytosis curves were generated. Here we present, to the authors' knowledge, the first example of using imaging flow cytometry for evaluating the subcellular transport of protein cargo, using the trafficking of ricin toxin in lung cells as a model. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

  5. Nucleotide sequence of pOLA52: a conjugative IncX1 plasmid from Escherichia coli which enables biofilm formation and multidrug efflux

    DEFF Research Database (Denmark)

    Norman, Anders; Hansen, Lars H.; She, Qunxin

    2008-01-01

    The large conjugative multidrug resistance (MDR) plasmid pOLA52 was sequenced and annotated. The plasmid encodes two phenotypes normally associated with the chromosomes of opportunistic pathogens, namely MDR via a resistance-nodulation-division (RND)-type efflux-pump (oqxAB), and the formation of...... and Tn6011) that seemed to originate from Klebsiella pneumoniae, thus demonstrating the capability of IncX1 plasmids of facilitating lateral transfer of gene cassettes between different Enterobacteriaceae.......The large conjugative multidrug resistance (MDR) plasmid pOLA52 was sequenced and annotated. The plasmid encodes two phenotypes normally associated with the chromosomes of opportunistic pathogens, namely MDR via a resistance-nodulation-division (RND)-type efflux-pump (oqxAB), and the formation...... of type 3 fimbriae (mrkABCDF). The plasmid was found to be 51,602 bp long with 68 putative genes. About half of the plasmid constituted a conserved IncX1-type backbone with predicted regions for conjugation, replication and partitioning, as well as a toxin/antitoxin (TA) plasmid addiction system...

  6. Anti-snake venom: use and adverse reaction in a snake bite study clinic in Bangladesh

    Directory of Open Access Journals (Sweden)

    MR Amin

    2008-01-01

    Full Text Available Snakebites can present local or systemic envenomation, while neurotoxicity and respiratory paralysis are the main cause of death. The mainstay of management is anti-snake venom (ASV, which is highly effective, but liable to cause severe adverse reactions including anaphylaxis. The types of adverse reaction to polyvalent anti-snake venom have not been previously studied in Bangladesh. In this prospective observational study carried out between 1999 and 2001, in the Snake Bite Study Clinic of Chittagong Medical College Hospital, 35 neurotoxic-snake-bite patients who had received polyvalent anti-snake venom were included while the ones sensitized to different antitoxins and suffering from atopy were excluded. The common neurotoxic features were ptosis (100%, external ophthalmoplegia (94.2%, dysphagia (77.1%, dysphonia (68.5% and broken neck sign (80%. The percentage of anti-snake venom reaction cases was 88.57%; pyrogenic reaction was 80.64%; and anaphylaxis was 64.51%. The common features of anaphylaxis were urticaria (80%; vomiting and wheezing (40%; and angioedema (10%. The anti-snake venom reaction was treated mainly with adrenaline for anaphylaxis and paracetamol suppository in pyrogenic reactions. The average recovery time was 4.5 hours. Due to the danger of reactions the anti-snake venom should not be withheld from a snakebite victim when indicated and appropriate guidelines should be followed for its administration.

  7. [Two horses with neurological symptoms: could this be equine botulism?].

    Science.gov (United States)

    Roest, H I J; de Bruijn, C M; Picavet, M T J E; Prins, B; Parmentier, D; de Zwart, G M A M; Dijkstra, Y E; van Zijderveld, F G

    2009-10-01

    Symptoms, diagnosis and therapy of equine botulism are discussed by the presentation of two detailed reports of horses with neurological symptoms and the results of laboratory investigations over the period 2003-2008 in the Netherlands. In addition a brief summary of the available literature is presented. Prevailing symptoms of botulism in horses include paralysis of the tongue, salvation, dysphagia and paresis and paralysis of the skeletal muscles, as well as signs of colic. Symptoms and prognosis vary with the amount of botulinum neurotoxin (BoNT) involved. For early clinical diagnosis of botulism thorough investigation of the facial nerves is important, for instance by the use of the 'Tongue Stress Test'. Laboratory results often remain negative, probably due to the sampling time, the high sensitivity of horses for botulinum neurotoxin or treatment with antitoxins. Most clinical cases in horses are caused by botulinum neurotoxin B (BoNT/B). For therapy to be successful antiserum needs to be administered in the earliest possible stage of the disease and this should be supported by symptomatic therapy. Botulism is a feed-related intoxication caused by either carcasses in the roughage or BoNT/B production after poor conservation of grass silage. This is the main source of botulism in horses due to the popularity of individually packed grass silage as feed for horses. As long as no vaccine is available in the Netherlands quality control of silage and haylage is strictly recommended in order to reduce the risk of botulism in horses.

  8. Escherichia coli Quorum-Sensing EDF, A Peptide Generated by Novel Multiple Distinct Mechanisms and Regulated by trans-Translation

    Directory of Open Access Journals (Sweden)

    Sathish Kumar

    2016-01-01

    Full Text Available Eshcerichia coli mazEF is a stress-induced toxin-antitoxin module mediating cell death and requiring a quorum-sensing (QS extracellular death factor (EDF, the pentapeptide NNWNN. Here we uncovered several distinct molecular mechanisms involved in its generation from the zwf mRNA encoding glucose-6-phosphate dehydrogenase. In particular, we show that, under stress conditions, the endoribonuclease MazF cleaves specific ACA sites, thereby generating a leaderless zwf mRNA which is truncated 30 codons after the EDF-encoding region. Since the nascent ribosome peptide exit tunnel can accommodate up to 40 amino acids, this arrangement allows the localization of the EDF residues inside the tunnel when the ribosome is stalled at the truncation site. Moreover, ribosome stalling activates the trans-translation system, which provides a means for the involvement of ClpPX in EDF generation. Furthermore, the trans-translation is described as a regulatory system that attenuated the generation of EDF, leading to low levels of EDF in the single cell. Therefore, the threshold EDF molecule concentration required is achieved only by the whole population, as expected for QS.

  9. Development of rocket electrophoresis technique as an analytical tool in preformulation study of tetanus vaccine formulation.

    Science.gov (United States)

    Ahire, V J; Sawant, K K

    2006-08-01

    Rocket Electrophoresis (RE) technique relies on the difference in charges of the antigen and antibodies at the selected pH. The present study involves optimization of RE run conditions for Tetanus Toxoid (TT). Agarose gel (1% w/v, 20 ml, pH 8.6), anti-TT IgG - 1 IU/ml, temperature 4-8 degrees C and run duration of 18 h was found to be optimum. Height of the rocket-shaped precipitate was proportional to TT concentration. The RE method was found to be linear in the concentration range of 2.5 to 30 Lf/mL. The method was validated and found to be accurate, precise, and reproducible when analyzed statistically using student's t-test. RE was used as an analytical method for analyzing TT content in plain and marketed formulations as well as for the preformulation study of vaccine formulation where formulation additives were tested for compatibility with TT. The optimized RE method has several advantages: it uses safe materials, is inexpensive, and easy to perform. RE results are less prone to operator's bias as compared to flocculation test and can be documented by taking photographs and scanned by densitometer; RE can be easily standardized for the required antigen concentration by changing antitoxin concentration. It can be used as a very effective tool for qualitative and quantitative analysis and in preformulation studies of antigens.

  10. The Effect of Various Species of Macroalgae on the Growth, Survival, and Toxicity of Karenia brevis

    Science.gov (United States)

    Gardner, K. G.; Lovko, V. J.; Henry, M. S.

    2016-02-01

    Harmful algal blooms (HABs) caused by the dinoflagellate Karenia brevis produce toxins that result in negative impacts to both humans and the environment. Little is known about the termination stages of these blooms, and few viable control mechanisms have been suggested. Natural, algae derived compounds have been proposed as a way to limit bloom growth and reduce brevetoxins in the water column. The work presented here examines the ability of macroalgae to inhibit the growth or survival of K. brevis, similar to what has been demonstrated with other red tide species. Additionally, we attempted to determine if macroalgae decreases water column brevetoxins which, to our knowledge, has not been tested with macroalgae but has been demonstrated in other studies with microalgal species. The macroalgae species Dictyota sp. and Gracilaria sp. caused 100% mortality of K. brevis in under 24 hours. Compared to the control, 7 other species significantly decreased the growth rate of K. brevis. The Dictyota treatments showed significant toxin reduction and increase of the antitoxin brevanol. These results indicate that some combination of compounds produced by macroalgae inhibit growth and survival of K. brevis and possibly limit their toxin production. Future studies will attempt to isolate and identify these compounds and test their effects on other marine organisms such as diatoms. Determining the interactions between HAB species K. brevis and macroalgal species will provide insights on the mechanism of bloom termination and a potential control method.

  11. Fatal injury in eastern Sri Lanka, with special reference to cardenolide self-poisoning with Cerbera manghas fruits.

    Science.gov (United States)

    Eddleston, Michael; Haggalla, Sapumal

    2008-09-01

    Self-poisoning with plant seeds or fruits is a common method of self-harm in South Asia. While most deaths follow ingestion of Thevetia peruviana (yellow oleander) seeds, other plants are locally common. During review of fatal injuries seen in a teaching hospital in eastern Sri Lanka, we noted cases of fatal self-poisoning with Cerbera manghas (sea mango, pink eyed cerbera, odollam tree) fruits. We reviewed the post-mortem records of the Batticaloa Teaching Hospital and extracted data on all cases of fatal injury. During 2001 and 2002, 315 post mortems for injury were performed in Batticaloa Teaching Hospital. Intentional self-harm was responsible for 48.6% of cases. While T. peruviana was responsible for 33 deaths, C. manghas self-poisoning caused seven deaths. C. manghas cases had typical features of cardenolide poisoning with cardiac dysrhythmias and hyperkalemia. In the absence of pacing facilities and anti-digoxin Fab, management involved administration of atropine and of insulin and dextrose to lower serum potassium concentrations. C. manghas self-poisoning has only previously been reported from Kerala and Tamil Nadu in south India. While uncommon in other parts of Sri Lanka, it has become a common method of self-harm in one east coast district, accounting for 20% of fatal self-harm with plants in one hospital. Management was inadequate with the available resources, emphasising the need for an affordable antitoxin for plant cardenolide poisoning.

  12. Effective humoral immunity against diphtheria and tetanus in patients with systemic lupus erythematosus or myasthenia gravis.

    Science.gov (United States)

    Csuka, Dorottya; Czirják, László; Hóbor, Renáta; Illes, Zsolt; Bánáti, Miklós; Rajczy, Katalin; Tordai, Attila; Füst, George

    2013-07-01

    Controversy exists about the effectiveness of vaccine-induced immune response in patients with immunoregulatory disorders. Our aim was to determine the antibody titers to diphtheria and tetanus in patients with either of two autoimmune diseases. 279 patients with SLE (205 females, aged 45.0 ± 13.8 years), 158 patients with myasthenia gravis (MG) (101 females, aged 55 ± 18.7 years) and 208 healthy subjects (122 females, aged 48 ± 14.6 years) were enrolled. Serum concentrations of diphtheria-antitoxin-IgG (A-DIPHTH) and tetanus-antitoxoid-IgG (A-TET) were determined with ELISA. Equal proportions of healthy subjects, as well as patients with SLE or MG exhibited proper antibody responses and immune protection against diphtheria and tetanus. In all three test groups, serum concentration of A-DIPHTH decreased significantly (p60-years-old) subjects. There were no significant differences among the groups in the age-related changes of A-TET and A-DIPHTH except that in diphtheria and tetanus infections in patients with SLE or MG is comparable to the healthy population. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Corynebacterium ulcerans cutaneous diphtheria.

    Science.gov (United States)

    Moore, Luke S P; Leslie, Asuka; Meltzer, Margie; Sandison, Ann; Efstratiou, Androulla; Sriskandan, Shiranee

    2015-09-01

    We describe the case of a patient with cutaneous diphtheria caused by toxigenic Corynebacterium ulcerans who developed a right hand flexor sheath infection and symptoms of sepsis such as fever, tachycardia, and elevated C-reactive protein, after contact with domestic cats and dogs, and a fox. We summarise the epidemiology, clinical presentation, microbiology, diagnosis, therapy, and public health aspects of this disease, with emphasis on improving recognition. In many European countries, C ulcerans has become the organism commonly associated with cutaneous diphtheria, usually seen as an imported tropical disease or resulting from contact with domestic and agricultural animals. Diagnosis relies on bacterial culture and confirmation of toxin production, with management requiring appropriate antimicrobial therapy and prompt administration of antitoxin, if necessary. Early diagnosis is essential for implementation of control measures and clear guidelines are needed to assist clinicians in managing clinical diphtheria. This case was a catalyst to the redrafting of the 2014 national UK interim guidelines for the public health management of diphtheria, released as final guidelines in March, 2015. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Mutagenic Deimmunization of Diphtheria Toxin for Use in Biologic Drug Development

    Directory of Open Access Journals (Sweden)

    Joerg U. Schmohl

    2015-10-01

    Full Text Available Background: Targeted toxins require multiple treatments and therefore must be deimmunized. We report a method of protein deimmunization based on the point mutation of highly hydrophilic R, K, D, E, and Q amino acids on the molecular surface of truncated diphtheria-toxin (DT390. Methods: Based on their surface position derived from an X-ray-crystallographic model, residues were chosen for point mutation that were located in prominent positions on the molecular surface and away from the catalytic site. Mice were immunized with a targeted toxin containing either a mutated DT390 containing seven critical point mutations or the non-mutated parental toxin form. Results: Serum analysis revealed a significant 90% reduction in anti-toxin antibodies in mice immunized with the mutant, but not the parental drug form despite multiple immunizations. The experiment was repeated in a second strain of mice with a different MHC-haplotype to address whether point mutation removed T or B cell epitopes. Findings were identical indicating that B cell epitopes were eliminated from DT. The mutant drug form lost only minimal activity in vitro as well as in vivo. Conclusion: These findings indicate that this method may be effective for deimmunizing of other proteins and that discovery of a deimmunized form of DT may lead to the development of more effective targeted toxin.

  15. Diphtheria: forgotten, but not gone.

    Science.gov (United States)

    Adler, N R; Mahony, A; Friedman, N D

    2013-02-01

    Diphtheria is an acute, highly infectious, vaccine-preventable and previously endemic disease whose etiologic agent is Corynebacterium diphtheriae. Diphtheria may manifest as an upper respiratory tract infection, a cutaneous infection or as an asymptomatic carrier state. The most common sites of infection are the pharynx and the tonsils, with common clinical manifestations that include sore throat, malaise, cervical lymphadenopathy and low-grade fever. Absorption and dissemination of C. diphtheriae from the respiratory tract can cause disseminated infection and may lead to cardiac or neurological toxicity. The cornerstone of treatment for diphtheria is diphtheria antitoxin. Early treatment is critical as the degree of protection is inversely proportional to the duration of the illness before its administration. Routine childhood vaccination virtually eliminated diphtheria in most industrialised countries. However, in the pre-vaccination era, diphtheria was the most common infectious cause of death in Australia. A case of diphtheria in Brisbane in April 2011 and two recent positive cultures in regional Victoria underscore the need for heightened awareness of C. diphtheriae as an important pathogen. In order to prevent the re-emergence of diphtheria in Australia, public health measures are required to increase immunity in early school leavers and the adult population, and to ensure that travellers to endemic regions are fully immunised. Health policy-makers and clinicians alike should not underestimate the importance of primary vaccination and booster vaccination against diphtheria among healthy adults and travellers. © 2013 The Authors; Internal Medicine Journal © 2013 Royal Australasian College of Physicians.

  16. Immunogenicity test of tetanus component in adsorbed vaccines by toxin binding inhibition test

    Directory of Open Access Journals (Sweden)

    Denise Cristina Souza Matos

    2002-09-01

    Full Text Available Samples from 20 lots of diphtheria-tetanus (adult use dT vaccine and from 20 lots of diphtheria-tetanus-pertussis (DTP vaccine were used to standardize and validate the in vitro toxin binding inhibition (ToBI test for the immunogenicity test of the tetanus component. The levels of tetanus antitoxin obtained by ToBI test were compared to those obtained using the toxin neutralization (TN test in mice routinely employed to perform the quality control of the tetanus component in adsorbed vaccines. The results ranged from 1.8 to 3.5 IU/ml for dT and 2 to 4 IU/ml for DTP by ToBI test and 1.4 to 3 IU/ml for dT and 1.8 to 3.5 IU/ml for DTP by TN in mice. These results were significantly correlated. From this study, it is concluded that the ToBI test is an alternative to the in vivo neutralization procedure in the immunogenicity test of the tetanus component in adsorbed vaccines. A substantial refinement and a reduction in use of animals can be achieved.

  17. Molecular structure of endotoxins from Gram-negative marine bacteria: an update.

    Science.gov (United States)

    Leone, Serena; Silipo, Alba; L Nazarenko, Evgeny; Lanzetta, Rosa; Parrilli, Michelangelo; Molinaro, Antonio

    2007-09-19

    Marine bacteria are microrganisms that have adapted, through millions of years, to survival in environments often characterized by one or more extreme physical or chemical parameters, namely pressure, temperature and salinity. The main interest in the research on marine bacteria is due to their ability to produce several biologically active molecules, such as antibiotics, toxins and antitoxins, antitumor and antimicrobial agents. Nonetheless, lipopolysaccharides (LPSs), or their portions, from Gram-negative marine bacteria, have often shown low virulence, and represent potential candidates in the development of drugs to prevent septic shock. Besides, the molecular architecture of such molecules is related to the possibility of thriving in marine habitats, shielding the cell from the disrupting action of natural stress factors. Over the last few years, the depiction of a variety of structures of lipids A, core oligosaccharides and O-specific polysaccharides from LPSs of marine microrganisms has been given. In particular, here we will examine the most recently encountered structures for bacteria belonging to the genera Shewanella, Pseudoalteromonas and Alteromonas, of the gamma-Proteobacteria phylum, and to the genera Flavobacterium, Cellulophaga, Arenibacter and Chryseobacterium, of the Cytophaga-Flavobacterium-Bacteroides phylum. Particular attention will be paid to the chemical features expressed by these structures (characteristic monosaccharides, non-glycidic appendages, phosphate groups), to the typifying traits of LPSs from marine bacteria and to the possible correlation existing between such features and the adaptation, over years, of bacteria to marine environments.

  18. Molecular characterization of the pSinB plasmid of the arsenite oxidizing, metallotolerant Sinorhizobium sp. M14 - insight into the heavy metal resistome of sinorhizobial extrachromosomal replicons.

    Science.gov (United States)

    Romaniuk, Krzysztof; Dziewit, Lukasz; Decewicz, Przemyslaw; Mielnicki, Sebastian; Radlinska, Monika; Drewniak, Lukasz

    2017-01-01

    Sinorhizobium sp. M14 is an As(III)-oxidizing, psychrotolerant strain, capable of growth in the presence of extremely high concentrations of arsenic and many other heavy metals. Metallotolerant abilities of the M14 strain depend upon the presence of two extrachromosomal replicons: pSinA (∼ 109 kb) and pSinB (∼ 300 kb). The latter was subjected to complex analysis. The performed analysis demonstrated that the plasmid pSinB is a narrow-host-range repABC-type replicon, which is fully stabilized by the phd-vapC-like toxin-antitoxin stabilizing system. In silico analysis showed that among the phenotypic gene clusters of the plasmid pSinB, eight modules are potentially involved in heavy metals resistance (HMR). These modules carry genes encoding efflux pumps, permeases, transporters and copper oxidases, which provide resistance to arsenic, cadmium, cobalt, copper, iron, mercury, nickel, silver and zinc. The functional analysis revealed that the HMR modules are active and have an effect on the minimal inhibitory concentration (MIC) values observed for the heterological host cells. The phenotype was manifested by an increase or decrease of the MICs of heavy metals and it was strain specific. The analysis of distribution of the heavy metal resistance genes, i.e. resistome, in Sinorhizobium spp. plasmids, revealed that the HMR modules are common in these replicons. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Veterinary immunology as colonial science: method and quantification in the investigation of horsesickness in South Africa, C. 1905-1945.

    Science.gov (United States)

    Gilfoyle, Daniel

    2006-01-01

    This article examines the practice of veterinary immunology in South Africa during the first half of the twentieth century through an analysis of research into a horsesickness vaccine at the Onderstepoort Veterinary Institute. From the early 1900s, Arnold Theiler prioritized research into horsesickness, by then defined as an insect-borne disease caused by an ultravisible virus. He succeeded in devising a means of prophylaxis using a simultaneous injection of infective blood and immune serum, but he discovered antigenically different strains of the virus, which could overcome the immunity produced by his treatment. The practical value of Theiler's methods was further limited by difficulties in standardizing the biological material used in immunization, the results of which remained too erratic for application on a large scale. No further advances were made until the 1930s, by which time Onderstepoort had been drawn more closely into international scientific networks. Using techniques derived from research into yellow fever in America and canine distemper in Britain, the Onderstepoort scientist Raymond Alexander invented a method of immunization that utilized the propagation of the horsesickness virus in the brains of mice. Alexander's methods, which were characterized by successful technical adaptation and innovation, depended upon methods of quantification first developed by Paul Ehrlich to standardize diphtheria antitoxin during the 1890s. During the 1940s, vaccination expanded rapidly in South Africa, and Onderstepoort later exported the vaccine and associated technology to other countries affected by horsesickness.

  20. Affinity-purified tetanus neurotoxin interaction with synaptic membranes: properties of a protease-sensitive receptor component

    International Nuclear Information System (INIS)

    Lazarovici, P.; Yavin, E.

    1986-01-01

    The pharmacokinetic interaction of an affinity-purified 125 I-labeled tetanotoxin fraction with guinea pig brain synaptosomal preparations was investigated. Binding of tetanotoxin was time- and temperature-dependent, was proportional to protein concentration, and was saturable at about 8 x 10 -9 M as estimated by a solid-surface binding assay. Binding was optimal at pH 6.5 under low ionic strength buffer and was almost entirely blocked by gangliosides or antitoxin. In analogy to intact nerve cells, binding of toxin to membranes resulted in a tight association operationally defined as sequestration. Binding and sequestration were abolished after membrane pretreatment with sialidase. The enzyme could not dissociate the membrane-bound toxin formed at 4 or 37 0 C under low ionic strength conditions, which is in part compatible with internalization as defined in nerve cell cultures. In the latter system the toxin could be removed at 4 0 C but not at 37 0 C. Binding was significantly reduced upon pretreatment of guinea pig brain membranes by a variety of hydrolytic enzymes. It is proposed that, in addition to a ganglioside, interaction of tetanotoxin with synaptic membranes is facilitated by a protein and may also require an appropriate lipid environment. These latter membrane constituents may play a pivotal role in the sequestration of the toxin

  1. A radioimmunoassay for tetanus-antibodies using protein A-containing Staphylococcus aureus

    International Nuclear Information System (INIS)

    Habermann, E.; Horvath, E.; Schaeg, W.

    1977-01-01

    To measure tetanus antibodies a trace amount of 125 I-labeled tetanus toxin is mixed with appropriate dilutions of human serum or blood. The labeled antigen-antibody complexes are adsorbed to heat-killed staphylococci (Cowan I) via their surface protein A. The radioactivity of the washed solid phase is a function of the initial antibody concentration. The test allows the measurement of 6 x 10 -0 U of tetanus antitoxin in a volume of 0.03 ml. In order to avoid possible interferences, serum has to be diluted 20-fold before use. Taking that into account the real border limit of sensitivity is 4 x 10 -3 U/ml serum. Antibodies may be measured in serum, in plasma, and even in heparinized blood. As to its sensitivity, the test compares well with the toxin neutralization procedure. It is superior to the previous radioimmunologic, enzymoimmunologic, and hemagglutination techniques with respect to sensitivity and reproducibility. It reflects the values obtained in the toxin neutralization test better than the other in vitro procedures, as shown by parallel assays of 17 sera. (orig.) [de

  2. [Tetanus after cat scratch and bites in a previously immunized patient].

    Science.gov (United States)

    Fica, Alberto; Gaínza, Daniela; Ortigosa, Pablo

    2017-04-01

    Tetanus is declining due to vaccination, professional labor management and appropriate wound care. Tetanus cases have been reported despite immunization. We report the case of a previously healthy 21 years old female patient that presented a mild generalized tetanus requiring admission after mild and recurrent cat scratch and bites. She had received six vaccine shots during childhood, and a booster dose five years earlier after a rabbit bite. Symptoms appeared seven weeks after the last contact, and included headache, muscle spasms and mild opisthotonus. Laboratory evaluation, including CSF analysis and microbiological investigation, as well as imaging studies were all normal. The patient received 6,000 IU of human antitoxin immunoglobulin. No autonomic manifestations or respiratory compromise were registered. Symptoms resolved rapidly and she was discharge after seven days with an order to complete a tetanus toxoid immunization schedule with three doses. Tetanus is possible in urban settings with a declining epidemiologic curve of disease in previously immunized patients. Severity of disease is modulated by previous vaccination.

  3. Distribution of Tritiated Tetanus Toxin Following an Intraperitoneal Injection in Immunized and Non-Immunized Mice

    International Nuclear Information System (INIS)

    Speirs, R.S.

    1962-01-01

    Tetanus toxin, purified by ultra-filtration and precipitation with ammonium sulphate, was lyophilized and exposed to 5 c of tritium gas (Wilzbach procedure) for 2½ d in a deep freeze cabinet at 0.38 atm of pressure. The toxin was then homogenized and the precipitated material removed by filtration through a HA millipore membrane. The filtrate was washed and concentrated in a membrane colloidal dialyzer. The resuspended material (particles estimated to be below 450 mpm in size) was highly toxic when injected into mice. Both the crude precipitate and the suspended toxin were injected into immunized mice and the animals autopsied at various times to determine the presence of radioactivity in the various inflammatory cells. These results were compared with those obtained when the toxins were neutralized and injected into non-immunized mice. In the inflammatory area produced by the injection of tritiated toxin, neutrophils containing radioactivity were found during the first 2 d, and macrophages containing radioactivity were found in the spleen as well as in the inflammatory area for as long as 15 d. Trace amounts of radioactivity were found in eosinophils. No radioactivity was found within mast cells in either the immunized or non-immunized animals. The significance of these results will be discussed in relation to initiation of antitoxin production. (author) [fr

  4. New insights into the mechanisms of acetic acid resistance in Acetobacter pasteurianus using iTRAQ-dependent quantitative proteomic analysis.

    Science.gov (United States)

    Xia, Kai; Zang, Ning; Zhang, Junmei; Zhang, Hong; Li, Yudong; Liu, Ye; Feng, Wei; Liang, Xinle

    2016-12-05

    Acetobacter pasteurianus is the main starter in rice vinegar manufacturing due to its remarkable abilities to resist and produce acetic acid. Although several mechanisms of acetic acid resistance have been proposed and only a few effector proteins have been identified, a comprehensive depiction of the biological processes involved in acetic acid resistance is needed. In this study, iTRAQ-based quantitative proteomic analysis was adopted to investigate the whole proteome of different acidic titers (3.6, 7.1 and 9.3%, w/v) of Acetobacter pasteurianus Ab3 during the vinegar fermentation process. Consequently, 1386 proteins, including 318 differentially expressed proteins (pacetic acid stress, where >150 proteins were differentially expressed. Specifically, proteins involved in amino acid metabolic processes and fatty acid biosynthesis were differentially expressed, which may contribute to the acetic acid resistance of Acetobacter. Transcription factors, two component systems and toxin-antitoxin systems were implicated in the modulatory network at multiple levels. In addition, the identification of proteins involved in redox homeostasis, protein metabolism, and the cell envelope suggested that the whole cellular system is mobilized in response to acid stress. These findings provide a differential proteomic profile of acetic acid resistance in Acetobacter pasteurianus and have potential application to highly acidic rice vinegar manufacturing. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Ethanol-induced stress response of Staphylococcus aureus.

    Science.gov (United States)

    Pando, Jasmine M; Pfeltz, Richard F; Cuaron, Jesus A; Nagarajan, Vijayaraj; Mishra, Mukti N; Torres, Nathanial J; Elasri, Mohamed O; Wilkinson, Brian J; Gustafson, John E

    2017-09-01

    Transcriptional profiles of 2 unrelated clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were analyzed following 10% (v/v) ethanol challenge (15 min), which arrested growth but did not reduce viability. Ethanol-induced stress (EIS) resulted in differential gene expression of 1091 genes, 600 common to both strains, of which 291 were upregulated. With the exception of the downregulation of genes involved with osmotic stress functions, EIS resulted in the upregulation of genes that contribute to stress response networks, notably those altered by oxidative stress, protein quality control in general, and heat shock in particular. In addition, genes involved with transcription, translation, and nucleotide biosynthesis were downregulated. relP, which encodes a small alarmone synthetase (RelP), was highly upregulated in both MRSA strains following ethanol challenge, and relP inactivation experiments indicated that this gene contributed to EIS growth arrest. A number of persistence-associated genes were also upregulated during EIS, including those that encode toxin-antitoxin systems. Overall, transcriptional profiling indicated that the MRSA investigated responded to EIS by entering a state of dormancy and by altering the expression of elements from cross protective stress response systems in an effort to protect preexisting proteins.

  6. Ca2+ influx and tyrosine kinases trigger Bordetella adenylate cyclase toxin (ACT endocytosis. Cell physiology and expression of the CD11b/CD18 integrin major determinants of the entry route.

    Directory of Open Access Journals (Sweden)

    Kepa B Uribe

    Full Text Available Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3, its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca(2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.

  7. Genomics of Clostridium tetani.

    Science.gov (United States)

    Brüggemann, Holger; Brzuszkiewicz, Elzbieta; Chapeton-Montes, Diana; Plourde, Lucile; Speck, Denis; Popoff, Michel R

    2015-05-01

    Genomic information about Clostridium tetani, the causative agent of the tetanus disease, is scarce. The genome of strain E88, a strain used in vaccine production, was sequenced about 10 years ago. One additional genome (strain 12124569) has recently been released. Here we report three new genomes of C. tetani and describe major differences among all five C. tetani genomes. They all harbor tetanus-toxin-encoding plasmids that contain highly conserved genes for TeNT (tetanus toxin), TetR (transcriptional regulator of TeNT) and ColT (collagenase), but substantially differ in other plasmid regions. The chromosomes share a large core genome that contains about 85% of all genes of a given chromosome. The non-core chromosome comprises mainly prophage-like genomic regions and genes encoding environmental interaction and defense functions (e.g. surface proteins, restriction-modification systems, toxin-antitoxin systems, CRISPR/Cas systems) and other fitness functions (e.g. transport systems, metabolic activities). This new genome information will help to assess the level of genome plasticity of the species C. tetani and provide the basis for detailed comparative studies. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  8. Generalized Tetanus in a Gyrfalcon ( Falco rusticolus ) with Pododermatitis.

    Science.gov (United States)

    Beaufrère, Hugues; Laniesse, Delphine; Stickings, Paul; Tierney, Robert; Sesardic, Thea; Slavic, Durda; Compo, Nicole; Smith, Dale A

    2016-12-01

    A 2-yr-old male gyrfalcon ( Falco rusticolus ) was presented for severe and generalized muscle spasticity and pododermatitis. The falcon had been treated for pododermatitis over the previous 4 mo. Muscle rigidity and spasms involved the entire bird but were more severe on the right leg. The bird was also tachypneic and hyperthermic at 45 C. While the plantar pododermatitis lesions had healed, there was still a small abscess on the lateral aspect of the right foot. Clinical signs were consistent with tetanus. Several bacteria were isolated from the abscess including Clostridium tetani . The isolate was confirmed to be toxigenic by PCR. Attempts to detect tetanus toxin in the bird's plasma were unsuccessful. The abscess was debrided. The gyrfalcon received equine tetanus antitoxin, intravenous metronidazole, methocarbamol, midazolam, a constant-rate infusion of Fentanyl, active cooling, and supportive care. Inhalant anesthesia with isoflurane was the only treatment that would lower the body temperature and reduce the clinical signs. The gyrfalcon died a few hours after admission. The characteristic clinical signs and isolation of toxigenic C. tetani from a wound were strong supportive evidence for a diagnosis of tetanus. This case constitutes the first reported natural occurrence of tetanus in an avian species. Further information is needed to determine whether gyrfalcons are more susceptible to tetanus than are other avian species and whether pododermatitis lesions may be risk factors.

  9. View from the front lines: an emergency medicine perspective on clostridial infections in injection drug users.

    Science.gov (United States)

    Gonzales y Tucker, Richard Diego; Frazee, Bradley

    2014-12-01

    Injection drug use (IDU), specifically non-intravenous "skin-popping" of heroin, seems to provide optimal conditions for Clostridial infection and toxin production. IDU is therefore a major risk factor for wound botulism and Clostridial necrotizing soft tissue infections (NSTI) and continues to be linked to cases of tetanus. Case clusters of all 3 diseases have occurred among IDUs in Western U.S. and Europe. Medical personnel who care for the IDU population must be thoroughly familiar with the clinical presentation and management of these diseases. Wound botulism presents with bulbar symptoms and signs that are easily overlooked; rapid acquisition and administration of antitoxin can prevent neuromuscular respiratory failure. In addition to Clostridium perfringens, IDU-related NSTIs can be caused by Clostridium sordellii and Clostridium novyi, which may share a distinct clinical presentation. Early definitive NSTI management, which decreases mortality, requires a low index of suspicion on the part of emergency physicians and low threshold for surgical exploration and debridement on the part of the surgeon. Tetanus should be preventable in the IDU population through careful attention to vaccination status. Published by Elsevier Ltd.

  10. Camelid-derived heavy-chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris.

    Science.gov (United States)

    Baghban, Roghayyeh; Gargari, Seyed Latif Mousavi; Rajabibazl, Masoumeh; Nazarian, Shahram; Bakherad, Hamid

    2016-01-01

    Botulinum neurotoxins (BoNTs) result in severe and often fatal disease, botulism. Common remedial measures such as equine antitoxin and human botulism immunoglobulin in turn are problematic and time-consuming. Therefore, diagnosis and therapy of BoNTs are vital. The variable domain of heavy-chain antibodies (VHH) has unique features, such as the ability to identify and bind specifically to target epitopes and ease of production in bacteria and yeast. The Pichia pastoris is suitable for expression of recombinant antibody fragments. Disulfide bond formation and correct folds of protein with a high yield are some of the advantages of this eukaryotic host. In this study, we have expressed and purified the camelid VHH against BoNT/E in P. pastoris. The final yield of P. pastoris-expressed antibody was estimated to be 16 mg/l, which is higher than that expressed by Escherichia coli. The nanobody expressed in P. pastoris neutralized 4LD50 of the BoNT/E upon i.p. injection in 25% of mice. The nanobody expressed in E. coli extended the mice's survival to 1.5-fold compared to the control. This experiment indicated that the quality of expressed protein in the yeast is superior to that of the bacterial expression. Favorable protein folding by P. pastoris seems to play a role in its better toxin-binding property. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  11. Ca2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route

    Science.gov (United States)

    Etxebarria, Aitor; González-Bullón, David; Gómez-Bilbao, Geraxane; Ostolaza, Helena

    2013-01-01

    Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT) which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3), its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface. PMID:24058533

  12. HASIL GUNA IMUNISASI DASAR DIFTERI DENGAN VAKSIN DPT 2 DOSIS

    Directory of Open Access Journals (Sweden)

    Dyah W. Isbagio

    2012-09-01

    Full Text Available A retrospective study on a basic diptheria immunization was done in Tulangan district of East Jawa, involving three hundred ninety two children with various immune status, that say 0,1 and 2 doses of DPT vaccine manufactured by Perum. Bio Farma, Bandung. The study was aimed to measure serological effectiviness 2 of doses of DPT vaccine for inducing immunity against diphtheria in real life situation. Diphtheria antitoxin titre was examined by Passive Haemagglutination Assay on 0,1 ml of capillary blood specimens. The result showed that 2 doses of DPT vaccine given at 1-3 months interval on children of 3—14 months old, were able to induce an adequate immune response on more than 80% of the children. Immune response with 0 and 1 doses of DPT vaccine has also been discussed. The potensial implication of this study result, supported by other study on a various of age, on the administration strategy of booster dose of Td ("adult type" vaccine among school children has also been suggested.

  13. PENETAPAN STANDAR NASIONAL VAKSIN DIFTERI, PERTUSSIS, TETANUS: Penetapan Standar Nasional Toksoid Serap Difteri

    Directory of Open Access Journals (Sweden)

    Muljati Prijanto

    2012-09-01

    Full Text Available To check the potency of DPT vaccine, standard preparations of the components, namely Adsorbed Diphtheria Toxoid, Pertussis Vaccine and Adsorbed Tetanus Toxoid, are needed. Since WHO International Standard Preparations are distributed only in limited amounts, WHO has suggested that each member country should develop a National Standard, which is matched against In­ternational Standard Preparation. An Indonesian National Standard of DPT vaccine (lot 1 has been prepared and lyophilized at the National Institute of Health in Tokyo. The potency of the National Standard of Adsorbed Diphtheria Toxoid is determined by antibody ti­tration in guinea pigs. Antitoxin titres are determined by cell culture method. After several experiments, the potency of the National Standard of Adsorbed Diphtheria Toxoid has been decided of being 59 IU/ml. Using the same standard preparations, namely the National Standards, it is hoped that from the same lot of DPT vaccine, similar results of potency could be achieved when determined by the Government Vaccine Quality Control laboratory and the Manufacturer's laboratory.

  14. Massive Activation of Archaeal Defense Genes during Viral Infection

    Science.gov (United States)

    Voet, Marleen; Sismeiro, Odile; Dillies, Marie-Agnes; Jagla, Bernd; Coppée, Jean-Yves; Sezonov, Guennadi; Forterre, Patrick; van der Oost, John; Lavigne, Rob

    2013-01-01

    Archaeal viruses display unusually high genetic and morphological diversity. Studies of these viruses proved to be instrumental for the expansion of knowledge on viral diversity and evolution. The Sulfolobus islandicus rod-shaped virus 2 (SIRV2) is a model to study virus-host interactions in Archaea. It is a lytic virus that exploits a unique egress mechanism based on the formation of remarkable pyramidal structures on the host cell envelope. Using whole-transcriptome sequencing, we present here a global map defining host and viral gene expression during the infection cycle of SIRV2 in its hyperthermophilic host S. islandicus LAL14/1. This information was used, in combination with a yeast two-hybrid analysis of SIRV2 protein interactions, to advance current understanding of viral gene functions. As a consequence of SIRV2 infection, transcription of more than one-third of S. islandicus genes was differentially regulated. While expression of genes involved in cell division decreased, those genes playing a role in antiviral defense were activated on a large scale. Expression of genes belonging to toxin-antitoxin and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems was specifically pronounced. The observed different degree of activation of various CRISPR-Cas systems highlights the specialized functions they perform. The information on individual gene expression and activation of antiviral defense systems is expected to aid future studies aimed at detailed understanding of the functions and interplay of these systems in vivo. PMID:23698312

  15. In vitro Determination of Extracellular Proteins from Xylella fastidiosa

    Science.gov (United States)

    Mendes, Juliano S.; Santiago, André S.; Toledo, Marcelo A. S.; Horta, Maria A. C.; de Souza, Alessandra A.; Tasic, Ljubica; de Souza, Anete P.

    2016-01-01

    The phytopathogen Xylella fastidiosa causes economic losses in important agricultural crops. Xylem vessel occlusion caused by biofilm formation is the major mechanism underlying the pathogenicity of distinct strains of X. fastidiosa. Here, we provide a detailed in vitro characterization of the extracellular proteins of X. fastidiosa. Based on the results, we performed a comparison with a strain J1a12, which cannot induce citrus variegated chlorosis symptoms when inoculated into citrus plants. We then extend this approach to analyze the extracellular proteins of X. fastidiosa in media supplemented with calcium. We verified increases in extracellular proteins concomitant with the days of growth and, consequently, biofilm development (3–30 days). Outer membrane vesicles carrying toxins were identified beginning at 10 days of growth in the 9a5c strain. In addition, a decrease in extracellular proteins in media supplemented with calcium was observed in both strains. Using mass spectrometry, 71 different proteins were identified during 30 days of X. fastidiosa biofilm development, including proteases, quorum-sensing proteins, biofilm formation proteins, hypothetical proteins, phage-related proteins, chaperones, toxins, antitoxins, and extracellular vesicle membrane components. PMID:28082960

  16. Global expression profile of biofilm resistance to antimicrobial compounds in the plant-pathogenic bacterium Xylella fastidiosa reveals evidence of persister cells.

    Science.gov (United States)

    Muranaka, Lígia S; Takita, Marco A; Olivato, Jacqueline C; Kishi, Luciano T; de Souza, Alessandra A

    2012-09-01

    Investigations of biofilm resistance response rarely focus on plant-pathogenic bacteria. Since Xylella fastidiosa is a multihost plant-pathogenic bacterium that forms biofilm in the xylem, the behavior of its biofilm in response to antimicrobial compounds needs to be better investigated. We analyzed here the transcriptional profile of X. fastidiosa subsp. pauca in response to inhibitory and subinhibitory concentrations of copper and tetracycline. Copper-based products are routinely used to control citrus diseases in the field, while antibiotics are more widely used for bacterial control in mammals. The use of antimicrobial compounds triggers specific responses to each compound, such as biofilm formation and phage activity for copper. Common changes in expression responses comprise the repression of genes associated with metabolic functions and movement and the induction of toxin-antitoxin systems, which have been associated with the formation of persister cells. Our results also show that these cells were found in the population at a ca. 0.05% density under inhibitory conditions for both antimicrobial compounds and that pretreatment with subinhibitory concentration of copper increases this number. No previous report has detected the presence of these cells in X. fastidiosa population, suggesting that this could lead to a multidrug tolerance response in the biofilm under a stressed environment. This is a mechanism that has recently become the focus of studies on resistance of human-pathogenic bacteria to antibiotics and, based on our data, it seems to be more broadly applicable.

  17. Specific Gene Loci of Clinical Pseudomonas putida Isolates

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    Molina, Lázaro; Udaondo, Zulema; Duque, Estrella; Fernández, Matilde; Bernal, Patricia; Roca, Amalia; de la Torre, Jesús; Ramos, Juan Luis

    2016-01-01

    Pseudomonas putida are ubiquitous inhabitants of soils and clinical isolates of this species have been seldom described. Clinical isolates show significant variability in their ability to cause damage to hosts because some of them are able to modulate the host’s immune response. In the current study, comparisons between the genomes of different clinical and environmental strains of P. putida were done to identify genetic clusters shared by clinical isolates that are not present in environmental isolates. We show that in clinical strains specific genes are mostly present on transposons, and that this set of genes exhibit high identity with genes found in pathogens and opportunistic pathogens. The set of genes prevalent in P. putida clinical isolates, and absent in environmental isolates, are related with survival under oxidative stress conditions, resistance against biocides, amino acid metabolism and toxin/antitoxin (TA) systems. This set of functions have influence in colonization and survival within human tissues, since they avoid host immune response or enhance stress resistance. An in depth bioinformatic analysis was also carried out to identify genetic clusters that are exclusive to each of the clinical isolates and that correlate with phenotypical differences between them, a secretion system type III-like was found in one of these clinical strains, a determinant of pathogenicity in Gram-negative bacteria. PMID:26820467

  18. Specific Gene Loci of Clinical Pseudomonas putida Isolates.

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    Lázaro Molina

    Full Text Available Pseudomonas putida are ubiquitous inhabitants of soils and clinical isolates of this species have been seldom described. Clinical isolates show significant variability in their ability to cause damage to hosts because some of them are able to modulate the host's immune response. In the current study, comparisons between the genomes of different clinical and environmental strains of P. putida were done to identify genetic clusters shared by clinical isolates that are not present in environmental isolates. We show that in clinical strains specific genes are mostly present on transposons, and that this set of genes exhibit high identity with genes found in pathogens and opportunistic pathogens. The set of genes prevalent in P. putida clinical isolates, and absent in environmental isolates, are related with survival under oxidative stress conditions, resistance against biocides, amino acid metabolism and toxin/antitoxin (TA systems. This set of functions have influence in colonization and survival within human tissues, since they avoid host immune response or enhance stress resistance. An in depth bioinformatic analysis was also carried out to identify genetic clusters that are exclusive to each of the clinical isolates and that correlate with phenotypical differences between them, a secretion system type III-like was found in one of these clinical strains, a determinant of pathogenicity in Gram-negative bacteria.

  19. Molecular characterization of a 21.4 kilobase antibiotic resistance plasmid from an α-hemolytic Escherichia coli O108:H- human clinical isolate.

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    Fay E Dawes

    Full Text Available This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-. DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes.

  20. pEOC01: a plasmid from Pediococcus acidilactici which encodes an identical streptomycin resistance (aadE) gene to that found in Campylobacter jejuni.

    Science.gov (United States)

    O'Connor, E B; O'Sullivan, O; Stanton, C; Danielsen, M; Simpson, P J; Callanan, M J; Ross, R P; Hill, C

    2007-09-01

    The complete nucleotide sequence of pEOC01, a plasmid (11,661 bp) from Pediococcus acidilactici NCIMB 6990 encoding resistance to clindamycin, erythromycin, and streptomycin was determined. The plasmid, which also replicates in Lactococcus and Lactobacillus species contains 16 putative open reading frames (ORFs), including regions annotated to encode replication, plasmid maintenance and multidrug resistance functions. Based on an analysis the plasmid replicates via a theta replicating mechanism closely related to those of many larger Streptococcus and Enterococcus plasmids. Interestingly, genes homologous to a toxin/antitoxin plasmid maintenance system are present and are highly similar to the omega-epsilon-zeta operon of Streptococcus plasmids. The plasmid contains two putative antibiotic resistance homologs, an ermB gene encoding erythromycin and clindamycin resistance, and a streptomycin resistance gene, aadE. Of particular note is the aadE gene which holds 100% identity to an aadE gene found in Campylobacter jejuni plasmid but which probably originated from a Gram-positive source. This observation is significant in that it provides evidence for recent horizontal transfer of streptomycin resistance from a lactic acid bacterium to a Gram-negative intestinal pathogen and as such infers a role for such plasmids for dissemination of antibiotic resistance genes possibly in the human gut.

  1. [Clinical analysis of 53 patients with Clostridium botulinum food poisoning].

    Science.gov (United States)

    Zhang, Peng; Dong, Jianguang; Bai, Lili; Qiu, Zewu

    2017-05-01

    .57±39.52 vs. 6.61±3.72, WBC (×10 9 /L): 13.01±6.44 vs. 6.85±2.07, NEUT: 0.85±0.07 vs. 0.63±0.14, RR (bpm): 32.14±4.33 vs. 15.18±1.70, HR (bpm): 132.29±5.19 vs. 75.54±8.24, FiO 2 : 0.32±0.05 vs. 0.21±0.00, ALB (g/L): 38.57±4.65 vs. 42.09±4.57, pH: 7.08±0.10 vs. 7.38±0.07, PaO 2 (mmHg, 1 mmHg = 0.133 kPa): 75.16±5.24 vs. 98.39±1.50, SaO 2 : 0.78±0.06 vs. 0.97±0.02, all P food borne botulism was dizziness, followed by fatigue, blurred vision, nausea, and other symptoms and signs were lower than 50%, and the occurrence of dizziness with rank one happen rate was significantly higher than blurred vision and nausea (χ 2 values were 7.209 and 10.502 respectively, and P values were 0.007 and 0.004 respectively). After the on time prescription of botulinum antitoxins treatment, the clinical symptoms of patients could be relived quickly. All the patients were discharged without deaths. In order to improve the recovery of the food borne botulism poisoning patients, adequate antitoxin and the related organ supports should be prescribed on time.

  2. Viability, biofilm formation, and MazEF expression in drug-sensitive and drug-resistant Mycobacterium tuberculosis strains circulating in Xinjiang, China.

    Science.gov (United States)

    Zhao, Ji-Li; Liu, Wei; Xie, Wan-Ying; Cao, Xu-Dong; Yuan, Li

    2018-01-01

    Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is one of the most common chronic infectious amphixenotic diseases worldwide. Prevention and control of TB are greatly difficult, due to the increase in drug-resistant TB, particularly multidrug-resistant TB. We speculated that there were some differences between drug-sensitive and drug-resistant MTB strains and that mazEF 3,6,9 toxin-antitoxin systems (TASs) were involved in MTB viability. This study aimed to investigate differences in viability, biofilm formation, and MazEF expression between drug-sensitive and drug-resistant MTB strains circulating in Xinjiang, China, and whether mazEF 3,6,9 TASs contribute to MTB viability under stress conditions. Growth profiles and biofilm-formation abilities of drug-sensitive, drug-resistant MTB strains and the control strain H37Rv were monitored. Using molecular biology experiments, the mRNA expression of the mazF 3, 6, and 9 toxin genes, the mazE 3, 6, and 9 antitoxin genes, and expression of the MazF9 protein were detected in the different MTB strains, H37RvΔ mazEF 3,6,9 mutants from the H37Rv parent strain were generated, and mutant viability was tested. Ex vivo culture analyses demonstrated that drug-resistant MTB strains exhibit higher survival rates than drug-sensitive strains and the control strain H37Rv. However, there was no statistical difference in biofilm-formation ability in the drug-sensitive, drug-resistant, and H37Rv strains. mazE 3,6 mRNA-expression levels were relatively reduced in the drug-sensitive and drug-resistant strains compared to H37Rv. Conversely, mazE 3,9 expression was increased in drug-sensitive strains compared to drug-resistant strains. Furthermore, compared with the H37Rv strain, mazF 3,6 expression was increased in drug-resistant strains, mazF 9 expression was increased in drug-sensitive strains, and mazF 9 exhibited reduced expression in drug-resistant strains compared with drug-sensitive strains. Protein expression of mazF9

  3. Analysis of pCERC7, a small antibiotic resistance plasmid from a commensal ST131 Escherichia coli, defines a diverse group of plasmids that include various segments adjacent to a multimer resolution site and encode the same NikA relaxase accessory protein enabling mobilisation.

    Science.gov (United States)

    Moran, Robert A; Hall, Ruth M

    2017-01-01

    The ampicillin resistance plasmid pCERC7, carrying transposon Tn2 with an IS4 insertion, was detected in the draft genome of a commensal Escherichia coli isolate. The genome data also revealed that this isolate belongs to ST131, clade B. pCERC7 is 9712bp comprised of a 3319bp backbone, Tn2::IS4 (6388bp) and 5bp of target site duplication, and was present at a copy number of 40. pCERC7 is related to several plasmids composed of only the backbone, or the backbone with the Tn2 insertion in the same position. These plasmids have been found previously in Escherichia coli or Salmonella enterica recovered in several different countries from as early as the 1970s. This group was named the NTP16 group after the best studied example. pCERC7 was annotated using available information about plasmids in this group and additional analyses. The backbone includes genes for RNA I and RNA II to initiate replication and the Tn2 interrupts a gene found here to encode a protein 66% identical to the Rom regulatory protein of ColE1. NTP16 family plasmids include a gene, previously designated mobA, that was found to encode a homologue (53% identical) of the NikA relaxase accessory protein of the conjugative IncI1 plasmid R64, which is known to bind to the R64 oriT. However, a nikB relaxase gene is not present, indicating that a relaxase must be supplied in trans for mobilisation by R64 to occur, as demonstrated previously for NTP16. Hence, MobA of NTP16 and relatives was renamed NikA. Upstream of nikA, we found a region closely related to the oriT of R64. pCERC7 and all members of the NTP16 family also include a multimer resolution site, nmr, similar to the cer site of ColE1. The backbone of the NTP16 family also includes genes for a demonstrated toxin-antitoxin system, LsoAB. Several more distantly related groups of plasmids that include a very closely related nmr-nikA-oriT segment (99.4-93.7% DNA identity) were identified in the GenBank non-redundant DNA database. All use an RNA I/RNA II

  4. Transcriptome Mapping of pAR060302, a blaCMY-2-Positive Broad-Host-Range IncA/C Plasmid

    Science.gov (United States)

    Lang, Kevin S.; Danzeisen, Jessica L.; Xu, Wayne

    2012-01-01

    The multidrug resistance-encoding plasmids belonging to the IncA/C incompatibility group have recently emerged among Escherichia coli and Salmonella enterica strains in the United States. These plasmids have a unique genetic structure compared to other enterobacterial plasmid types, a broad host range, and a propensity to acquire large numbers of antimicrobial resistance genes via their accessory regions. Using E. coli strain DH5α harboring the prototype IncA/C plasmid pAR060302, we sought to define the baseline transcriptome of IncA/C plasmids under laboratory growth and in the face of selective pressure. The effects of ampicillin, florfenicol, or streptomycin exposure were compared to those on cells left untreated at logarithmic phase using Illumina platform-based RNA sequencing (RNA-Seq). Under growth in Luria-Bertani broth lacking antibiotics, much of the backbone of pAR060302 was transcriptionally inactive, including its putative transfer regions. A few plasmid backbone genes of interest were highly transcribed, including genes of a putative toxin-antitoxin system and an H-NS-like transcriptional regulator. In contrast, numerous genes within the accessory regions of pAR060302 were highly transcribed, including the resistance genes floR, blaCMY-2, aadA, and aacA. Treatment with ampicillin or streptomycin resulted in no genes being differentially expressed compared to controls lacking antibiotics, suggesting that many of the resistance-associated genes are not differentially expressed due to exposure to these antibiotics. In contrast, florfenicol treatment resulted in the upregulation of floR and numerous chromosomal genes. Overall, the transcriptome mapping of pAR060302 suggests that it mitigates the fitness costs of carrying resistance-associated genes through global regulation with its transcriptional regulators. PMID:22344651

  5. A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes.

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    Yongfeng Fan

    Full Text Available Human botulism is primarily caused by botulinum neurotoxin (BoNT serotypes A, B and E, with around 1% caused by serotype F (BoNT/F. BoNT/F comprises at least seven different subtypes with the amino acid sequence difference between subtypes as high as 36%. The sequence differences present a significant challenge for generating monoclonal antibodies (mAbs that can bind, detect and neutralize all BoNT/F subtypes. We used repertoire cloning of immune mouse antibody variable (V regions and yeast display to generate a panel of 33 lead single chain Fv (scFv mAbs that bound one or more BoNT/F subtypes with a median equilibrium dissociation constant (KD of 4.06 × 10-9 M. By diversifying the V-regions of the lead mAbs and selecting for cross reactivity we generated five mAbs that bound each of the seven subtypes. Three scFv binding non-overlapping epitopes were converted to IgG that had KD for the different BoNT/F subtypes ranging from 2.2×10-8 M to 1.47×10-12 pM. An equimolar combination of the mAbs was able to potently neutralize BoNT/F1, F2, F4 and F7 in the mouse neutralization assay. The mAbs have potential utility as diagnostics capable of recognizing the known BoNT/F subtypes and could be developed as antitoxins to prevent and treat type F botulism.

  6. A Nonsynonymous SNP Catalog of Mycobacterium tuberculosis Virulence Genes and Its Use for Detecting New Potentially Virulent Sublineages.

    Science.gov (United States)

    Mikheecheva, Natalya E; Zaychikova, Marina V; Melerzanov, Alexander V; Danilenko, Valery N

    2017-04-01

    Mycobacterium tuberculosis is divided into several distinct lineages, and various genetic markers such as IS-elements, VNTR, and SNPs are used for lineage identification. We propose an M. tuberculosis classification approach based on functional polymorphisms in virulence genes. An M. tuberculosis virulence genes catalog has been established, including 319 genes from various protein groups, such as proteases, cell wall proteins, fatty acid and lipid metabolism proteins, sigma factors, toxin-antitoxin systems. Another catalog of 1,573 M. tuberculosis isolates of different lineages has been developed. The developed SNP-calling program has identified 3,563 nonsynonymous SNPs. The constructed SNP-based phylogeny reflected the evolutionary relationship between lineages and detected new sublineages. SNP analysis of sublineage F15/LAM4/KZN revealed four lineage-specific mutations in cyp125, mce3B, vapC25, and vapB34. The Ural lineage has been divided into two geographical clusters based on different SNPs in virulence genes. A new sublineage, B0/N-90, was detected inside the Beijing-B0/W-148 by SNPs in irtB, mce3F and vapC46. We have found 27 members of B0/N-90 among the 227 available genomes of the Beijing-B0/W-148 sublineage. Whole-genome sequencing of strain B9741, isolated from an HIV-positive patient, was demonstrated to belong to the new B0/N-90 group. A primer set for PCR detection of B0/N-90 lineage-specific mutations has been developed. The prospective use of mce3 mutant genes as genetically engineered vaccine is discussed. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  7. Conserved inhibitory mechanism and competent ATP binding mode for adenylyltransferases with Fic fold.

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    Arnaud Goepfert

    Full Text Available The ubiquitous FIC domain is evolutionarily conserved from bacteria to human and has been shown to catalyze AMP transfer onto protein side-chain hydroxyl groups. Recently, it was predicted that most catalytically competent Fic proteins are inhibited by the presence of an inhibitory helix αinh that is provided by a cognate anti-toxin (class I, or is part of the N- or C-terminal part of the Fic protein itself (classes II and III. In vitro, inhibition is relieved by mutation of a conserved glutamate of αinh to glycine. For the class III bacterial Fic protein NmFic from Neisseria meningitidis, the inhibitory mechanism has been elucidated. Here, we extend above study by including bacterial class I and II Fic proteins VbhT from Bartonella schoenbuchensis and SoFic from Shewanella oneidensis, respectively, and the respective E->G mutants. Comparative enzymatic and crystallographic analyses show that, in all three classes, the ATP substrate binds to the wild-type FIC domains, but with the α-phosphate in disparate and non-competent orientations. In the E->G mutants, however, the tri-phosphate moiety is found reorganized to the same tightly bound structure through a unique set of hydrogen bonds with Fic signature motif residues. The γ-phosphate adopts the location that is taken by the inhibitory glutamate in wild-type resulting in an α-phosphate orientation that can be attacked in-line by a target side-chain hydroxyl group. The latter is properly registered to the Fic active center by main-chain β-interactions with the β-hairpin flap. These data indicate that the active site motif and the exposed edge of the flap are both required to form an adenylylation-competent Fic protein.

  8. Terminology of allergic phenomena.

    Science.gov (United States)

    Ring, Johannes

    2014-01-01

    Over the last 2,000 years a variety of terms have been used for the description of phenomena possibly related to allergy. Many have been forgotten, while some of them have remained. In Greco-Roman literature the term 'idiosyncrasy' was used to describe an individual characterization of a health condition, possibly comparable to 'constitution'. The same term was also used to describe individual reaction patterns, and the term 'antipathy' was used in a similar sense. 'Hypersensitivity' originated from the German word 'Überempfindlichkeit' and was first used in a medical sense by Emil von Behring when he described untoward reactions to his antitoxin containing serum therapy. 'Anaphylaxis' was coined by Richet and Portier to describe the new phenomenon of a life-threatening general pathogenic reaction after repeated injection of antigen. In 1906, Clemens von Pirquet introduced the term 'allergy' in order to bring more clarity to the confusing debate regarding protective and harmful immunity. In order to characterize the familial occurrence of hypersensitivity reactions such as asthma, hay fever and others, the American allergists A.F. Coca and R.A. Cooke introduced the term 'atopy'. Contrary to anaphylaxis, which was experimentally induced, this type of 'hypersensitiveness' occurred spontaneously. The nature of the pathogenic factor was called the 'atopic reagin' and was found to be transferable with serum by Prausnitz and Küstner. After the detection of immunoglobulin (Ig) E as the carrier of this type of hypersensitivity, the term 'atopy' gained a new sense, since IgE is a characteristic - yet not exclusive - parameter of the so-called atopic diseases. Clinically similar diseases such as asthma, rhinoconjunctivitis or eczema can be found in the absence of IgE, and are then called 'intrinsic' variants of the same disease. © 2014 S. Karger AG, Basel.

  9. Antibacterial and antifungal activities of new acylated derivatives of epigallocatechin gallate

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    Yoshimi eMatsumoto

    2012-02-01

    Full Text Available (--Epigallocatechin-3-O-gallate (EGCG has useful antiviral, antimicrobial, antitoxin, and antitumor properties. Previously, Mori, S. et al. (Bioorg Med Chem Lett 18:4249-4252, 2008 found that addition of long acyl chains (C16–18 to EGCG enhanced its anti-influenza virus activity up to 44-fold. The chemical stability of EGCG against oxidative degradation was also enhanced by acylation. We further evaluated the in vitro activity spectrum of the EGCG derivatives against a wide range of bacteria and fungi. A series of EGCG O-acyl derivatives were synthesized by lipase-catalyzed transesterification. These derivatives exhibited several-fold higher activities than EGCG, particularly against Gram-positive organisms. Antifungal activities of the derivatives were also 2 to 4-fold superior to those of EGCG. The activities of the EGCG derivatives against Gram-negative bacteria were not distinguishable from those of EGCG. Among the derivatives evaluated, MICs of dioctanoate, palmitate (C16, palmitoleate, and linolenate for 17 Staphylococcus aureus strains were 4–32 μg/ml, although MIC of EGCG for these 17 strains was >128 μg/ml. C16 demonstrated rapid bactericidal activity against MRSA at 25 μg/ml. The enhanced activity of C16 against S. aureus was supported by its increased membrane permeabilizing activity determined by increased SYTOX Green uptake. The EGCG derivatives were exported by the efflux pump AcrAB-TolC of Escherichia coli. The tolC deletion mutant exhibited higher sensitivity to C16 than to EGCG. Addition of long alkyl chains to EGCG significantly enhanced its activities against various bacteria and fungi, particularly against S. aureus including MRSA. C16 would be an alternative to antibiotics and disinfectants.

  10. A palindromic CpG-containing phosphodiester oligodeoxynucleotide as a mucosal adjuvant stimulates plasmacytoid dendritic cell-mediated T(H1 immunity.

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    Jun-ichi Maeyama

    Full Text Available BACKGROUND: CpG oligodeoxynucleotides (ODNs, resembling bacterial DNA, are currently tested in clinical trials as vaccine adjuvants. They have the nuclease-resistant phosphorothioate bond; the immune responses elicited differ according to the CpG ODN sequence and vaccination method. To develop a CpG ODN that can induce plasmacytoid dendritic cell (pDC-mediated T(H1 immunity through the mucosa, we constructed phosphodiester G9.1 comprising one palindromic CpG motif with unique polyguanosine-runs that allows degradation similar to naturally occurring bacterial DNA. METHODS: T(H1 and T(H2 immunity activation was evaluated by cytokine production pattern and T-bet/GATA-3 ratio in human peripheral blood mononuclear cells and mouse bone marrow cells. Adjuvanticity was evaluated in mice administered G9.1 with diphtheria toxoid (DT through nasal vaccination. RESULTS: G9.1 exhibited stronger IFN-α-inducing activity than A-class CpG ODN2216 and increased T-bet/GATA-3 ratio by enhancing T-bet expression. Nasally administered G9.1 plus DT induced DT-specific mucosal IgA and serum IgG, but not IgE, responses with antitoxin activity in C57BL/6 and BALB/c mice, possibly due to IFN/BAFF production. Induction of T(H1, but not T(H2-type Abs depended completely on pDCs, the first in vivo demonstration by CpG ODNs. CONCLUSIONS: G9.1 is a promising mucosal adjuvant for induction of pDC-mediated T(H1 immunity.

  11. Insights on the Emergence of Mycobacterium tuberculosis from the Analysis of Mycobacterium kansasii

    Science.gov (United States)

    Wang, Joyce; McIntosh, Fiona; Radomski, Nicolas; Dewar, Ken; Simeone, Roxane; Enninga, Jost; Brosch, Roland; Rocha, Eduardo P.; Veyrier, Frédéric J.; Behr, Marcel A.

    2015-01-01

    By phylogenetic analysis, Mycobacterium kansasii is closely related to Mycobacterium tuberculosis. Yet, although both organisms cause pulmonary disease, M. tuberculosis is a global health menace, whereas M. kansasii is an opportunistic pathogen. To illuminate the differences between these organisms, we have sequenced the genome of M. kansasii ATCC 12478 and its plasmid (pMK12478) and conducted side-by-side in vitro and in vivo investigations of these two organisms. The M. kansasii genome is 6,432,277 bp, more than 2 Mb longer than that of M. tuberculosis H37Rv, and the plasmid contains 144,951 bp. Pairwise comparisons reveal conserved and discordant genes and genomic regions. A notable example of genomic conservation is the virulence locus ESX-1, which is intact and functional in the low-virulence M. kansasii, potentially mediating phagosomal disruption. Differences between these organisms include a decreased predicted metabolic capacity, an increased proportion of toxin–antitoxin genes, and the acquisition of M. tuberculosis-specific genes in the pathogen since their common ancestor. Consistent with their distinct epidemiologic profiles, following infection of C57BL/6 mice, M. kansasii counts increased by less than 10-fold over 6 weeks, whereas M. tuberculosis counts increased by over 10,000-fold in just 3 weeks. Together, these data suggest that M. kansasii can serve as an image of the environmental ancestor of M. tuberculosis before its emergence as a professional pathogen, and can be used as a model organism to study the switch from an environmental opportunistic pathogen to a professional host-restricted pathogen. PMID:25716827

  12. Comparative genomics evidence that only protein toxins are tagging bad bugs

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    Kalliopi eGeorgiades

    2011-10-01

    Full Text Available The term toxin was introduced by Roux and Yersin and describes macromolecular substances that, when produced during infection or when introduced parenterally or orally, cause an impairment of physiological functions that lead to disease or to the death of the infected organism. Long after the discovery of toxins, early genetic studies on bacterial virulence demonstrated that removing a certain number of genes from pathogenic bacteria decreases their capacity to infect hosts. Each of the removed factors was therefore referred to as a virulence factor, and it was speculated that non-pathogenic bacteria lack such supplementary factors. However, many recent comparative studies demonstrate that the specialization of bacteria to eukaryotic hosts is associated with massive gene loss. We recently demonstrated that the only features that seem to characterize 12 epidemic bacteria are toxin-antitoxin (TA modules, which are addiction molecules in host bacteria. In this study, we investigated if protein toxins are indeed the only molecules specific to pathogenic bacteria by comparing 14 epidemic bacterial killers (bad bugs with their 14 closest non-epidemic relatives (controls. We found protein toxins in significantly more elevated numbers in all of the bad bugs. For the first time, statistical principal components analysis, including genome size, GC%, TA modules, restriction enzymes and toxins, revealed that toxins are the only proteins other than TA modules that are correlated with the pathogenic character of bacteria. Moreover, intracellular toxins appear to be more correlated with the pathogenic character of bacteria than secreted toxins. In conclusion, we hypothesize that the only truly identifiable phenomena, witnessing the convergent evolution of the most pathogenic bacteria for humans are the loss of metabolic activities, i.e., the outcome of the loss of regulatory and transcription factors and the presence of protein toxins, alone or coupled as TA

  13. Draft genome sequence of Actinotignum schaalii DSM 15541T: Genetic insights into the lifestyle, cell fitness and virulence.

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    Atteyet F Yassin

    Full Text Available The permanent draft genome sequence of Actinotignum schaalii DSM 15541T is presented. The annotated genome includes 2,130,987 bp, with 1777 protein-coding and 58 rRNA-coding genes. Genome sequence analysis revealed absence of genes encoding for: components of the PTS systems, enzymes of the TCA cycle, glyoxylate shunt and gluconeogensis. Genomic data revealed that A. schaalii is able to oxidize carbohydrates via glycolysis, the nonoxidative pentose phosphate and the Entner-Doudoroff pathways. Besides, the genome harbors genes encoding for enzymes involved in the conversion of pyruvate to lactate, acetate and ethanol, which are found to be the end products of carbohydrate fermentation. The genome contained the gene encoding Type I fatty acid synthase required for de novo FAS biosynthesis. The plsY and plsX genes encoding the acyltransferases necessary for phosphatidic acid biosynthesis were absent from the genome. The genome harbors genes encoding enzymes responsible for isoprene biosynthesis via the mevalonate (MVA pathway. Genes encoding enzymes that confer resistance to reactive oxygen species (ROS were identified. In addition, A. schaalii harbors genes that protect the genome against viral infections. These include restriction-modification (RM systems, type II toxin-antitoxin (TA, CRISPR-Cas and abortive infection system. A. schaalii genome also encodes several virulence factors that contribute to adhesion and internalization of this pathogen such as the tad genes encoding proteins required for pili assembly, the nanI gene encoding exo-alpha-sialidase, genes encoding heat shock proteins and genes encoding type VII secretion system. These features are consistent with anaerobic and pathogenic lifestyles. Finally, resistance to ciprofloxacin occurs by mutation in chromosomal genes that encode the subunits of DNA-gyrase (GyrA and topisomerase IV (ParC enzymes, while resistant to metronidazole was due to the frxA gene, which encodes NADPH

  14. Clostridium botulinum type D/C intoxication in a dairy cow stock in Saxony-Anhalt (Germany)--report on an innovative diagnostic approach.

    Science.gov (United States)

    Dlabola, Janine; Hashish, Emad; Pauly, Birgit; Kubisiak, Bernd; Behm, Ingrid; Heseler, Rüdiger; Schliephake, Annette; Wieler, Lothar H; Neubauer, Heinrich; Seyboldt, Christian

    2016-01-01

    Botulism in cattle is a rare but serious disease. In Germany there is no obligation to report botulism in animals and therefore a precise morbidity rate is not available. In this manuscript we describe an outbreak of Clostridium (C.) botulinum neurotoxin (BoNT) intoxication in a Saxony-Anhalt dairy cow stock of 286 Holstein-Friesian cows and offspring in spring/summer 2009 and its diagnostic approach. 122 animals showed clinical signs of BoNT intoxication. 115 of the affected animals (40.2% of the herd) independent of age died or had to be euthanized. Therapeutic attempts failed in almost all diseased cows, only four calves and three heifers recovered. Diagnostic samples of several animals (n = 4) (liver, ruminal and intestinal contents) and feed (n = 6) were tested for BoNT genes by polymerase chain reaction (PCR). BoNT gene type D was found in several (n = 8) organ samples. The PCR results allowed a preselection of samples for BoNT that were then tested by the mouse bioassay. Thus, the number of mice being inoculated in the mouse bioassay could be reduced. The mouse bioassay turned out positive (wasp-waist) in three preselected organ samples and the neutralization test of one sample with type-specific antitoxin confirmed the presence of BoNT type D. We succeeded in isolating a C. botulinum strain from a liver sample which was typed as a D/C mosaic strain by sequence analysis of the toxin gene. However, the source of the BoNT intoxication could not be traced back.

  15. Using probiotics to improve swine gut health and nutrient utilization

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    Shengfa F. Liao

    2017-12-01

    Full Text Available To maintain a healthy gut is definitely key for a pig to digest and absorb dietary nutrients efficiently. A balanced microbiota (i.e., a healthy micro-ecosystem is an indispensable constituent of a healthy gut. Probiotics, the live microorganisms which, when administered in adequate amounts, confer good health benefits onto the host, are a category of feed additives that can be used to replenish the gut microbial population while recuperating the host immune system. Besides their antitoxin and diarrhea reduction effects, dietary supplementation of probiotics can improve gut health, nutrient digestibilities and, therefore, benefit nutrient utilization and growth performance of pigs. Current knowledge in the literature pertinent to the beneficial effects of utilizing various probiotics for swine production has been comprehensively reviewed, and the safety and the risk issues related to probiotic usage have also been discussed in this paper. Considering that the foremost cost in a swine operation is feed cost, feed efficiency holds a very special, if not the paramount, significance in commercial swine production. Globally, the swine industry along with other animal industries is moving towards restricting and eventually a total ban on the usage of antibiotic growth promoters. Therefore, selection of an ideal alternative to the in-feed antibiotics to compensate for the lost benefits due to the ban on the antibiotic usage is urgently needed to support the industry for profitable and sustainable swine production. As is understood, a decision on this selection is not easy to make. Thus, this review paper aims to provide some much needed up-to-date knowledge and comprehensive references for swine nutritionists and producers to refer to before making prudent decisions and for scientists and researchers to develop better commercial products.

  16. Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H

    Directory of Open Access Journals (Sweden)

    Guorui Yao

    2017-03-01

    Full Text Available Botulinum neurotoxins (BoNTs, which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin treatment and intensive care over a long period of time. In addition to the existing seven established BoNT serotypes (BoNT/A–G, a new mosaic toxin type termed BoNT/HA (aka type FA or H was reported recently. Sequence analyses indicate that the receptor-binding domain (HC of BoNT/HA is ~84% identical to that of BoNT/A1. However, BoNT/HA responds differently to some potent BoNT/A-neutralizing antibodies (e.g., CR2 that target the HC. Therefore, it raises a serious concern as to whether BoNT/HA poses a new threat to our biosecurity. In this study, we report the first high-resolution crystal structure of BoNT/HA-HC at 1.8 Å. Sequence and structure analyses reveal that BoNT/HA and BoNT/A1 are different regarding their binding to cell-surface receptors including both polysialoganglioside (PSG and synaptic vesicle glycoprotein 2 (SV2. Furthermore, the new structure also provides explanations for the ~540-fold decreased affinity of antibody CR2 towards BoNT/HA compared to BoNT/A1. Taken together, these new findings advance our understanding of the structure and function of this newly identified toxin at the molecular level, and pave the way for the future development of more effective countermeasures.

  17. Prevalence of diphtheria and tetanus antibodies among adults in Singapore: a national serological study to identify most susceptible population groups.

    Science.gov (United States)

    Ang, L W; James, L; Goh, K T

    2016-03-01

    In view of waning antitoxin titres over time after the last vaccine dose against diphtheria and tetanus, we determined the immunity levels in adults to identify most susceptible groups for protection in Singapore. Our study involved residual sera from 3293 adults aged 18-79 who had participated in a national health survey in 2010. IgG antibody levels were determined using commercial enzyme-linked immunosorbent assay. Overall, 92.0% (95% confidence interval [CI]: 91.1-92.9%) had at least basic protection against diphtheria (antibody levels ≥0.01 IU/ml), while 71.4% (95% CI: 69.8-72.9%) had at least short-term protection against tetanus (antibody levels >0.1 IU/ml). The seroprevalence declined significantly with age for both diseases; the drop was most marked in the 50- to 59-year age group for diphtheria and 60- to 69-year age group for tetanus. There was a significant difference in seroprevalence by residency for diphtheria (92.8% among Singapore citizens versus 87.1% among permanent residents; P = 0.001). The seroprevalence for tetanus was significantly higher among males (83.2%) than females (62.4%) (P < 0.0005). It may be of value to consider additional vaccination efforts to protect older adults at higher risk for exposure against diphtheria and tetanus, particularly those travelling to areas where diphtheria is endemic or epidemic. © The Author 2015. Published by Oxford University Press on behalf of Faculty of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Mobile Genetic Elements and Evolution of CRISPR-Cas Systems: All the Way There and Back

    Science.gov (United States)

    Makarova, Kira S.

    2017-01-01

    Abstract The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-CRISPR-associated proteins (Cas) systems of bacterial and archaeal adaptive immunity show multifaceted evolutionary relationships with at least five classes of mobile genetic elements (MGE). First, the adaptation module of CRISPR-Cas that is responsible for the formation of the immune memory apparently evolved from a Casposon, a self-synthesizing transposon that employs the Cas1 protein as the integrase and might have brought additional cas genes to the emerging immunity loci. Second, a large subset of type III CRISPR-Cas systems recruited a reverse transcriptase from a Group II intron, providing for spacer acquisition from RNA. Third, effector nucleases of Class 2 CRISPR-Cas systems that are responsible for the recognition and cleavage of the target DNA were derived from transposon-encoded TnpB nucleases, most likely, on several independent occasions. Fourth, accessory nucleases in some variants of types I and III toxin and type VI effectors RNases appear to be ultimately derived from toxin nucleases of microbial toxin–antitoxin modules. Fifth, the opposite direction of evolution is manifested in the recruitment of CRISPR-Cas systems by a distinct family of Tn7-like transposons that probably exploit the capacity of CRISPR-Cas to recognize unique DNA sites to facilitate transposition as well as by bacteriophages that employ them to cope with host defense. Additionally, individual Cas proteins, such as the Cas4 nuclease, were recruited by bacteriophages and transposons. The two-sided evolutionary connection between CRISPR-Cas and MGE fits the “guns for hire” paradigm whereby homologous enzymatic machineries, in particular nucleases, are shuttled between MGE and defense systems and are used alternately as means of offense or defense. PMID:28985291

  19. Plasmids of carotenoid-producing Paracoccus spp. (Alphaproteobacteria) - structure, diversity and evolution.

    Science.gov (United States)

    Maj, Anna; Dziewit, Lukasz; Czarnecki, Jakub; Wlodarczyk, Miroslawa; Baj, Jadwiga; Skrzypczyk, Grazyna; Giersz, Dorota; Bartosik, Dariusz

    2013-01-01

    Plasmids are components of many bacterial genomes. They enable the spread of a large pool of genetic information via lateral gene transfer. Many bacterial strains contain mega-sized replicons and these are particularly common in Alphaproteobacteria. Considerably less is known about smaller alphaproteobacterial plasmids. We analyzed the genomes of 14 such plasmids residing in 4 multireplicon carotenoid-producing strains of the genus Paracoccus (Alphaproteobacteria): P. aestuarii DSM 19484, P. haeundaensis LG P-21903, P. marcusii DSM 11574 and P. marcusii OS22. Comparative analyses revealed mosaic structures of the plasmids and recombinational shuffling of diverse genetic modules involved in (i) plasmid replication, (ii) stabilization (including toxin-antitoxin systems of the relBE/parDE, tad-ata, higBA, mazEF and toxBA families) and (iii) mobilization for conjugal transfer (encoding relaxases of the MobQ, MobP or MobV families). A common feature of the majority of the plasmids is the presence of AT-rich sequence islets (located downstream of exc1-like genes) containing genes, whose homologs are conserved in the chromosomes of many bacteria (encoding e.g. RelA/SpoT, SMC-like proteins and a retron-type reverse transcriptase). The results of this study have provided insight into the diversity and plasticity of plasmids of Paracoccus spp., and of the entire Alphaproteobacteria. Some of the identified plasmids contain replication systems not described previously in this class of bacteria. The composition of the plasmid genomes revealed frequent transfer of chromosomal genes into plasmids, which significantly enriches the pool of mobile DNA that can participate in lateral transfer. Many strains of Paracoccus spp. have great biotechnological potential, and the plasmid vectors constructed in this study will facilitate genetic studies of these bacteria.

  20. Plasmids of carotenoid-producing Paracoccus spp. (Alphaproteobacteria - structure, diversity and evolution.

    Directory of Open Access Journals (Sweden)

    Anna Maj

    Full Text Available Plasmids are components of many bacterial genomes. They enable the spread of a large pool of genetic information via lateral gene transfer. Many bacterial strains contain mega-sized replicons and these are particularly common in Alphaproteobacteria. Considerably less is known about smaller alphaproteobacterial plasmids. We analyzed the genomes of 14 such plasmids residing in 4 multireplicon carotenoid-producing strains of the genus Paracoccus (Alphaproteobacteria: P. aestuarii DSM 19484, P. haeundaensis LG P-21903, P. marcusii DSM 11574 and P. marcusii OS22. Comparative analyses revealed mosaic structures of the plasmids and recombinational shuffling of diverse genetic modules involved in (i plasmid replication, (ii stabilization (including toxin-antitoxin systems of the relBE/parDE, tad-ata, higBA, mazEF and toxBA families and (iii mobilization for conjugal transfer (encoding relaxases of the MobQ, MobP or MobV families. A common feature of the majority of the plasmids is the presence of AT-rich sequence islets (located downstream of exc1-like genes containing genes, whose homologs are conserved in the chromosomes of many bacteria (encoding e.g. RelA/SpoT, SMC-like proteins and a retron-type reverse transcriptase. The results of this study have provided insight into the diversity and plasticity of plasmids of Paracoccus spp., and of the entire Alphaproteobacteria. Some of the identified plasmids contain replication systems not described previously in this class of bacteria. The composition of the plasmid genomes revealed frequent transfer of chromosomal genes into plasmids, which significantly enriches the pool of mobile DNA that can participate in lateral transfer. Many strains of Paracoccus spp. have great biotechnological potential, and the plasmid vectors constructed in this study will facilitate genetic studies of these bacteria.

  1. A Supercluster of Neutralizing Epitopes at the Interface of Ricin’s Enzymatic (RTA and Binding (RTB Subunits

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    Amanda Y. Poon

    2017-11-01

    Full Text Available As part of an effort to engineer ricin antitoxins and immunotherapies, we previously produced and characterized a collection of phage-displayed, heavy chain-only antibodies (VHHs from alpacas that had been immunized with ricin antigens. In our initial screens, we identified nine VHHs directed against ricin toxin’s binding subunit (RTB, but only one, JIZ-B7, had toxin-neutralizing activity. Linking JIZ-B7 to different VHHs against ricin’s enzymatic subunit (RTA resulted in several bispecific antibodies with potent toxin-neutralizing activity in vitro and in vivo. JIZ-B7 may therefore be an integral component of a future VHH-based neutralizing agent (VNA for ricin toxin. In this study, we now localize, using competitive ELISA, JIZ-B7’s epitope to a region of RTB’s domain 2 sandwiched between the high-affinity galactose/N-acetylgalactosamine (Gal/GalNAc-binding site and the boundary of a neutralizing hotspot on RTA known as cluster II. Analysis of additional RTB (n = 8- and holotoxin (n = 4-specific VHHs from a recent series of screens identified a “supercluster” of neutralizing epitopes at the RTA-RTB interface. Among the VHHs tested, toxin-neutralizing activity was most closely associated with epitope proximity to RTA, and not interference with RTB’s ability to engage Gal/GalNAc receptors. We conclude that JIZ-B7 is representative of a larger group of potent toxin-neutralizing antibodies, possibly including many described in the literature dating back several decades, that recognize tertiary and possibly quaternary epitopes located at the RTA-RTB interface and that target a region of vulnerability on ricin toxin.

  2. Neutralization of Clostridium difficile Toxin B Mediated by Engineered Lactobacilli That Produce Single-Domain Antibodies

    Science.gov (United States)

    Andersen, Kasper Krogh; Strokappe, Nika M.; Hultberg, Anna; Truusalu, Kai; Smidt, Imbi; Mikelsaar, Raik-Hiio; Mikelsaar, Marika; Verrips, Theo; Hammarström, Lennart

    2015-01-01

    Clostridium difficile is the primary cause of nosocomial antibiotic-associated diarrhea in the Western world. The major virulence factors of C. difficile are two exotoxins, toxin A (TcdA) and toxin B (TcdB), which cause extensive colonic inflammation and epithelial damage manifested by episodes of diarrhea. In this study, we explored the basis for an oral antitoxin strategy based on engineered Lactobacillus strains expressing TcdB-neutralizing antibody fragments in the gastrointestinal tract. Variable domain of heavy chain-only (VHH) antibodies were raised in llamas by immunization with the complete TcdB toxin. Four unique VHH fragments neutralizing TcdB in vitro were isolated. When these VHH fragments were expressed in either secreted or cell wall-anchored form in Lactobacillus paracasei BL23, they were able to neutralize the cytotoxic effect of the toxin in an in vitro cell-based assay. Prophylactic treatment with a combination of two strains of engineered L. paracasei BL23 expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3) delayed killing in a hamster protection model where the animals were challenged with spores of a TcdA− TcdB+ strain of C. difficile (P survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized with C. difficile for up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients. PMID:26573738

  3. Genome sequence of Lactobacillus pentosus KCA1: vaginal isolate from a healthy premenopausal woman.

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    Kingsley C Anukam

    Full Text Available The vaginal microbiota, in particular Lactobacillus species, play an important role in female health through modulation of immunity, countering pathogens and maintaining a pH below 4.7. We report the isolation and genome sequence of Lactobacillus pentosus strain KCA1 (formally known as L. plantarum from the vagina of a healthy Nigerian woman. The genome was sequenced using Illumina GA II technology. The resulting 16,920,226 paired-end reads were assembled with the Velvet tool. Contigs were annotated using the RAST server, and manually curated. A comparative analysis with the available genomes of L. pentosus IG1 and L. plantarum WCFS1 showed that over 15% of the predicted functional activities are found only in this strain. The strain has a chromosome sequence of 3,418,159 bp with a G+C content of 46.4%, and is devoid of plasmids. Novel gene clusters or variants of known genes relative to the reference genomes were found. In particular, the strain has loci encoding additional putative mannose phosphotransferase systems. Clusters of genes include those for utilization of hydantoin, isopropylmalate, malonate, rhamnosides, and genes for assimilation of polyglycans, suggesting the metabolic versatility of L. pentosus KCA1. Loci encoding putative phage defense systems were also found including clustered regularly interspaced short palindromic repeats (CRISPRs, abortive infection (Abi systems and toxin-antitoxin systems (TA. A putative cluster of genes for biosynthesis of a cyclic bacteriocin precursor, here designated as pentocin KCA1 (penA were identified. These findings add crucial information for understanding the genomic and geographic diversity of vaginal lactobacilli.

  4. Immunogenicity of a reduced schedule of meningococcal group C conjugate vaccine given concomitantly with the Prevenar and Pediacel vaccines in healthy infants in the United Kingdom.

    Science.gov (United States)

    Southern, Jo; Borrow, Ray; Andrews, Nick; Morris, Rhonwen; Waight, Pauline; Hudson, Michael; Balmer, Paul; Findlow, Helen; Findlow, Jamie; Miller, Elizabeth

    2009-02-01

    This study investigated the use of two doses of three different meningococcal group C conjugate (MCC) vaccines when given for primary immunization with a seven-valent pneumococcal conjugate vaccine (PCV7) and Pediacel, a combination product containing five acellular pertussis components, diphtheria and tetanus toxoids, Haemophilus influenzae type b (Hib) conjugate, and inactivated-poliovirus vaccine. The immune response after a single dose of MCC is also presented. Infants were randomized to receive two doses of one of the MCC vaccines and PCV7 at 2 and 3 months or at 2 and 4 months of age. Meningococcal group C serum bactericidal antibody (SBA) geometric mean titers, Hib-polyribosylribitol phosphate (PRP) immunoglobulin G (IgG) geometric mean concentrations (GMCs), and diphtheria and tetanus antitoxin GMCs, together with the proportions of infants achieving putative protective levels, were determined. A total of 393 infants were recruited. Following the first dose of NeisVac-C (MCC conjugated to tetanus toxoid), 97% of infants achieved protective levels (SBA titer of >or=8), compared with 80% and 53%, respectively, for Menjugate and Meningitec (both of which are conjugated to CRM(197)). SBA responses to MCC vaccines were not significantly different when administered at 2 and 3 or 2 and 4 months of age. Following two doses of each MCC, 98 to 100% of infants achieved protective levels. Both PRP IgG and tetanus responses were significantly enhanced when Pediacel was coadministered with NeisVac-C. This study demonstrates that NeisVac-C and Menjugate generate good immunogenicity after the first dose at 2 months of age when coadministered with PCV7 and Pediacel and merit further investigation in single-dose priming strategies.

  5. Protein translation and cell death: the role of rare tRNAs in biofilm formation and in activating dormant phage killer genes.

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    Rodolfo García-Contreras

    2008-06-01

    Full Text Available We discovered previously that the small Escherichia coli proteins Hha (hemolysin expression modulating protein and the adjacent, poorly-characterized YbaJ are important for biofilm formation; however, their roles have been nebulous. Biofilms are intricate communities in which cell signaling often converts single cells into primitive tissues. Here we show that Hha decreases biofilm formation dramatically by repressing the transcription of rare codon tRNAs which serves to inhibit fimbriae production and by repressing to some extent transcription of fimbrial genes fimA and ihfA. In vivo binding studies show Hha binds to the rare codon tRNAs argU, ileX, ileY, and proL and to two prophage clusters D1P12 and CP4-57. Real-time PCR corroborated that Hha represses argU and proL, and Hha type I fimbriae repression is abolished by the addition of extra copies of argU, ileY, and proL. The repression of transcription of rare codon tRNAs by Hha also leads to cell lysis and biofilm dispersal due to activation of prophage lytic genes rzpD, yfjZ, appY, and alpA and due to induction of ClpP/ClpX proteases which activate toxins by degrading antitoxins. YbaJ serves to mediate the toxicity of Hha. Hence, we have identified that a single protein (Hha can control biofilm formation by limiting fimbriae production as well as by controlling cell death. The mechanism used by Hha is the control of translation via the availability of rare codon tRNAs which reduces fimbriae production and activates prophage lytic genes. Therefore, Hha acts as a toxin in conjunction with co-transcribed YbaJ (TomB that attenuates Hha toxicity.

  6. Tissue-specific gene-expression patterns of genes associated with thymol/carvacrol biosynthesis in thyme (Thymus vulgaris L.) and their differential changes upon treatment with abiotic elicitors.

    Science.gov (United States)

    Majdi, Mohammad; Malekzadeh-Mashhady, Atefe; Maroufi, Asad; Crocoll, Christoph

    2017-06-01

    Thyme (Thymus vulgaris L.) is known to produce a variety of phenolic monoterpenes such as thymol and carvacrol. Thymol and carvacrol are health-promoting, biocide and antitoxin compounds and have been considered as the main constituents of essential oils in T. vulgaris. To improve our understanding of the regulation of monoterpene biosynthesis in thyme, the expression of genes related to thymol and carvacrol biosynthesis in different tissues and in response to abiotic elicitors was analyzed. Methyl jasmonate (MeJA), salicylic acid (SA), trans-cinnamic acid (tCA) and UV-C irradiation were applied to T. vulgare leaves and transcript levels of early (DXR) and late (TvTPS1, CYP71D178 and CYP71D180) biosynthetic genes of thymol and carvacrol were measured. The results showed that early step and late step genes in thymol/carvacrol biosynthesis are differentially regulated. DXR was not found to be exclusively expressed in glandular trichomes; in contrast, biosynthetic genes including γ-terpinene synthase (TvTPS1) and two cytochrome P450s, CYP71D178 and CYP71D180, were preferentially expressed in glandular secretory trichomes. The high expression of late biosynthetic genes in glandular trichomes, which also contain the highest concentration of thymol and carvacrol, suggests that glandular trichomes are the structure in which thymol/carvacrol biosynthesis and accumulation occur. Our results indicate that in addition to abiotic elicitors, developmental and spatial factors also play a key role in the biosynthesis of thymol and carvacrol, most likely relating to glandular trichome density and/or activity. Hence optimization of these factors could be considered as a useful strategy to achieve high yield of valuable compounds in T. vulgare or other closely related plant species. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  7. Botulinum Neurotoxins: Qualitative and Quantitative Analysis Using the Mouse Phrenic Nerve Hemidiaphragm Assay (MPN).

    Science.gov (United States)

    Bigalke, Hans; Rummel, Andreas

    2015-11-25

    The historical method for the detection of botulinum neurotoxin (BoNT) is represented by the mouse bioassay (MBA) measuring the animal survival rate. Since the endpoint of the MBA is the death of the mice due to paralysis of the respiratory muscle, an ex vivo animal replacement method, called mouse phrenic nerve (MPN) assay, employs the isolated N. phrenicus-hemidiaphragm tissue. Here, BoNT causes a dose-dependent characteristic decrease of the contraction amplitude of the indirectly stimulated muscle. Within the EQuATox BoNT proficiency 13 test samples were analysed using the MPN assay by serial dilution to a bath concentration resulting in a paralysis time within the range of calibration curves generated with BoNT/A, B and E standards, respectively. For serotype identification the diluted samples were pre-incubated with polyclonal anti-BoNT/A, B or E antitoxin or a combination of each. All 13 samples were qualitatively correctly identified thereby delivering superior results compared to single in vitro methods like LFA, ELISA and LC-MS/MS. Having characterized the BoNT serotype, the final bath concentrations were calculated using the calibration curves and then multiplied by the respective dilution factor to obtain the sample concentration. Depending on the source of the BoNT standards used, the quantitation of ten BoNT/A containing samples delivered a mean z-score of 7 and of three BoNT/B or BoNT/E containing samples z-scores <2, respectively.

  8. Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H.

    Science.gov (United States)

    Yao, Guorui; Lam, Kwok-Ho; Perry, Kay; Weisemann, Jasmin; Rummel, Andreas; Jin, Rongsheng

    2017-03-08

    Botulinum neurotoxins (BoNTs), which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin treatment and intensive care over a long period of time. In addition to the existing seven established BoNT serotypes (BoNT/A-G), a new mosaic toxin type termed BoNT/HA (aka type FA or H) was reported recently. Sequence analyses indicate that the receptor-binding domain (H C ) of BoNT/HA is ~84% identical to that of BoNT/A1. However, BoNT/HA responds differently to some potent BoNT/A-neutralizing antibodies (e.g., CR2) that target the H C . Therefore, it raises a serious concern as to whether BoNT/HA poses a new threat to our biosecurity. In this study, we report the first high-resolution crystal structure of BoNT/HA-H C at 1.8 Å. Sequence and structure analyses reveal that BoNT/HA and BoNT/A1 are different regarding their binding to cell-surface receptors including both polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Furthermore, the new structure also provides explanations for the ~540-fold decreased affinity of antibody CR2 towards BoNT/HA compared to BoNT/A1. Taken together, these new findings advance our understanding of the structure and function of this newly identified toxin at the molecular level, and pave the way for the future development of more effective countermeasures.

  9. Nosocomial Outbreak of Extensively Drug-Resistant Acinetobacter baumannii Isolates ContainingblaOXA-237Carried on a Plasmid.

    Science.gov (United States)

    Hujer, Andrea M; Higgins, Paul G; Rudin, Susan D; Buser, Genevieve L; Marshall, Steven H; Xanthopoulou, Kyriaki; Seifert, Harald; Rojas, Laura J; Domitrovic, T Nicholas; Cassidy, P Maureen; Cunningham, Margaret C; Vega, Robert; Furuno, Jon P; Pfeiffer, Christopher D; Beldavs, Zintars G; Wright, Meredith S; Jacobs, Michael R; Adams, Mark D; Bonomo, Robert A

    2017-11-01

    Carbapenem antibiotics are among the mainstays for treating infections caused by Acinetobacter baumannii , especially in the Northwest United States, where carbapenem-resistant A. baumannii remains relatively rare. However, between June 2012 and October 2014, an outbreak of carbapenem-resistant A. baumannii occurred in 16 patients from five health care facilities in the state of Oregon. All isolates were defined as extensively drug resistant. Multilocus sequence typing revealed that the isolates belonged to sequence type 2 (international clone 2 [IC2]) and were >95% similar as determined by repetitive-sequence-based PCR analysis. Multiplex PCR revealed the presence of a bla OXA carbapenemase gene, later identified as bla OXA-237 Whole-genome sequencing of all isolates revealed a well-supported separate branch within a global A. baumannii phylogeny. Pacific Biosciences (PacBio) SMRT sequencing was also performed on one isolate to gain insight into the genetic location of the carbapenem resistance gene. We discovered that bla OXA-237 , flanked on either side by IS Aba1 elements in opposite orientations, was carried on a 15,198-bp plasmid designated pORAB01-3 and was present in all 16 isolates. The plasmid also contained genes encoding a TonB-dependent receptor, septicolysin, a type IV secretory pathway (VirD4 component, TraG/TraD family) ATPase, an integrase, a RepB family plasmid DNA replication initiator protein, an alpha/beta hydrolase, and a BrnT/BrnA type II toxin-antitoxin system. This is the first reported outbreak in the northwestern United States associated with this carbapenemase. Particularly worrisome is that bla OXA-237 was carried on a plasmid and found in the most prominent worldwide clonal group IC2, potentially giving pORAB01-3 great capacity for future widespread dissemination. Copyright © 2017 American Society for Microbiology.

  10. Retrospective evaluation of 155 adult equids and 21 foals with tetanus from Western, Northern, and Central Europe (2000-2014). Part 2: Prognostic assessment.

    Science.gov (United States)

    van Galen, Gaby; Rijckaert, Joke; Mair, Tim; Amory, Helene; Armengou, Lara; Bezdekova, Barbora; Durie, Inge; Findshøj Delany, Rikke; Fouché, Nathalie; Haley, Laura; Hewetson, Michael; van den Hoven, Rene; Kendall, Anna; Malalana, Fernando; Muller Cavalleri, Jessika; Picavet, Tresemiek; Roscher, Katja; Verwilghen, Denis; Westermann, Cornélie; Saegerman, Claude

    2017-11-01

    To identify prognostic variables for adult equids and foals with tetanus. Multicenter retrospective study (2000-2014). Twenty Western, Northern, and Central European university teaching hospitals and private referral centers. One hundred fifty-five adult equids and 21 foals with tetanus. None. Variables from history and clinical examination were statistically compared between survivors and nonsurvivors (adults: 49 survivors, 85 nonsurvivors; foals: 7 survivors, 10 nonsurvivors). Cases euthanized for financial reasons were excluded. Mortality rates in adults and foals were 68.4% and 66.7%, respectively. Variables associated with survival in adults included: standing, normal intestinal sounds and defecation, voluntarily drinking, eating soft or normal food, lower heart and respiratory rates, high base excess on admission, longer diagnosis time, treatment and hospitalization delay, and mild severity grade. Variables associated with death included: anorexia, dysphagia, dyspnea, low blood potassium concentration on admission, moderate and severe disease grading, development of dysphagia, dyspnea, recumbency and seizures during hospitalization, treatment with glycerol guaiacolate, intravenous fluids, and intravenous glucose solutions. Variables associated with survival in foals included standing on admission, voluntarily eating soft food and drinking, older age, and longer hospitalization delay. Outcome was not different between different tetanus antitoxin (TAT) dosages, although there was a trend of increasing survival rate with increasing TAT dosages. Cases with appropriate vaccination prior to development of tetanus were rare, but had improved outcome and shorter hospitalization. Prognosis for equine tetanus is poor with similar outcome and prognostic factors in foals and adults. The prognostic assessment of cases with tetanus provides clinicians with new evidence-based information related to patient management. Several prognostic indicators relate to the ability to

  11. Tn5253 family integrative and conjugative elements carrying mef(I) and catQ determinants in Streptococcus pneumoniae and Streptococcus pyogenes.

    Science.gov (United States)

    Mingoia, Marina; Morici, Eleonora; Morroni, Gianluca; Giovanetti, Eleonora; Del Grosso, Maria; Pantosti, Annalisa; Varaldo, Pietro E

    2014-10-01

    The linkage between the macrolide efflux gene mef(I) and the chloramphenicol inactivation gene catQ was first described in Streptococcus pneumoniae (strain Spn529), where the two genes are located in a module designated IQ element. Subsequently, two different defective IQ elements were detected in Streptococcus pyogenes (strains Spy029 and Spy005). The genetic elements carrying the three IQ elements were characterized, and all were found to be Tn5253 family integrative and conjugative elements (ICEs). The ICE from S. pneumoniae (ICESpn529IQ) was sequenced, whereas the ICEs from S. pyogenes (ICESpy029IQ and ICESpy005IQ, the first Tn5253-like ICEs reported in this species) were characterized by PCR mapping, partial sequencing, and restriction analysis. ICESpn529IQ and ICESpy029IQ were found to share the intSp 23FST81 integrase gene and an identical Tn916 fragment, whereas ICESpy005IQ has int5252 and lacks Tn916. All three ICEs were found to lack the linearized pC194 plasmid that is usually associated with Tn5253-like ICEs, and all displayed a single copy of a toxin-antitoxin operon that is typically contained in the direct repeats flanking the excisable pC194 region when this region is present. Two different insertion sites of the IQ elements were detected, one in ICESpn529IQ and ICESpy029IQ, and another in ICESpy005IQ. The chromosomal integration of the three ICEs was site specific, depending on the integrase (intSp 23FST81 or int5252). Only ICESpy005IQ was excised in circular form and transferred by conjugation. By transformation, mef(I) and catQ were cotransferred at a high frequency from S. pyogenes Spy005 and at very low frequencies from S. pneumoniae Spn529 and S. pyogenes Spy029. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  12. Paul Ehrlich: pathfinder in cell biology. 1. Chronicle of his life and accomplishments in immunology, cancer research, and chemotherapy.

    Science.gov (United States)

    Kasten, F H

    1996-01-01

    The paper reviews the life of Paul Ehrlich and his biomedical accomplishments in immunology, cancer research, and chemotherapy. Ehrlich achieved renown as an organic chemist, histologist, hematologist, immunologist, and pharmacologist. He disliked the formality of school but managed to excel in Latin and mathematics. His role model was an older cousin, Carl Weigert, who became a lifelong friend. Ehrlich studied medicine at Breslau, Strasbourg, Freiburg, and Leipzig, coming under the influence of Wilhelm Waldeyer, Julius Cohnheim, Rudolf Heidenhein, and Ferdinand Cohn. As a medical student, Ehrlich was captivated by structural organic chemistry and dyes. When he was 23, his first paper was published on selective staining. His doctoral thesis, "Contribution to the Theory and Practice of Histological Staining" contained most of the germinal ideas that would guide his future career. Most of his early work was centered in Berlin at Charite Hospital, where he did pioneering studies on blood and intravital staining, and at Robert Koch's Institute for Infectious Diseases, where he undertook important investigations in immunology. Ehrlich became an authority on antitoxin standardization and developed the "side-chain theory" of antibody formation for which he was later awarded the Nobel Prize. He became director of an Institute for Experimental Therapy in Frankfurt where he continued research in immunology and carried out routine serum testing. He developed new lines of investigation in cancer research and originated the field of chemotherapy. Using principles developed in his early work with dyes, he successfully treated certain experimental trypanosomal infections with azo dyes. His crowning accomplishment was discovering that the compound Salvarsan could control human syphilis. Ehrlich's legacy in immunology and chemotherapy is discussed and an intimate portrait is drawn of Ehrlich the person.

  13. A camelid single-domain antibody neutralizes botulinum neurotoxin A by blocking host receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Guorui; Lam, Kwok-ho; Weisemann, Jasmin; Peng, Lisheng; Krez, Nadja; Perry, Kay; Shoemaker, Charles B.; Dong, Min; Rummel, Andreas; Jin, Rongsheng (BCH); (Cornell); (Tufts CTSI); (UCI); (MHH)

    2017-08-07

    Antibody treatment is currently the only available countermeasure for botulism, a fatal illness caused by flaccid paralysis of muscles due to botulinum neurotoxin (BoNT) intoxication. Among the seven major serotypes of BoNT/A-G, BoNT/A poses the most serious threat to humans because of its high potency and long duration of action. Prior to entering neurons and blocking neurotransmitter release, BoNT/A recognizes motoneurons via a dual-receptor binding process in which it engages both the neuron surface polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Previously, we identified a potent neutralizing antitoxin against BoNT/A1 termed ciA-C2, derived from a camelid heavy-chain-only antibody (VHH). In this study, we demonstrate that ciA-C2 prevents BoNT/A1 intoxication by inhibiting its binding to neuronal receptor SV2. Furthermore, we determined the crystal structure of ciA-C2 in complex with the receptor-binding domain of BoNT/A1 (HCA1) at 1.68 Å resolution. The structure revealed that ciA-C2 partially occupies the SV2-binding site on HCA1, causing direct interference of HCA1 interaction with both the N-glycan and peptide-moiety of SV2. Interestingly, this neutralization mechanism is similar to that of a monoclonal antibody in clinical trials, despite that ciA-C2 is more than 10-times smaller. Taken together, these results enlighten our understanding of BoNT/A1 interactions with its neuronal receptor, and further demonstrate that inhibiting toxin binding to the host receptor is an efficient countermeasure strategy.

  14. Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Guorui; Lam, Kwok-ho; Perry, Kay; Weisemann, Jasmin; Rummel, Andreas; Jin, Rongsheng (Cornell); (Dusseldorf); (UCI)

    2017-03-01

    Botulinum neurotoxins (BoNTs), which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin treatment and intensive care over a long period of time. In addition to the existing seven established BoNT serotypes (BoNT/A–G), a new mosaic toxin type termed BoNT/HA (aka type FA or H) was reported recently. Sequence analyses indicate that the receptor-binding domain (HC) of BoNT/HA is ~84% identical to that of BoNT/A1. However, BoNT/HA responds differently to some potent BoNT/A-neutralizing antibodies (e.g., CR2) that target the HC. Therefore, it raises a serious concern as to whether BoNT/HA poses a new threat to our biosecurity. In this study, we report the first high-resolution crystal structure of BoNT/HA-HC at 1.8 Å. Sequence and structure analyses reveal that BoNT/HA and BoNT/A1 are different regarding their binding to cell-surface receptors including both polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Furthermore, the new structure also provides explanations for the ~540-fold decreased affinity of antibody CR2 towards BoNT/HA compared to BoNT/A1. Taken together, these new findings advance our understanding of the structure and function of this newly identified toxin at the molecular level, and pave the way for the future development of more effective countermeasures

  15. Regulatory RNAs in the Less Studied Streptococcal Species: from Nomenclature to Identification

    Directory of Open Access Journals (Sweden)

    Mohamed Amine Zorgani

    2016-07-01

    Full Text Available Streptococcal species are Gram-positive bacteria involved in severe and invasive diseases in humans and animals. Although this group includes different pathogenic species involved in life-threatening infections for humans, it also includes beneficial species, such as Streptococcus thermophilus, which is used in yogurt production. In bacteria virulence factors are controlled by various regulatory networks including regulatory RNAs. For clearness and to develop logical thinking, we start this review with a revision of regulatory RNAs nomenclature. Previous reviews are mostly dealing with Streptococcus pyogenes and Streptococcus pneumoniae regulatory RNAs. We especially focused our analysis on regulatory RNAs in Streptococcus agalactiae, Streptococcus mutans, Streptococcus thermophilus and other less studied Streptococcus species. Although S. agalactiae RNome remains largely unknown, sRNAs (small RNAs are supposed to mediate regulation during environmental adaptation and host infection. In the case of S. mutans, sRNAs are suggested to be involved in competence regulation, carbohydrate metabolism and Toxin-Antitoxin systems. A new category of miRNA-size small RNAs (msRNAs was also identified for the first time in this species. The analysis of S. thermophilus sRNome shows that many sRNAs are associated to the bacterial immune system known as CRISPR-Cas system. Only few of the other different Streptococcus species have been the subject of studies pointed toward the characterization of regulatory RNAs. Finally, understanding bacterial sRNome can constitute one step forward to the elaboration of new strategies in therapy such as substitution of antibiotics in the management of S. agalactiae neonatal infections, prevention of S. mutans dental caries or use of S. thermophilus CRISPR-Cas system in genome editing applications.

  16. Molecular and Epidemiological Review of Toxigenic Diphtheria Infections in England between 2007 and 2013

    Science.gov (United States)

    Both, Leonard; Collins, Sarah; de Zoysa, Aruni; White, Joanne; Mandal, Sema

    2014-01-01

    Human infections caused by toxigenic corynebacteria occur sporadically across Europe. In this report, we undertook the epidemiological and molecular characterization of all toxigenic corynebacterium strains isolated in England between January 2007 and December 2013. Epidemiological aspects include case demographics, risk factors, clinical presentation, treatment, and outcome. Molecular characterization was performed using multilocus sequence typing (MLST) alongside traditional phenotypic methods. In total, there were 20 cases of toxigenic corynebacteria; 12 (60.0%) were caused by Corynebacterium ulcerans, where animal contact was the predominant risk factor. The remaining eight (40.0%) were caused by Corynebacterium diphtheriae strains; six were biovar mitis, which were associated with recent travel abroad. Adults 45 years and older were particularly affected (55.0%; 11/20), and typical symptoms included sore throat and fever. Respiratory diphtheria with the absence of a pharyngeal membrane was the most common presentation (50.0%; 10/20). None of the eight C. diphtheriae cases were fully immunized. Diphtheria antitoxin was issued in two (9.5%) cases; both survived. Two (9.5%) cases died, one due to a C. diphtheriae infection and one due to C. ulcerans. MLST demonstrated that the majority (87.5%; 7/8) of C. diphtheriae strains represented new sequence types (STs). By adapting several primer sequences, the MLST genes in C. ulcerans were also amplified, thereby providing the basis for extension of the MLST scheme, which is currently restricted to C. diphtheriae. Despite high population immunity, occasional toxigenic corynebacterium strains are identified in England and continued surveillance is required. PMID:25502525

  17. Seroprevalence of diphtheria toxoid IgG antibodies in children, adolescents and adults in Poland.

    Science.gov (United States)

    Zasada, Aleksandra A; Rastawicki, Waldemar; Rokosz, Natalia; Jagielski, Marek

    2013-11-19

    Recommendations for diphtheria immunization are to apply an effective primary immunization in infancy and to maintain immunity throughout life. Immunity against diphtheria depends primarily on antibody to the diphtheria toxin. This study evaluated the seroprevalence of IgG diphtheria antitoxin in sera of healthy children, adolescents and adults in Poland. A total of 1387 serum samples collected between 2010 and 2012 from individuals with ages ranging from 1 month to 85 years were investigated. Antibody concentrations were measured with an enzyme-linked immunosorbent assay (Anti-Diphtheria Toxoid ELISA IgG, Euroimmun, Germany). The results showed that among 1387 individuals examined, 547 (39.4%) had anti-diphtheria toxoid IgG antibody levels below 0.1 IU/ml (36.9% ≤ 18 years and 40.5% >18 years old, respectively). The 212 (50.8%) children and 542 (55.9%) adults showed only basic protection (0.1-1.0 IU/ml) and need immediate booster. High levels of anti-diphtheria toxoid IgG antibodies (>1.0 IU/ml) were found more often in children and adolescent (12.2%) than in adults (3.6%) and this was statistically significant (P 60 years old. Characteristically, in individuals > 40 years old high levels of anti-diphtheria toxoid IgG antibodies (>1.0 IU/ml) were not seen. There were no statistically significant differences in results in relation to gender. The present study showed inadequate immunity levels to diphtheria amongst the Polish population, especially in adults > 40 years old and children ≤ 2 years old. To prevent reemergence of diphtheria an information campaign reminding people about recommendations concerning diphtheria booster vaccination in adults should be conducted. Moreover, the immunogenicity of the DTP vaccine used in Poland should be verified.

  18. International External Quality Assurance for Laboratory Diagnosis of Diphtheria ▿

    Science.gov (United States)

    Neal, S. E.; Efstratiou, A.

    2009-01-01

    The diphtheria surveillance network (DIPNET) encompassing National Diphtheria Reference Centers from 25 European countries is a Dedicated Surveillance Network recognized by the European Commission. A key DIPNET objective is the quality assessment of microbiological procedures for diphtheria across the European Union and beyond. A detailed questionnaire on the level of reference laboratory services and an external quality assessment (EQA) panel comprising six simulated throat specimens were sent to 34 centers. Twenty-three centers are designated National Diphtheria Reference Centers, with the laboratory in the United Kingdom being the only WHO Collaborating Centre. A variety of screening and identification tests were used, including the cysteinase test (20/34 centers), pyrazinamidase test (17/34 centers), and commercial kits (25/34 centers). The classic Elek test for toxigenicity testing is mostly used (28/34 centers), with variations in serum sources and antitoxin concentrations. Many laboratories reported problems obtaining Elek reagents or media. Only six centers produced acceptable results for all six specimens. Overall, 21% of identification and 13% of toxigenicity reports were unacceptable. Many centers could not isolate the target organism, and most found difficulties with the specimens that contained Corynebacterium striatum as a commensal contaminant. Nineteen centers generated either false-positive or negative toxigenic results, which may have caused inappropriate medical management. The discrepancies in this diphtheria diagnostics EQA alarmingly reflect the urgent need to improve laboratory performance in diphtheria diagnostics in Europe, standardize feasible and robust microbiological methods, and build awareness among public health authorities. Therefore, DIPNET recommends that regular workshops and EQA distributions for diphtheria diagnostics should be supported and maintained. PMID:19828749

  19. Durability of Vaccine-Induced Immunity Against Tetanus and Diphtheria Toxins: A Cross-sectional Analysis

    Science.gov (United States)

    Hammarlund, Erika; Thomas, Archana; Poore, Elizabeth A.; Amanna, Ian J.; Rynko, Abby E.; Mori, Motomi; Chen, Zunqiu; Slifka, Mark K.

    2016-01-01

    Background. Many adult immunization schedules recommend that tetanus and diphtheria vaccination be performed every 10 years. In light of current epidemiological trends of disease incidence and rates of vaccine-associated adverse events, the 10-year revaccination schedule has come into question. Methods. We performed cross-sectional analysis of serum antibody titers in 546 adult subjects stratified by age or sex. All serological results were converted to international units after calibration with international serum standards. Results. Approximately 97% of the population was seropositive to tetanus and diphtheria as defined by a protective serum antibody titer of ≥0.01 IU/mL. Mean antibody titers were 3.6 and 0.35 IU/mL against tetanus and diphtheria, respectively. Antibody responses to tetanus declined with an estimated half-life of 14 years (95% confidence interval, 11–17 years), whereas antibody responses to diphtheria were more long-lived and declined with an estimated half-life of 27 years (18–51 years). Mathematical models combining antibody magnitude and duration predict that 95% of the population will remain protected against tetanus and diphtheria for ≥30 years without requiring further booster vaccination. Conclusions. These studies demonstrate that durable levels of protective antitoxin immunity exist in the majority of vaccinated individuals. Together, this suggests that it may no longer be necessary to administer booster vaccinations every 10 years and that the current adult vaccination schedule for tetanus and diphtheria should be revisited. PMID:27060790

  20. The Structural Diversity of Carbohydrate Antigens of Selected Gram-Negative Marine Bacteria

    Directory of Open Access Journals (Sweden)

    Elena P. Ivanova

    2011-10-01

    Full Text Available Marine microorganisms have evolved for millions of years to survive in the environments characterized by one or more extreme physical or chemical parameters, e.g., high pressure, low temperature or high salinity. Marine bacteria have the ability to produce a range of biologically active molecules, such as antibiotics, toxins and antitoxins, antitumor and antimicrobial agents, and as a result, they have been a topic of research interest for many years. Among these biologically active molecules, the carbohydrate antigens, lipopolysaccharides (LPSs, O-antigens found in cell walls of Gram-negative marine bacteria, show great potential as candidates in the development of drugs to prevent septic shock due to their low virulence. The structural diversity of LPSs is thought to be a reflection of the ability for these bacteria to adapt to an array of habitats, protecting the cell from being compromised by exposure to harsh environmental stress factors. Over the last few years, the variety of structures of core oligosaccharides and O-specific polysaccharides from LPSs of marine microrganisms has been discovered. In this review, we discuss the most recently encountered structures that have been identified from bacteria belonging to the genera Aeromonas, Alteromonas, Idiomarina, Microbulbifer, Pseudoalteromonas, Plesiomonas and Shewanella of the Gammaproteobacteria phylum; Sulfitobacter and Loktanella of the Alphaproteobactera phylum and to the genera Arenibacter, Cellulophaga, Chryseobacterium, Flavobacterium, Flexibacter of the Cytophaga-Flavobacterium-Bacteroides phylum. Particular attention is paid to the particular chemical features of the LPSs, such as the monosaccharide type, non-sugar substituents and phosphate groups, together with some of the typifying traits of LPSs obtained from marine bacteria. A possible correlation is then made between such features and the environmental adaptations undertaken by marine bacteria.

  1. The structural diversity of carbohydrate antigens of selected gram-negative marine bacteria.

    Science.gov (United States)

    Nazarenko, Evgeny L; Crawford, Russell J; Ivanova, Elena P

    2011-01-01

    Marine microorganisms have evolved for millions of years to survive in the environments characterized by one or more extreme physical or chemical parameters, e.g., high pressure, low temperature or high salinity. Marine bacteria have the ability to produce a range of biologically active molecules, such as antibiotics, toxins and antitoxins, antitumor and antimicrobial agents, and as a result, they have been a topic of research interest for many years. Among these biologically active molecules, the carbohydrate antigens, lipopolysaccharides (LPSs, O-antigens) found in cell walls of gram-negative marine bacteria, show great potential as candidates in the development of drugs to prevent septic shock due to their low virulence. The structural diversity of LPSs is thought to be a reflection of the ability for these bacteria to adapt to an array of habitats, protecting the cell from being compromised by exposure to harsh environmental stress factors. Over the last few years, the variety of structures of core oligosaccharides and O-specific polysaccharides from LPSs of marine microrganisms has been discovered. In this review, we discuss the most recently encountered structures that have been identified from bacteria belonging to the genera Aeromonas, Alteromonas, Idiomarina, Microbulbifer, Pseudoalteromonas, Plesiomonas and Shewanella of the Gammaproteobacteria phylum; Sulfitobacter and Loktanella of the Alphaproteobactera phylum and to the genera Arenibacter, Cellulophaga, Chryseobacterium, Flavobacterium, Flexibacter of the Cytophaga-Flavobacterium-Bacteroides phylum. Particular attention is paid to the particular chemical features of the LPSs, such as the monosaccharide type, non-sugar substituents and phosphate groups, together with some of the typifying traits of LPSs obtained from marine bacteria. A possible correlation is then made between such features and the environmental adaptations undertaken by marine bacteria.

  2. Composition and Predictive Functional Analysis of Bacterial Communities in Seawater, Sediment and Sponges in the Spermonde Archipelago, Indonesia.

    Science.gov (United States)

    Cleary, Daniel F R; de Voogd, Nicole J; Polónia, Ana R M; Freitas, Rossana; Gomes, Newton C M

    2015-11-01

    were predicted to be enriched for KOs related to the toxin-antitoxin (TA) system, nutrient starvation and heavy metal export.

  3. 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.

    Science.gov (United States)

    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.

  4. Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme.

    Science.gov (United States)

    Hancock, Steven J; Phan, Minh-Duy; Peters, Kate M; Forde, Brian M; Chong, Teik Min; Yin, Wai-Fong; Chan, Kok-Gan; Paterson, David L; Walsh, Timothy R; Beatson, Scott A; Schembri, Mark A

    2017-02-01

    Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including bla CMY and bla NDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a bla NDM-1 -positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of bla NDM -positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this bla NDM -containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies. Copyright © 2017 American Society for Microbiology.

  5. Comparative genomics of the IncA/C multidrug resistance plasmid family.

    Science.gov (United States)

    Fricke, W Florian; Welch, Timothy J; McDermott, Patrick F; Mammel, Mark K; LeClerc, J Eugene; White, David G; Cebula, Thomas A; Ravel, Jacques

    2009-08-01

    Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.

  6. Transcriptome mapping of pAR060302, a blaCMY-2-positive broad-host-range IncA/C plasmid.

    Science.gov (United States)

    Lang, Kevin S; Danzeisen, Jessica L; Xu, Wayne; Johnson, Timothy J

    2012-05-01

    The multidrug resistance-encoding plasmids belonging to the IncA/C incompatibility group have recently emerged among Escherichia coli and Salmonella enterica strains in the United States. These plasmids have a unique genetic structure compared to other enterobacterial plasmid types, a broad host range, and a propensity to acquire large numbers of antimicrobial resistance genes via their accessory regions. Using E. coli strain DH5α harboring the prototype IncA/C plasmid pAR060302, we sought to define the baseline transcriptome of IncA/C plasmids under laboratory growth and in the face of selective pressure. The effects of ampicillin, florfenicol, or streptomycin exposure were compared to those on cells left untreated at logarithmic phase using Illumina platform-based RNA sequencing (RNA-Seq). Under growth in Luria-Bertani broth lacking antibiotics, much of the backbone of pAR060302 was transcriptionally inactive, including its putative transfer regions. A few plasmid backbone genes of interest were highly transcribed, including genes of a putative toxin-antitoxin system and an H-NS-like transcriptional regulator. In contrast, numerous genes within the accessory regions of pAR060302 were highly transcribed, including the resistance genes floR, bla(CMY-2), aadA, and aacA. Treatment with ampicillin or streptomycin resulted in no genes being differentially expressed compared to controls lacking antibiotics, suggesting that many of the resistance-associated genes are not differentially expressed due to exposure to these antibiotics. In contrast, florfenicol treatment resulted in the upregulation of floR and numerous chromosomal genes. Overall, the transcriptome mapping of pAR060302 suggests that it mitigates the fitness costs of carrying resistance-associated genes through global regulation with its transcriptional regulators.

  7. Evolutionary blueprint for host- and niche-adaptation in Staphylococcus aureus clonal complex CC30

    Directory of Open Access Journals (Sweden)

    Martin John Mcgavin

    2012-04-01

    Full Text Available Staphylococcus aureus clonal complex CC30 has caused infectious epidemics for more than 60 years, and therefore provides a model system to evaluate how evolution has influenced the disease potential of closely related strains. In previous multiple genome comparisons, phylogenetic analyses established three major branches that evolved from a common ancestor. Clade 1, comprised of historic pandemic phage type 80/81 methicillin susceptible S. aureus (MSSA, and Clade 2 comprised of contemporary community acquired methicillin resistant S. aureus (CA-MRSA were hyper-virulent in murine infection models. Conversely, Clade 3 strains comprised of contemporary hospital associated MRSA (HA-MRSA and clinical MSSA exhibited attenuated virulence, due to common single nucleotide polymorphisms (SNP’s that abrogate production of α-hemolysin Hla, and interfere with signaling of the accessory gene regulator agr. We have now completed additional in silico genome comparisons of fifteen additional CC30 genomes in the public domain, to assess the hypothesis that Clade 3 has evolved to favor niche adaptation. In addition to SNP’s that influence agr and hla, other common traits of Clade 3 include tryptophan auxotrophy due to a di-nucleotide deletion within trpD, a premature stop codon within isdH encoding an immunogenic cell surface protein involved in iron acquisition, loss of a genomic toxin-antitoxin addiction module, acquisition of S. aureus pathogenicity islands SaPI4, and SaPI2 encoding toxic shock syndrome toxin tst, and increased copy number of insertion sequence ISSau2, which appears to target transcription terminators. Compared to other Clade 3 MSSA, S. aureus MN8, which is associated with Staphylococcal toxic shock syndrome, exhibited a unique ISSau2 insertion, and enhanced production of toxic shock syndrome toxin encoded by SaPI2. Cumulatively, our data support the notion that Clade 3 strains are following an evolutionary blueprint towards niche-adaptation.

  8. Prova de hemaglutinação passiva para a evidenciação da toxina do C. diphtherias na lesão diftérica

    Directory of Open Access Journals (Sweden)

    Dacio de Almeida Christovão

    1967-12-01

    Full Text Available Foi demonstrada a presença da toxina do C. diphtheriae, através de prova de hemaglutinação passiva, usando-se hemácias sensibilizadas com antitoxina diftérica, na lesão da garganta de 47,7% dos casos suspeitos de difteria examinados no presente trabalho. De 53,0% dos mesmos pacientes pôde ser isolado bacilo diftérico toxígeno. A prova de hemaglutinação passiva foi a única positiva em 13,0% dos casos e a cultura, a única positiva em 18,9%. Em 42 crianças normais ou portadoras de faringite ou amigdalite sem nenhuma característica clínica de difteria, a prova de hemaglutinação passiva foi negativa. O processo descrito, de execução muito simples, pode acusar o resultado em menos de 2 horas e oferece grande possibilidade de aplicação vantajosa no diagnóstico da difteria.The presense of C. diphteriae toxin was demonstrated in throat lesions of 47.7 per cent of case with probable diagnosis of diphtheria, by passive hemagglutination test using erithrocytes previously sensitized by diphtheria anti-toxin. From 53.0 per cent of the same patients toxigenous bacilli were isolated. Hemagglutination test was the only positive one in 13.6 per cent of cases and cultivation in 18.9 per cent. The passive hemagglutination tests performed on 42 children, normal or with pharyngitis or tonsillitis but without clinical characteristics of diphtheria were all negative. The procedure described in this paper, of very simple execution can show results in less than two hours and offers great possibility of advantageous application in the diagnosis of diphtheria.

  9. Unusual evolutionary history of the tRNA splicing endonuclease EndA: relationship to the LAGLIDADG and PD-(D/E)XK deoxyribonucleases.

    Science.gov (United States)

    Bujnicki, J M; Rychlewski, L

    2001-03-01

    The tRNA splicing endoribonuclease EndA from Methanococcus jannaschii is a homotetramer formed via heterologous interaction between the two pairs of homodimers. Each monomer consists of two alpha/beta domains, the N-terminal domain (NTD) and the C-terminal domain (CTD) containing the RNase A-like active site. Comparison of the EndA coordinates with the publicly available protein structure database revealed the similarity of both domains to site-specific deoxyribonucleases: the NTD to the LAGLIDADG family and the CTD to the PD-(D/E)XK family. Superposition of the NTD on the catalytic domain of LAGLIDADG homing endonucleases allowed a suggestion to be made about which amino acid residues of the tRNA splicing nuclease might participate in formation of a presumptive cryptic deoxyribonuclease active site. On the other hand, the CTD and PD-(D/E)XK endonucleases, represented by restriction enzymes and a phage lambda exonuclease, were shown to share extensive similarities of the structural framework, to which entirely different active sites might be attached in two alternative locations. These findings suggest that EndA evolved from a fusion protein with at least two distinct endonuclease activities: the ribonuclease, which made it an essential "antitoxin" for the cells whose RNA genes were interrupted by introns, and the deoxyribonuclease, which provided the means for homing-like mobility. The residues of the noncatalytic CTDs from the positions corresponding to the catalytic side chains in PD-(D/E)XK deoxyribonucleases map to the surface at the opposite side to the tRNA binding site, for which no function has been implicated. Many restriction enzymes from the PD-(D/E)XK superfamily might have the potential to maintain an additional active or binding site at the face opposite the deoxyribonuclease active site, a property that can be utilized in protein engineering.

  10. Sequencing and characterizing the genome of Estrella lausannensis as an undergraduate project: training students and biological insights.

    Science.gov (United States)

    Bertelli, Claire; Aeby, Sébastien; Chassot, Bérénice; Clulow, James; Hilfiker, Olivier; Rappo, Samuel; Ritzmann, Sébastien; Schumacher, Paolo; Terrettaz, Céline; Benaglio, Paola; Falquet, Laurent; Farinelli, Laurent; Gharib, Walid H; Goesmann, Alexander; Harshman, Keith; Linke, Burkhard; Miyazaki, Ryo; Rivolta, Carlo; Robinson-Rechavi, Marc; van der Meer, Jan Roelof; Greub, Gilbert

    2015-01-01

    With the widespread availability of high-throughput sequencing technologies, sequencing projects have become pervasive in the molecular life sciences. The huge bulk of data generated daily must be analyzed further by biologists with skills in bioinformatics and by "embedded bioinformaticians," i.e., bioinformaticians integrated in wet lab research groups. Thus, students interested in molecular life sciences must be trained in the main steps of genomics: sequencing, assembly, annotation and analysis. To reach that goal, a practical course has been set up for master students at the University of Lausanne: the "Sequence a genome" class. At the beginning of the academic year, a few bacterial species whose genome is unknown are provided to the students, who sequence and assemble the genome(s) and perform manual annotation. Here, we report the progress of the first class from September 2010 to June 2011 and the results obtained by seven master students who specifically assembled and annotated the genome of Estrella lausannensis, an obligate intracellular bacterium related to Chlamydia. The draft genome of Estrella is composed of 29 scaffolds encompassing 2,819,825 bp that encode for 2233 putative proteins. Estrella also possesses a 9136 bp plasmid that encodes for 14 genes, among which we found an integrase and a toxin/antitoxin module. Like all other members of the Chlamydiales order, Estrella possesses a highly conserved type III secretion system, considered as a key virulence factor. The annotation of the Estrella genome also allowed the characterization of the metabolic abilities of this strictly intracellular bacterium. Altogether, the students provided the scientific community with the Estrella genome sequence and a preliminary understanding of the biology of this recently-discovered bacterial genus, while learning to use cutting-edge technologies for sequencing and to perform bioinformatics analyses.

  11. A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes.

    Science.gov (United States)

    Garcia-Rodriguez, Consuelo; Razai, Ali; Geren, Isin N; Lou, Jianlong; Conrad, Fraser; Wen, Wei-Hua; Farr-Jones, Shauna; Smith, Theresa J; Brown, Jennifer L; Skerry, Janet C; Smith, Leonard A; Marks, James D

    2018-03-01

    Human botulism is most commonly caused by botulinum neurotoxin (BoNT) serotypes A, B, and E. For this work, we sought to develop a human monoclonal antibody (mAb)-based antitoxin capable of binding and neutralizing multiple subtypes of BoNT/E. Libraries of yeast-displayed single chain Fv (scFv) antibodies were created from the heavy and light chain variable region genes of humans immunized with pentavalent-toxoid- and BoNT/E-binding scFv isolated by Fluorescence-Activated Cell Sorting (FACS). A total of 10 scFv were isolated that bound one or more BoNT/E subtypes with nanomolar-level equilibrium dissociation constants (K D ). By diversifying the V-regions of the lead mAbs and selecting for cross-reactivity, we generated three scFv that bound all four BoNT/E subtypes tested at three non-overlapping epitopes. The scFvs were converted to IgG that had K D values for the different BoNT/E subtypes ranging from 9.7 nM to 2.28 pM. An equimolar combination of the three mAbs was able to potently neutralize BoNT/E1, BoNT/E3, and BoNT/E4 in a mouse neutralization assay. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing multiple BoNT/E subtypes. A derivative of the three-antibody combination (NTM-1633) is in pre-clinical development with an investigational new drug (IND) application filing expected in 2018.

  12. Botulinum Neurotoxins: Qualitative and Quantitative Analysis Using the Mouse Phrenic Nerve Hemidiaphragm Assay (MPN

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    Hans Bigalke

    2015-11-01

    Full Text Available The historical method for the detection of botulinum neurotoxin (BoNT is represented by the mouse bioassay (MBA measuring the animal survival rate. Since the endpoint of the MBA is the death of the mice due to paralysis of the respiratory muscle, an ex vivo animal replacement method, called mouse phrenic nerve (MPN assay, employs the isolated N. phrenicus-hemidiaphragm tissue. Here, BoNT causes a dose-dependent characteristic decrease of the contraction amplitude of the indirectly stimulated muscle. Within the EQuATox BoNT proficiency 13 test samples were analysed using the MPN assay by serial dilution to a bath concentration resulting in a paralysis time within the range of calibration curves generated with BoNT/A, B and E standards, respectively. For serotype identification the diluted samples were pre-incubated with polyclonal anti-BoNT/A, B or E antitoxin or a combination of each. All 13 samples were qualitatively correctly identified thereby delivering superior results compared to single in vitro methods like LFA, ELISA and LC-MS/MS. Having characterized the BoNT serotype, the final bath concentrations were calculated using the calibration curves and then multiplied by the respective dilution factor to obtain the sample concentration. Depending on the source of the BoNT standards used, the quantitation of ten BoNT/A containing samples delivered a mean z-score of 7 and of three BoNT/B or BoNT/E containing samples z-scores <2, respectively.

  13. Envenenamento ofídico em crianças: frequência de reações precoces ao antiveneno em pacientes que receberam pré-tratamento com antagonistas H1 e H2 da histamina e hidrocortisona Snake envenomation in children: early reactions frequency at antivenom in patients pretreated with histamine antagonists HI and H2 and hydrocortisone

    Directory of Open Access Journals (Sweden)

    Fábio Bucarethi

    1994-10-01

    Full Text Available Foram estudadas 24 crianças, com idade entre 2 a 14 anos, de 1989 a 1993, vítimas de acidentes ofídicos, submetidas a pré-tratamento com antagonistas H1 (dextroclorfeniramina e H2 (cimetidine ou ranitidina da histamina e hidrocortisona, com objetivo de avaliar a frequência e o tipo de reações precoces (RP ao antiveneno (AV. Em nenhum paciente havia antecedente de atopia ou uso prévio de algum tipo de antiveneno ou antitoxina heteróloga. Das 24 crianças 15 receberam AV botrópico (RP em 5, 7 AV crotálico (RP em 5, 1 AV crotálico e AV botrópico-crotálico e 1 AV elapídico (RP. Foram observadas RP graves em 3 crianças, as 3 classificadas como acidente crotálico grave. A análise dos resultados sugere que o pré-tratamento realizado não ofereceu uma proteção segura quanto ao aparecimento de RP.Type and frequency of early reactions (ER were studied in 24 children aging 2-14 years victims of snake bites who received pretreatment with histamine antagonists H1 (dextrochlorfenirarnine and H2 (cimetidine or ranitidine and hydrocortisone from 1989 to 1993. None of them had atopy nor received any type of anti-venoms(AV and antitoxins before. Of 24 children, 15 received bothropic AV (ER in 5, 7 crotalic AV (ER in 5, 1 crotalic plus crotalic-bothropic AV, and 1 elapidic AV (ER in 1. In 3 children severe early reactions were observed and they were classified as severe crotalic accident. Results suggest that pre-treatment did not offer safety protection at the appearance of early reactions.

  14. Microevolutionary Events Involving Narrow Host Plasmids Influences Local Fixation of Vancomycin-Resistance in Enterococcus Populations

    Science.gov (United States)

    Freitas, Ana R.; Novais, Carla; Tedim, Ana P.; Francia, María Victoria; Baquero, Fernando; Peixe, Luísa; Coque, Teresa M.

    2013-01-01

    Vancomycin-resistance in enterococci (VRE) is associated with isolates within ST18, ST17, ST78 Enterococcus faecium (Efm) and ST6 Enterococcus faecalis (Efs) human adapted lineages. Despite of its global spread, vancomycin resistance rates in enterococcal populations greatly vary temporally and geographically. Portugal is one of the European countries where Tn1546 (vanA) is consistently found in a variety of environments. A comprehensive multi-hierarchical analysis of VRE isolates (75 Efm and 29 Efs) from Portuguese hospitals and aquatic surroundings (1996–2008) was performed to clarify the local dynamics of VRE. Clonal relatedness was established by PFGE and MLST while plasmid characterization comprised the analysis of known relaxases, rep initiator proteins and toxin-antitoxin systems (TA) by PCR-based typing schemes, RFLP comparison, hybridization and sequencing. Tn1546 variants were characterized by PCR overlapping/sequencing. Intra- and inter-hospital dissemination of Efm ST18, ST132 and ST280 and Efs ST6 clones, carrying rolling-circle (pEFNP1/pRI1) and theta-replicating (pCIZ2-like, Inc18, pHTβ-like, two pRUM-variants, pLG1-like, and pheromone-responsive) plasmids was documented. Tn1546 variants, mostly containing ISEf1 or IS1216, were located on plasmids (30–150 kb) with a high degree of mosaicism and heterogeneous RFLP patterns that seem to have resulted from the interplay between broad host Inc18 plasmids (pIP501, pRE25, pEF1), and narrow host RepA_N plasmids (pRUM, pAD1-like). TAs of Inc18 (ω-ε-ζ) and pRUM (Axe-Txe) plasmids were infrequently detected. Some plasmid chimeras were persistently recovered over years from different clonal lineages. This work represents the first multi-hierarchical analysis of VRE, revealing a frequent recombinatorial diversification of a limited number of interacting clonal backgrounds, plasmids and transposons at local scale. These interactions provide a continuous process of parapatric clonalization driving a full

  15. Sequence and Role in Virulence of the Three Plasmid Complement of the Model Tumor-Inducing Bacterium Pseudomonas savastanoi pv. savastanoi NCPPB 3335

    Science.gov (United States)

    Bardaji, Leire; Pérez-Martínez, Isabel; Rodríguez-Moreno, Luis; Rodríguez-Palenzuela, Pablo; Sundin, George W.; Ramos, Cayo; Murillo, Jesús

    2011-01-01

    Pseudomonas savastanoi pv. savastanoi NCPPB 3335 is a model for the study of the molecular basis of disease production and tumor formation in woody hosts, and its draft genome sequence has been recently obtained. Here we closed the sequence of the plasmid complement of this strain, composed of three circular molecules of 78,357 nt (pPsv48A), 45,220 nt (pPsv48B), and 42,103 nt (pPsv48C), all belonging to the pPT23A-like family of plasmids widely distributed in the P. syringae complex. A total of 152 coding sequences were predicted in the plasmid complement, of which 38 are hypothetical proteins and seven correspond to putative virulence genes. Plasmid pPsv48A contains an incomplete Type IVB secretion system, the type III secretion system (T3SS) effector gene hopAF1, gene ptz, involved in cytokinin biosynthesis, and three copies of a gene highly conserved in plant-associated proteobacteria, which is preceded by a hrp box motif. A complete Type IVA secretion system, a well conserved origin of transfer (oriT), and a homolog of the T3SS effector gene hopAO1 are present in pPsv48B, while pPsv48C contains a gene with significant homology to isopentenyl-diphosphate delta-isomerase, type 1. Several potential mobile elements were found on the three plasmids, including three types of MITE, a derivative of IS801, and a new transposon effector, ISPsy30. Although the replication regions of these three plasmids are phylogenetically closely related, their structure is diverse, suggesting that the plasmid architecture results from an active exchange of sequences. Artificial inoculations of olive plants with mutants cured of plasmids pPsv48A and pPsv48B showed that pPsv48A is necessary for full virulence and for the development of mature xylem vessels within the knots; we were unable to obtain mutants cured of pPsv48C, which contains five putative toxin-antitoxin genes. PMID:22022435

  16. Co-occurrence of resistance genes to antibiotics, biocides and metals reveals novel insights into their co-selection potential.

    Science.gov (United States)

    Pal, Chandan; Bengtsson-Palme, Johan; Kristiansson, Erik; Larsson, D G Joakim

    2015-11-17

    Antibacterial biocides and metals can co-select for antibiotic resistance when bacteria harbour resistance or tolerance genes towards both types of compounds. Despite numerous case studies, systematic and quantitative data on co-occurrence of such genes on plasmids and chromosomes is lacking, as is knowledge on environments and bacterial taxa that tend to carry resistance genes to such compounds. This effectively prevents identification of risk scenarios. Therefore, we aimed to identify general patterns for which biocide/metal resistance genes (BMRGs) and antibiotic resistance genes (ARGs) that tend to occur together. We also aimed to quantify co-occurrence of resistance genes in different environments and taxa, and investigate to what extent plasmids carrying both types of genes are conjugative and/or are carrying toxin-antitoxin systems. Co-occurrence patterns of resistance genes were derived from publicly available, fully sequenced bacterial genomes (n = 2522) and plasmids (n = 4582). The only BMRGs commonly co-occurring with ARGs on plasmids were mercury resistance genes and the qacE∆1 gene that provides low-level resistance to quaternary ammonium compounds. Novel connections between cadmium/zinc and macrolide/aminoglycoside resistance genes were also uncovered. Several clinically important bacterial taxa were particularly prone to carry both BMRGs and ARGs. Bacteria carrying BMRGs more often carried ARGs compared to bacteria without (p bacterial genomes, and co-occurred with ARGs in 17 % of the cases. In contrast, co-occurrences of BMRGs and ARGs were rare on plasmids from all external environments (resistance genes. This is the first large-scale identification of compounds, taxa and environments of particular concern for co-selection of resistance against antibiotics, biocides and metals. Genetic co-occurrences suggest that plasmids provide limited opportunities for biocides and metals to promote horizontal transfer of antibiotic resistance through co

  17. Genetic plasticity of the Shigella virulence plasmid is mediated by intra- and inter-molecular events between insertion sequences.

    Science.gov (United States)

    Pilla, Giulia; McVicker, Gareth; Tang, Christoph M

    2017-09-01

    Acquisition of a single copy, large virulence plasmid, pINV, led to the emergence of Shigella spp. from Escherichia coli. The plasmid encodes a Type III secretion system (T3SS) on a 30 kb pathogenicity island (PAI), and is maintained in a bacterial population through a series of toxin:antitoxin (TA) systems which mediate post-segregational killing (PSK). The T3SS imposes a significant cost on the bacterium, and strains which have lost the plasmid and/or genes encoding the T3SS grow faster than wild-type strains in the laboratory, and fail to bind the indicator dye Congo Red (CR). Our aim was to define the molecular events in Shigella flexneri that cause loss of Type III secretion (T3S), and to examine whether TA systems exert positional effects on pINV. During growth at 37°C, we found that deletions of regions of the plasmid including the PAI lead to the emergence of CR-negative colonies; deletions occur through intra-molecular recombination events between insertion sequences (ISs) flanking the PAI. Furthermore, by repositioning MvpAT (which belongs to the VapBC family of TA systems) near the PAI, we demonstrate that the location of this TA system alters the rearrangements that lead to loss of T3S, indicating that MvpAT acts both globally (by reducing loss of pINV through PSK) as well as locally (by preventing loss of adjacent sequences). During growth at environmental temperatures, we show for the first time that pINV spontaneously integrates into different sites in the chromosome, and this is mediated by inter-molecular events involving IS1294. Integration leads to reduced PAI gene expression and impaired secretion through the T3SS, while excision of pINV from the chromosome restores T3SS function. Therefore, pINV integration provides a reversible mechanism for Shigella to circumvent the metabolic burden imposed by pINV. Intra- and inter-molecular events between ISs, which are abundant in Shigella spp., mediate plasticity of S. flexneri pINV.

  18. Sequences of two related multiple antibiotic resistance virulence plasmids sharing a unique IS26-related molecular signature isolated from different Escherichia coli pathotypes from different hosts.

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    Carola Venturini

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC and atypical enteropathogenic E. coli (aEPEC are important zoonotic pathogens that increasingly are becoming resistant to multiple antibiotics. Here we describe two plasmids, pO26-CRL125 (125 kb from a human O26:H- EHEC, and pO111-CRL115 (115kb from a bovine O111 aEPEC, that impart resistance to ampicillin, kanamycin, neomycin, streptomycin, sulfathiazole, trimethoprim and tetracycline and both contain atypical class 1 integrons with an identical IS26-mediated deletion in their 3´-conserved segment. Complete sequence analysis showed that pO26-CRL125 and pO111-CRL115 are essentially identical except for a 9.7 kb fragment, present in the backbone of pO26-CRL125 but absent in pO111-CRL115, and several indels. The 9.7 kb fragment encodes IncI-associated genes involved in plasmid stability during conjugation, a putative transposase gene and three imperfect repeats. Contiguous sequence identical to regions within these pO26-CRL125 imperfect repeats was identified in pO111-CRL115 precisely where the 9.7 kb fragment is missing, suggesting it may be mobile. Sequences shared between the plasmids include a complete IncZ replicon, a unique toxin/antitoxin system, IncI stability and maintenance genes, a novel putative serine protease autotransporter, and an IncI1 transfer system including a unique shufflon. Both plasmids carry a derivate Tn21 transposon with an atypical class 1 integron comprising a dfrA5 gene cassette encoding resistance to trimethoprim, and 24 bp of the 3´-conserved segment followed by Tn6026, which encodes resistance to ampicillin, kanymycin, neomycin, streptomycin and sulfathiazole. The Tn21-derivative transposon is linked to a truncated Tn1721, encoding resistance to tetracycline, via a region containing the IncP-1α oriV. Absence of the 5 bp direct repeats flanking Tn3-family transposons, indicates that homologous recombination events played a key role in the formation of this complex

  19. Allodemic distribution of plasmids co-harbouring blaCTX-M-15/aac(6')-Ib-cr/qnrB in Klebsiella pneumoniae is the main source of extended-spectrum β-lactamases in Uruguay's paediatric hospital.

    Science.gov (United States)

    Garcia-Fulgueiras, V; Araujo, L; Bado, I; Cordeiro, N F; Mota, M I; Laguna, G; Algorta, G; Vignoli, R

    2017-06-01

    This study aimed to describe the characteristics of clinical isolates of extended-spectrum β-lactamase (ESBL)-producing enterobacteria (EPE) in Uruguay's paediatric hospital. ESBLs, qnr alleles and aac(6')-Ib-cr were sought and characterised in EPE isolated between March 2010 and March 2012. Transfer of resistance determinants was assessed by conjugation. Incompatibility (Inc) groups, plasmid toxin-antitoxin systems (TAS) and plasmid size were determined in transconjugants. Clonality was analysed by pulsed-field gel electrophoresis. Multilocus sequence typing was done for ESBL-producing Klebsiella pneumoniae. A total of 77 EPE isolates were characterised, comprising 43% K. pneumoniae, 19.5% Serratia marcescens, 19.5% Escherichia coli, 17% Enterobacter cloacae and 1% Klebsiella oxytoca. ESBLs belonged mainly to the bla CTX-M family (69.6%) [bla CTX-M-15 (45%) and bla CTX-M-2 (31%)]. The aac(6')-Ib-cr/qnrB duplex was the most frequently detected plasmid-mediated quinolone resistance mechanism; this association was detected in K. pneumoniae harbouring bla CTX-M-15 . Transconjugants were obtained for 71% of the EPE. Amongst transconjugants, certain combinations were found between ESBLs and Inc group, e.g. IncA/C-bla CTX-M-2 , IncHI1/HI2-bla CTX-M-9 and IncHI1/HI2-bla SHV-12 . In addition, the combination ccdAB-bla CTX-M-15 was also found. K. pneumoniae isolates harbouring bla CTX-M-15 /aac(6')-Ib-cr/qnrB showed allodemic behaviour, with a predominance of ST14, ST45 and ST48. In this study, epidemiological changes in ESBL distribution could be explained by the spread of K. pneumoniae harbouring bla CTX-M-15 /aac(6')-Ib-cr/qnrB, encoded mainly on conjugative plasmids featuring ccdAB TAS. Since reports of TAS in K. pneumoniae plasmids are scarce, new strategies are needed to combat intrinsic selection pressure exerted by the association, in conjugative plasmids, of resistance mechanisms with TAS. Copyright © 2017 International Society for Chemotherapy of Infection and

  20. Prioritizing drug targets in Clostridium botulinum with a computational systems biology approach.

    Science.gov (United States)

    Muhammad, Syed Aun; Ahmed, Safia; Ali, Amjad; Huang, Hui; Wu, Xiaogang; Yang, X Frank; Naz, Anam; Chen, Jake

    2014-07-01

    A computational and in silico system level framework was developed to identify and prioritize the antibacterial drug targets in Clostridium botulinum (Clb), the causative agent of flaccid paralysis in humans that can be fatal in 5 to 10% of cases. This disease is difficult to control due to the emergence of drug-resistant pathogenic strains and the only available treatment antitoxin which can target the neurotoxin at the extracellular level and cannot reverse the paralysis. This study framework is based on comprehensive systems-scale analysis of genomic sequence homology and phylogenetic relationships among Clostridium, other infectious bacteria, host and human gut flora. First, the entire 2628-annotated genes of this bacterial genome were categorized into essential, non-essential and virulence genes. The results obtained showed that 39% of essential proteins that functionally interact with virulence proteins were identified, which could be a key to new interventions that may kill the bacteria and minimize the host damage caused by the virulence factors. Second, a comprehensive comparative COGs and blast sequence analysis of these proteins and host proteins to minimize the risks of side effects was carried out. This revealed that 47% of a set of C. botulinum proteins were evolutionary related with Homo sapiens proteins to sort out the non-human homologs. Third, orthology analysis with other infectious bacteria to assess broad-spectrum effects was executed and COGs were mostly found in Clostridia, Bacilli (Firmicutes), and in alpha and beta Proteobacteria. Fourth, a comparative phylogenetic analysis was performed with human microbiota to filter out drug targets that may also affect human gut flora. This reduced the list of candidate proteins down to 131. Finally, the role of these putative drug targets in clostridial biological pathways was studied while subcellular localization of these candidate proteins in bacterial cellular system exhibited that 68% of the

  1. Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System.

    Science.gov (United States)

    Bradshaw, Marite; Tepp, William H; Whitemarsh, Regina C M; Pellett, Sabine; Johnson, Eric A

    2014-12-01

    Clostridium botulinum subtype A4 neurotoxin (BoNT/A4) is naturally expressed in the dual-toxin-producing C. botulinum strain 657Ba at 100× lower titers than BoNT/B. In this study, we describe purification of recombinant BoNT/A4 (rBoNT/A4) expressed in a nonsporulating and nontoxigenic C. botulinum expression host strain. The rBoNT/A4 copurified with nontoxic toxin complex components provided in trans by the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT/A1, and the recombinant BoNT/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT/A4 light chain (LC) expressed in Escherichia coli has indicated reduced stability and activity of BoNT/A4 LC compared to BoNT/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT/A4, a recombinant full-length BoNT/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of the in vitro, cellular, and in vivo activities of rBoNT/A4 and rBoNT/A4-L260F I264R showed 1,000-fold-lower activity than BoNT/A1 in both the mutated and nonmutated BoNT/A4. This indicates that these mutations do not alter the activity of BoNT/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  2. Variable Persister Gene Interactions with (pppGpp for Persister Formation in Escherichia coli

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    Shuang Liu

    2017-09-01

    Full Text Available Persisters comprise a group of phenotypically heterogeneous metabolically quiescent bacteria with multidrug tolerance and contribute to the recalcitrance of chronic infections. Although recent work has shown that toxin-antitoxin (TA system HipAB depends on stringent response effector (pppGppin persister formation, whether other persister pathways are also dependent on stringent response has not been explored. Here we examined the relationship of (pppGpp with 15 common persister genes (dnaK, clpB, rpoS, pspF, tnaA, sucB, ssrA, smpB, recA, umuD, uvrA, hipA, mqsR, relE, dinJ using Escherichia coli as a model. By comparing the persister levels of wild type with their single gene knockout and double knockout mutants with relA, we divided their interactions into five types, namely A “dependent” (dnaK, recA, B “positive reinforcement” (rpoS, pspF, ssrA, recA, C “antagonistic” (clpB, sucB, umuD, uvrA, hipA, mqsR, relE, dinJ, D “epistasis” (clpB, rpoS, tnaA, ssrA, smpB, hipA, and E “irrelevant” (dnaK, clpB, rpoS, tnaA, sucB, smpB, umuD, uvrA, hipA, mqsR, relE, dinJ. We found that the persister gene interactions are intimately dependent on bacterial culture age, cell concentrations (diluted versus undiluted culture, and drug classifications, where the same gene may belong to different groups with varying antibiotics, culture age or cell concentrations. Together, this study represents the first attempt to systematically characterize the intricate relationships among the different mechanisms of persistence and as such provide new insights into the complexity of the persistence phenomenon at the level of persister gene network interactions.

  3. Screening of bifidobacteria and lactobacilli able to antagonise the cytotoxic effect of Clostridium difficile upon intestinal epithelial HT29 monolayer

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    Lorena eValdés-Varela

    2016-04-01

    toxigenic supernatant. Image analysis showed that this strain prevents HT29 cell rounding; this was achieved by preserving the F-actin microstructure and tight-junctions between adjacent cells, thus keeping the typical epithelium-like morphology. Besides, preliminary evidence showed that the viability of B. longum IPLA20022 is needed to exert the protective effect and that secreted factors seems to have anti-toxin activity.

  4. Developing Universal Genetic Tools for Rapid and Efficient Deletion Mutation in Vibrio Species Based on Suicide T-Vectors Carrying a Novel Counterselectable Marker, vmi480.

    Science.gov (United States)

    Luo, Peng; He, Xiangyan; Liu, Qiuting; Hu, Chaoqun

    2015-01-01

    Despite that Vibrio spp. have a significant impact on the health of humans and aquatic animals, the molecular basis of their pathogenesis is little known, mainly due to the limited genetic tools for the functional research of genes in Vibrio. In some cases, deletion of target DNAs in Vibrio can be achieved through the use of suicide vectors. However, these strategies are time-consuming and lack universality, and the widely used counterselectable gene sacB does not work well in Vibrio cells. In this study, we developed universal genetic tools for rapid and efficient deletion mutations in Vibrio species based on suicide T-Vectors carrying a novel counterselectable marker, vmi480. We explored two uncharacterized genes, vmi480 and vmi470, in a genomic island from Vibrio mimicus VM573 and confirmed that vmi480 and vmi470 constitute a two-component toxin-antitoxin system through deletion and expression of vmi480 and vmi470. The product of vmi480 exhibited strong toxicity to Escherichia coli cells. Based on vmi480 and the PBAD or PTAC promoter system, we constructed two suicide T-vectors, pLP11 and pLP12, and each of these vectors contained a multiple cloning region with two AhdI sites. Both vectors linearized by AhdI digestion could be stored and directly ligated with purified PCR products without a digestion step. By using pLP11 and pLP12 coupled with a highly efficient conjugation system provided by E. coli β2163, six genes from four representative Vibrio species were easily deleted. By using the counterselective marker vmi480, we obtained 3-12 positive colonies (deletion mutants) among no more than 20 colonies randomly selected on counterselection plates. The strategy does not require the digestion of PCR products and suicide vectors every time, and it avoids large-scale screening colonies on counterselective plates. These results demonstrate that we successfully developed universal genetic tools for rapid and efficient gene deletion in Vibrio species.

  5. Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A

    Science.gov (United States)

    Benoit, Roger M.; Frey, Daniel; Hilbert, Manuel; Kevenaar, Josta T.; Wieser, Mara M.; Stirnimann, Christian U.; McMillan, David; Ceska, Tom; Lebon, Florence; Jaussi, Rolf; Steinmetz, Michel O.; Schertler, Gebhard F. X.; Hoogenraad, Casper C.; Capitani, Guido; Kammerer, Richard A.

    2014-01-01

    Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral β-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open β-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties.

  6. Isolation and functional characterization of the novel Clostridium botulinum neurotoxin A8 subtype.

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    Skadi Kull

    toxin function might pave the way for the development of novel therapeutics and tailor-made antitoxins.

  7. Characterization of Metagenomes in Urban Aquatic Compartments Reveals High Prevalence of Clinically Relevant Antibiotic Resistance Genes in Wastewaters

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    Charmaine Ng

    2017-11-01

    Full Text Available The dissemination of antimicrobial resistance (AMR is an escalating problem and a threat to public health. Comparative metagenomics was used to investigate the occurrence of antibiotic resistant genes (ARGs in wastewater and urban surface water environments in Singapore. Hospital and municipal wastewater (n = 6 were found to have higher diversity and average abundance of ARGs (303 ARG subtypes, 197,816 x/Gb compared to treated wastewater effluent (n = 2, 58 ARG subtypes, 2,692 x/Gb and surface water (n = 5, 35 subtypes, 7,985 x/Gb. A cluster analysis showed that the taxonomic composition of wastewaters was highly similar and had a bacterial community composition enriched in gut bacteria (Bacteroides, Faecalibacterium, Bifidobacterium, Blautia, Roseburia, Ruminococcus, the Enterobacteriaceae group (Klebsiella, Aeromonas, Enterobacter and opportunistic pathogens (Prevotella, Comamonas, Neisseria. Wastewater, treated effluents and surface waters had a shared resistome of 21 ARGs encoding multidrug resistant efflux pumps or resistance to aminoglycoside, macrolide-lincosamide-streptogramins (MLS, quinolones, sulfonamide, and tetracycline resistance which suggests that these genes are wide spread across different environments. Wastewater had a distinctively higher average abundance of clinically relevant, class A beta-lactamase resistant genes (i.e., blaKPC, blaCTX-M, blaSHV, blaTEM. The wastewaters from clinical isolation wards, in particular, had a exceedingly high levels of blaKPC-2 genes (142,200 x/Gb, encoding for carbapenem resistance. Assembled scaffolds (16 and 30 kbp from isolation ward wastewater samples indicated this gene was located on a Tn3-based transposon (Tn4401, a mobilization element found in Klebsiella pneumonia plasmids. In the longer scaffold, transposable elements were flanked by a toxin–antitoxin (TA system and other metal resistant genes that likely increase the persistence, fitness and propagation of the plasmid in the

  8. Clusters of orthologous genes for 41 archaeal genomes and implications for evolutionary genomics of archaea

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    Wolf Yuri I

    2007-11-01

    that, in addition to the core archaeal functions, encoded more idiosyncratic systems, e.g., the CASS systems of antivirus defense and some toxin-antitoxin systems. Conclusion The arCOGs provide a convenient, flexible framework for functional annotation of archaeal genomes, comparative genomics and evolutionary reconstructions. Genomic reconstructions suggest that the last common ancestor of archaea might have been (nearly as advanced as the modern archaeal hyperthermophiles. ArCOGs and related information are available at: ftp://ftp.ncbi.nih.gov/pub/koonin/arCOGs/. Reviewers This article was reviewed by Peer Bork, Patrick Forterre, and Purificacion Lopez-Garcia.

  9. Encapsulated living choroid plexus cells: potential long-term treatments for central nervous system disease and trauma

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    Skinner, S. J. M.; Geaney, M. S.; Lin, H.; Muzina, M.; Anal, A. K.; Elliott, R. B.; Tan, P. L. J.

    2009-12-01

    In neurodegenerative disease and in acute brain injury, there is often local up-regulation of neurotrophin production close to the site of the lesion. Treatment by direct injection of neurotrophins and growth factors close to these lesion sites has repeatedly been demonstrated to improve recovery. It has therefore been proposed that transplanting viable neurotrophin-producing cells close to the trauma lesion, or site of degenerative disease, might provide a novel means for continuous delivery of these molecules directly to the site of injury or to a degenerative region. The aim of this paper is to summarize recent published information and present new experimental data that indicate that long-lasting therapeutic implants of choroid plexus (CP) neuroepithelium may be used to treat brain disease. CP produces and secretes numerous biologically active neurotrophic factors (NT). New gene microarray and proteomics data presented here indicate that many other anti-oxidant, anti-toxin and neuronal support proteins are also produced and secreted by CP cells. In the healthy brain, these circulate in the cerebrospinal fluid through the brain and spinal cord, maintaining neuronal networks and associated cells. Recent publications describe how transplanted CP cells and tissue, either free or in an immunoprotected encapsulated form, can effectively deliver therapeutic molecules when placed near the lesion or site of degenerative disease in animal models. Using simple techniques, CP neuroepithelial cell clusters in suspension culture were very durable, remaining viable for 6 months or more in vitro. The cell culture conditions had little effect on the wide range and activity of genes expressed and proteins secreted. Recently, completed experiments show that implanting CP within alginate-poly-ornithine capsules effectively protected these xenogeneic cells from the host immune system and allowed their survival for 6 months or more in the brains of rats, causing no adverse effects

  10. Thetet39Determinant and themsrE-mphEGenes in Acinetobacter Plasmids Are Each Part of Discrete Modules Flanked by Inversely Oriented pdif(XerC-XerD) Sites.

    Science.gov (United States)

    Blackwell, Grace A; Hall, Ruth M

    2017-08-01

    The tet39 tetracycline resistance determinant and the macrolide resistance genes msrE and mphE were found in an 18.2-kb plasmid, pS30-1, recovered from a global clone 2 (GC2) Acinetobacter baumannii isolate from Singapore, that conferred resistance to tetracycline and erythromycin. pS30-1 also contains mobA and mobC genes encoding MOB Q family proteins, but attempts to mobilize pS30-1 utilizing a coresident conjugative repAci6 plasmid were unsuccessful. Eight p dif sites, consisting of inversely oriented binding sites for the XerC and XerD recombinases separated by 6 bp, were detected in pS30-1. The tet39 determinant and the msrE-mphE gene pair are each surrounded by two p dif sites in inverse orientation. Identical regions in different contexts and many previously unnoticed p dif sites were found in a number of different plasmids in GenBank, showing that the tet39 and msrE-mphE dif modules are mobile. A putative toxin/antitoxin system, a gene encoding a serine recombinase, and open reading frames of unknown function were also part of dif modules in pS30-1. In general, modules with internal XerC or XerD sites alternate. Two copies of IS Ajo2-1 (94% identical to IS Ajo2 ) in pS30-1 were inserted 5 bp from a XerC site, and this appears to be the preferred insertion site for this insertion sequence (IS) group. Apparently, Acinetobacter plasmids exploit the Acinetobacter XerC-XerD recombinases to mobilize DNA units containing resistance and other genes, via an uncharacterized mechanism. The tet39 and msrE-mphE dif modules add to the oxa24 module and the oxa58 module redefined here, bringing the total of resistance gene-containing dif modules in Acinetobacter plasmids to four. Copyright © 2017 American Society for Microbiology.

  11. Multilevel population genetic analysis of vanA and vanB Enterococcus faecium causing nosocomial outbreaks in 27 countries (1986-2012).

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    Freitas, Ana R; Tedim, Ana P; Francia, Maria V; Jensen, Lars B; Novais, Carla; Peixe, Luísa; Sánchez-Valenzuela, Antonio; Sundsfjord, Arnfinn; Hegstad, Kristin; Werner, Guido; Sadowy, Ewa; Hammerum, Anette M; Garcia-Migura, Lourdes; Willems, Rob J; Baquero, Fernando; Coque, Teresa M

    2016-12-01

    Vancomycin-resistant Enterococcus faecium (VREfm) have been increasingly reported since the 1980s. Despite the high number of published studies about VRE epidemiology, the dynamics and evolvability of these microorganisms are still not fully understood. A multilevel population genetic analysis of VREfm outbreak strains since 1986, representing the first comprehensive characterization of plasmid content in E. faecium, was performed to provide a detailed view of potential transmissible units. From a comprehensive MeSH search, we identified VREfm strains causing hospital outbreaks (1986-2012). In total, 53 VanA and 18 VanB isolates (27 countries, 5 continents) were analysed and 82 vancomycin-susceptible E. faecium (VSEfm) were included for comparison. Clonal relatedness was established by PFGE and MLST (goeBURST/Bayesian Analysis of Population Structure, BAPS). Characterization of van transposons (PCR mapping, RFLP, sequencing), plasmids (transfer, ClaI-RFLP, PCR typing of relaxases, replication-initiation proteins and toxin-antitoxin systems, hybridization, sequencing), bacteriocins and virulence determinants (PCR, hybridization, sequencing) was performed. VREfm were mainly associated with major human lineages ST17, ST18 and ST78. VREfm and VSEfm harboured plasmids of different families [RCR, small theta plasmids, RepA_N (pRUM/pLG1) and Inc18] able to yield mosaic elements. Tn1546-vanA was mainly located on pRUM/Axe-Txe (USA) and Inc18-pIP186 (Europe) plasmids. The VanB2 type (Tn5382/Tn1549) was predominant among VanB strains (chromosome and plasmids). Both strains and plasmids contributed to the spread and persistence of vancomycin resistance among E. faecium. Horizontal gene transfer events among genetic elements from different clonal lineages (same or different species) result in chimeras with different stability and host range, complicating the surveillance of epidemic plasmids. © The Author 2016. Published by Oxford University Press on behalf of the British

  12. Adding to Yersinia enterocolitica Gene Pool Diversity: Two Cryptic Plasmids from a Biotype 1A Isolate

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    Daniela Lepka

    2009-01-01

    Full Text Available We report the nucleotide sequence of two novel cryptic plasmids (4357 and 14 662 base pairs carried by a Yersinia enterocolitica biotype 1A strain isolated from pork. As distinguished from most biotype 1A strains, this isolate, designated 07-04449, exhibited adherence to eukaryotic cells. The smaller plasmid pYe4449-1 carries five attributable open reading frames (ORFs encoding the first CcdA/CcdB-like antitoxin/toxin system described for a Yersinia plasmid, a RepA-like replication initiation protein, and mobilizing factors MobA and MobC. The deduced amino acid sequences showed highest similarity to proteins described in Salmonella (CcdA/B, Klebsiella (RepA, and Plesiomonas (MobA/C indicating genomic fluidity among members of the Enterobacteriaceae. One additional ORF with unknown function, termed ORF5, was identified with an ancestry distinct from the rest of the plasmid. While the C+G content of ORF5 is 38.3%, the rest of pYe4449-1 shows a C+G content of 55.7%. The C+G content of the larger plasmid pYe4449-2 (54.9% was similar to that of pYe4449-1 (53.7% and differed from that of the Y. enterocolitica genome (47.3%. Of the 14 ORFs identified on pYe4449-2, only six ORFs showed significant similarity to database entries. For three of these ORFs likely functions could be ascribed: a TnpR-like resolvase and a phage replication protein, localized each on a low C+G island, and DNA primase TraC. Two ORFs of pYe4449-2, ORF3 and ORF7, seem to encode secretable proteins. Epitope-tagging of ORF3 revealed protein expression at 4°C but not at or above 27°C suggesting adaptation to a habitat outside swine. The hypothetical protein encoded by ORF7 is the member of a novel repeat protein family sharing the DxxGN(xnDxxGN motif. Our findings illustrate the exceptional gene pool diversity within the species Y. enterocolitica driven by horizontal gene transfer events.

  13. Mobility and generation of mosaic non-autonomous transposons by Tn3-derived inverted-repeat miniature elements (TIMEs).

    Science.gov (United States)

    Szuplewska, Magdalena; Ludwiczak, Marta; Lyzwa, Katarzyna; Czarnecki, Jakub; Bartosik, Dariusz

    2014-01-01

    Functional transposable elements (TEs) of several Pseudomonas spp. strains isolated from black shale ore of Lubin mine and from post-flotation tailings of Zelazny Most in Poland, were identified using a positive selection trap plasmid strategy. This approach led to the capture and characterization of (i) 13 insertion sequences from 5 IS families (IS3, IS5, ISL3, IS30 and IS1380), (ii) isoforms of two Tn3-family transposons--Tn5563a and Tn4662a (the latter contains a toxin-antitoxin system), as well as (iii) non-autonomous TEs of diverse structure, ranging in size from 262 to 3892 bp. The non-autonomous elements transposed into AT-rich DNA regions and generated 5- or 6-bp sequence duplications at the target site of transposition. Although these TEs lack a transposase gene, they contain homologous 38-bp-long terminal inverted repeat sequences (IRs), highly conserved in Tn5563a and many other Tn3-family transposons. The simplest elements of this type, designated TIMEs (Tn3 family-derived Inverted-repeat Miniature Elements) (262 bp), were identified within two natural plasmids (pZM1P1 and pLM8P2) of Pseudomonas spp. It was demonstrated that TIMEs are able to mobilize segments of plasmid DNA for transposition, which results in the generation of more complex non-autonomous elements, resembling IS-driven composite transposons in structure. Such transposon-like elements may contain different functional genetic modules in their core regions, including plasmid replication systems. Another non-autonomous element "captured" with a trap plasmid was a TIME derivative containing a predicted resolvase gene and a res site typical for many Tn3-family transposons. The identification of a portable site-specific recombination system is another intriguing example confirming the important role of non-autonomous TEs of the TIME family in shuffling genetic information in bacterial genomes. Transposition of such mosaic elements may have a significant impact on diversity and evolution, not

  14. Mobility and generation of mosaic non-autonomous transposons by Tn3-derived inverted-repeat miniature elements (TIMEs.

    Directory of Open Access Journals (Sweden)

    Magdalena Szuplewska

    Full Text Available Functional transposable elements (TEs of several Pseudomonas spp. strains isolated from black shale ore of Lubin mine and from post-flotation tailings of Zelazny Most in Poland, were identified using a positive selection trap plasmid strategy. This approach led to the capture and characterization of (i 13 insertion sequences from 5 IS families (IS3, IS5, ISL3, IS30 and IS1380, (ii isoforms of two Tn3-family transposons--Tn5563a and Tn4662a (the latter contains a toxin-antitoxin system, as well as (iii non-autonomous TEs of diverse structure, ranging in size from 262 to 3892 bp. The non-autonomous elements transposed into AT-rich DNA regions and generated 5- or 6-bp sequence duplications at the target site of transposition. Although these TEs lack a transposase gene, they contain homologous 38-bp-long terminal inverted repeat sequences (IRs, highly conserved in Tn5563a and many other Tn3-family transposons. The simplest elements of this type, designated TIMEs (Tn3 family-derived Inverted-repeat Miniature Elements (262 bp, were identified within two natural plasmids (pZM1P1 and pLM8P2 of Pseudomonas spp. It was demonstrated that TIMEs are able to mobilize segments of plasmid DNA for transposition, which results in the generation of more complex non-autonomous elements, resembling IS-driven composite transposons in structure. Such transposon-like elements may contain different functional genetic modules in their core regions, including plasmid replication systems. Another non-autonomous element "captured" with a trap plasmid was a TIME derivative containing a predicted resolvase gene and a res site typical for many Tn3-family transposons. The identification of a portable site-specific recombination system is another intriguing example confirming the important role of non-autonomous TEs of the TIME family in shuffling genetic information in bacterial genomes. Transposition of such mosaic elements may have a significant impact on diversity and

  15. The IncF plasmid pRSB225 isolated from a municipal wastewater treatment plant's on-site preflooder combining antibiotic resistance and putative virulence functions is highly related to virulence plasmids identified in pathogenic E. coli isolates.

    Science.gov (United States)

    Wibberg, Daniel; Szczepanowski, Rafael; Eikmeyer, Felix; Pühler, Alfred; Schlüter, Andreas

    2013-03-01

    The IncF antibiotic resistance and virulence plasmid pRSB225, isolated from an unknown bacterium released with the purified wastewater from a municipal sewage treatment plant into the environment has been analysed at the genomic level by pyrosequencing. The 164,550bp plasmid comprises 210 coding sequences (cds). It is composed of three replicons (RepFIA, RepFIB, and RepFII) and encodes further plasmid-specific functions for stable maintenance and inheritance and conjugative plasmid transfer. The plasmid is self-transmissible and shows a narrow host range limited to the family Enterobacteriaceae. The accessory modules of the plasmid mainly comprise genes conferring resistance to ampicillin (bla(TEM-1b)), chloramphenicol (catA1), erythromycin (mphA), kanamycin and neomycin (aphA1), streptomycin (strAB), sulphonamides (sul2), tetracycline (tetA(B)) and trimethoprim (dfrA14), as well as mercuric ions (mer genes). In addition, putative virulence-associated genes coding for iron uptake (iutA/iucABCD, sitABCD, and a putative high-affinity Fe²⁺ uptake system) and for a toxin/antitoxin system (vagCD) were identified on the plasmid. All antibiotic and heavy metal resistance genes are located either on class 1 (Tn10-remnant, Tn4352B) and class 2 transposons (Tn2-remnant, Tn21, Tn402-remnant) or a class 1 integron, whereas almost all putative virulence genes are associated with IS elements (IS1, IS26), indicating that transposition and/or recombination events were responsible for acquisition of the accessory pRSB225 modules. Particular modules of plasmid pRSB225 are related to corresponding segments of different virulence plasmids harboured by pathogenic Escherichia coli strains. Moreover, pRSB225 modules were also detected in entero-aggregative-haemorrhagic E. coli (EAHEC) draft genome sequences suggesting that IncF plasmids related to pRSB225 mediated gene transfer into pathogenic E. coli derivatives. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. A cross-sectional survey of Thoroughbred stud farm management in the North Island of New Zealand.

    Science.gov (United States)

    Rogers, C W; Gee, E K; Firth, E C

    2007-12-01

    To obtain initial baseline data on the management of Thoroughbred stud farms in the North Island of New Zealand. Data on the management of Thoroughbred stud farms were collected from a sample of 22 stud farms located in the south Auckland/Waikato region (n=15) and lower North Island (n=7) of New Zealand, using a face-to-face survey. The studmaster provided information on the size, scope and management of the farms during the 2004/2005 breeding season. Analysis was based on the location of the farm and size of the breeding operation (number of resident mares). Effective farm size ranged from 20 to 526 ha and averaged 167 (standard error (SE) 36) and 88 (SE 49) ha in the south Auckland/Waikato and lower North Island areas, respectively. Some farms in the Auckland/Waikato region stood shuttle stallions. The median number of stallions per farm was three (range 0-9), and the median mare-to-stallion ratio was 43 (range 10-250). The farms had a mean of 50 (range 7-180) wet mares and 21 (range 0-100) dry mares. The number of mares per breeding stallion increased with increasing size of breeding operation (p=0.04), being 28 (95% confidence interval (CI) = 10-56) vs 40 (95% CI=16-74) vs 74 (95% CI=44-113) for moderate (or=200 mares in total) operations, respectively. Seventy-one percent of farms aimed to breed dry mares early in the breeding season, and used a combination of lights, hormone therapy, and rising plane of nutrition to achieve this. Foaling took place in foaling paddocks monitored using a night foaling attendant (17/22) or with foaling alarms (5/22). At birth, 17/22 studmasters routinely administered antibiotics, 14/22 administered tetanus antitoxin, 9/22 administered an enema to foals, and 2/22 did not routinely administer prophylactic treatments. Weaning occurred at 5 (range 3.7-7) months of age, and foals were confined to a box for 1-2 weeks on 16/22 farms. Weaned foals were drenched with anthelmintics every 7 (range 4-9) weeks, and were fed 2.9 (range 1

  17. Vaccines for women to prevent neonatal tetanus.

    Science.gov (United States)

    Demicheli, Vittorio; Barale, Antonella; Rivetti, Alessandro

    2013-05-31

    Tetanus is an acute, often fatal, disease caused by an exotoxin produced by Clostridium tetani. It occurs in newborn infants born to mothers who do not have sufficient circulating antibodies to protect the infant passively, by transplacental transfer. Prevention may be possible by the vaccination of pregnant or non-pregnant women, or both, with tetanus toxoid, and the provision of clean delivery services. Tetanus toxoid consists of a formaldehyde-treated toxin which stimulates the production of antitoxin. To assess the effectiveness of tetanus toxoid, administered to women of childbearing age or pregnant women, to prevent cases of, and deaths from, neonatal tetanus. We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (31 October 2012), The Cochrane Library (2012, Issue 10), PubMed (1966 to 31 October 2012), EMBASE (1974 to 31 October 2012). We also used the results from handsearching and consultations with manufacturers and authors. Randomised or quasi-randomised trials evaluating the effects of tetanus toxoid in pregnant women or women of childbearing age on numbers of neonatal tetanus cases and deaths. Three review authors independently assessed trials for inclusion and trial quality, and extracted data. Two trials (10,560 infants) were included. It should be noted that these trials are very old,1966 and 1980 respectively, and one trial randomised exclusively non-pregnant women. The main outcomes were measured on infants born to a subset of those randomised women who became pregnant during the course of the studies. One study (1919 infants) assessed the effectiveness of tetanus toxoid in comparison with influenza vaccine in preventing neonatal tetanus deaths. After a single dose, the risk ratio (RR) was 0.57 (95% confidence interval (CI) 0.26 to 1.24), and the vaccine effectiveness was 43%. With a two- or three-dose course, the RR was 0.02 (95% CI 0.00 to 0.30); vaccine effectiveness was 98%. No effect was detected on causes of death other

  18. Vaccines for women for preventing neonatal tetanus.

    Science.gov (United States)

    Demicheli, Vittorio; Barale, Antonella; Rivetti, Alessandro

    2015-07-06

    Tetanus is an acute, often fatal, disease caused by an exotoxin produced by Clostridium tetani. It occurs in newborn infants born to mothers who do not have sufficient circulating antibodies to protect the infant passively, by transplacental transfer. Prevention may be possible by the vaccination of pregnant or non-pregnant women, or both, with tetanus toxoid, and the provision of clean delivery services. Tetanus toxoid consists of a formaldehyde-treated toxin that stimulates the production of antitoxin. To assess the effectiveness of tetanus toxoid, administered to women of reproductive age or pregnant women, to prevent cases of, and deaths from, neonatal tetanus. We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (31 January 2015), CENTRAL (The Cochrane Library 2015, Issue 1), PubMed (1966 to 28 January 2015), EMBASE (1974 to 28 January 2015) and reference lists of retrieved studies. Randomised or quasi-randomised trials evaluating the effects of tetanus toxoid in pregnant women or women of reproductive age on numbers of neonatal tetanus cases and deaths. Two review authors independently assessed trials for inclusion and risk of bias, extracted data and checked them for accuracy. Two effectiveness trials (9823 infants) and one safety trial (48 mothers) were included. The main outcomes were measured on infants born to a subset of those randomised women who became pregnant during the course of the studies. For our primary outcomes, there was no high-quality evidence according to GRADE assessments.One study (1182 infants) assessed the effectiveness of tetanus toxoid in comparison with influenza vaccine in preventing neonatal tetanus deaths. A single dose did not provide significant protection against neonatal tetanus deaths, (risk ratio (RR) 0.57, 95% confidence interval (CI) 0.26 to 1.24; 494 infants; GRADE: low-quality evidence). However, a two- or three-dose course did provide protection against neonatal deaths, (RR 0.02, 95% CI 0.00 to 0

  19. Doctor’s identity in modern Western society

    Directory of Open Access Journals (Sweden)

    KIM Ock-Joo

    2005-06-01

    Full Text Available Two centuries ago doctors perceived themselves quite differently as they do today Doctor’s identity in modern Western society shaped from the modernization of medicine starting in the nineteenth century Modern medicine as practiced today was established from 1800 to the World War I In the eighteenth century three medical groups (physicians surgeons and apothecaries struggled to elevate their position and to organize their education Surgery and surgical education in hospitals developed greatly while physicians tried to theorize their own medical system in the eighteenth century In the early nineteenth century hospital medicine emerged hospitals moved from the place for the poor and the social inadequate to the center of medical education and research Especially French hospitals became the birth places of clinico-pathology new diagnosis with stethoscope careful observation and the numerical method The influence of the hospital medicine spread from France to England America and other parts of Europe After the birth of clinic in France laboratory medicine emerged in Germany France Britain and the United States Surpassing other nations Germany developed university-centered laboratory research system Most of all the reward and status of the laboratory researchers were established so that they could concentrate on their research Although other countries were influenced by German system and knowledge they did not develop research system at the same degree as Germany Rise of scientific medicine transformed self-perception of doctors Science made a great impact not only on the doctors’ practice of medicine but also on the public’s perception of medicine and doctors In the late nineteenth century new discoveries and new armament of scientific medicine marched through antiseptic surgery tropical medicine new laboratories antitoxin therapies from immunology the rise of pharmaceutical industry and the discovery of X-ray Payment system also was changed